Sample records for early vertebrate embryos

  1. HNK-1 immunoreactivity during early morphogenesis of the head region in a nonmodel vertebrate, crocodile embryo

    NASA Astrophysics Data System (ADS)

    Kundrát, Martin

    2008-11-01

    The present study examines HNK-1 immunoidentification of a population of the neural crest (NC) during early head morphogenesis in the nonmodel vertebrate, the crocodile ( Crocodylus niloticus) embryos. Although HNK-1 is not an exclusive NC marker among vertebrates, temporospatial immunoreactive patterns found in the crocodile are almost consistent with NC patterns derived from gene expression studies known in birds (the closest living relatives of crocodiles) and mammals. In contrast to birds, the HNK-1 epitope is immunoreactive in NC cells at the neural fold level in crocodile embryos and therefore provides sufficient base to assess early migratory events of the cephalic NC. I found that crocodile NC forms three classic migratory pathways in the head: mandibular, hyoid, and branchial. Further, I demonstrate that, besides this classic phenotype, there is also a forebrain-derived migratory population, which consolidates into a premandibular stream in the crocodile. In contrast to the closely related chick model, crocodilian premandibular and mandibular NC cells arise from the open neural tube suggesting that species-specific heterochronic behavior of NC may be involved in the formation of different vertebrate facial phenotypes.

  2. Closure of the vertebral canal in human embryos and fetuses.

    PubMed

    Mekonen, Hayelom K; Hikspoors, Jill P J M; Mommen, Greet; Kruepunga, Nutmethee; Köhler, S Eleonore; Lamers, Wouter H

    2017-08-01

    The vertebral column is the paradigm of the metameric architecture of the vertebrate body. Because the number of somites is a convenient parameter to stage early human embryos, we explored whether the closure of the vertebral canal could be used similarly for staging embryos between 7 and 10 weeks of development. Human embryos (5-10 weeks of development) were visualized using Amira 3D ® reconstruction and Cinema 4D ® remodelling software. Vertebral bodies were identifiable as loose mesenchymal structures between the dense mesenchymal intervertebral discs up to 6 weeks and then differentiated into cartilaginous structures in the 7th week. In this week, the dense mesenchymal neural processes also differentiated into cartilaginous structures. Transverse processes became identifiable at 6 weeks. The growth rate of all vertebral bodies was exponential and similar between 6 and 10 weeks, whereas the intervertebral discs hardly increased in size between 6 and 8 weeks and then followed vertebral growth between 8 and 10 weeks. The neural processes extended dorsolaterally (6th week), dorsally (7th week) and finally dorsomedially (8th and 9th weeks) to fuse at the midthoracic level at 9 weeks. From there, fusion extended cranially and caudally in the 10th week. Closure of the foramen magnum required the development of the supraoccipital bone as a craniomedial extension of the exoccipitals (neural processes of occipital vertebra 4), whereas a growth burst of sacral vertebra 1 delayed closure until 15 weeks. Both the cranial- and caudal-most vertebral bodies fused to form the basioccipital (occipital vertebrae 1-4) and sacrum (sacral vertebrae 1-5). In the sacrum, fusion of its so-called alar processes preceded that of the bodies by at least 6 weeks. In conclusion, the highly ordered and substantial changes in shape of the vertebral bodies leading to the formation of the vertebral canal make the development of the spine an excellent, continuous staging system for

  3. Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos.

    PubMed

    Uchida, Yui; Uesaka, Masahiro; Yamamoto, Takayoshi; Takeda, Hiroyuki; Irie, Naoki

    2018-01-01

    Understanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this "mid-embryonic lethality hypothesis" by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations. By subjecting zebrafish ( Danio rerio ), African clawed frog ( Xenopus laevis ), and chicken ( Gallus gallus ) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species. In opposition to the "mid-embryonic lethality hypothesis," our results consistently showed that the stage with the highest lethality was not around the

  4. Establishment of left–right asymmetry in vertebrate development: the node in mouse embryos

    PubMed Central

    Komatsu, Yoshihiro

    2014-01-01

    Establishment of vertebrate left–right asymmetry is a critical process for normal embryonic development. After the discovery of genes expressed asymmetrically along the left–right axis in chick embryos in the mid 1990s, the molecular mechanisms responsible for left–right patterning in vertebrate embryos have been studied extensively. In this review article, we discuss the mechanisms by which the initial symmetry along the left–right axis is broken in the mouse embryo. We focus on the role of primary cilia and molecular mechanisms of ciliogenesis at the node when symmetry is broken and left–right asymmetry is established. The node is considered a signaling center for early mouse embryonic development, and the results we review here have led to a better understanding of how the node functions and establishes left–right asymmetry. PMID:23771646

  5. Establishment of left-right asymmetry in vertebrate development: the node in mouse embryos.

    PubMed

    Komatsu, Yoshihiro; Mishina, Yuji

    2013-12-01

    Establishment of vertebrate left-right asymmetry is a critical process for normal embryonic development. After the discovery of genes expressed asymmetrically along the left-right axis in chick embryos in the mid 1990s, the molecular mechanisms responsible for left-right patterning in vertebrate embryos have been studied extensively. In this review article, we discuss the mechanisms by which the initial symmetry along the left-right axis is broken in the mouse embryo. We focus on the role of primary cilia and molecular mechanisms of ciliogenesis at the node when symmetry is broken and left-right asymmetry is established. The node is considered a signaling center for early mouse embryonic development, and the results we review here have led to a better understanding of how the node functions and establishes left-right asymmetry.

  6. Efficient harvesting methods for early-stage snake and turtle embryos.

    PubMed

    Matsubara, Yoshiyuki; Kuroiwa, Atsushi; Suzuki, Takayuki

    2016-04-01

    Reptile development is an intriguing research target for understating the unique morphogenesis of reptiles as well as the evolution of vertebrates. However, there are numerous difficulties associated with studying development in reptiles. The number of available reptile eggs is usually quite limited. In addition, the reptile embryo is tightly adhered to the eggshell, making it a challenge to isolate reptile embryos intact. Furthermore, there have been few reports describing efficient procedures for isolating intact embryos especially prior to pharyngula stage. Thus, the aim of this review is to present efficient procedures for obtaining early-stage reptilian embryos intact. We first describe the method for isolating early-stage embryos of the Japanese striped snake. This is the first detailed method for obtaining embryos prior to oviposition in oviparous snake species. Second, we describe an efficient strategy for isolating early-stage embryos of the soft-shelled turtle. © 2016 Japanese Society of Developmental Biologists.

  7. Heat shock during early somitogenesis induces caudal vertebral column defects in Atlantic salmon (Salmo salar).

    PubMed

    Wargelius, Anna; Fjelldal, Per Gunnar; Hansen, Tom

    2005-07-01

    In several terrestrial vertebrates, heat shock (HS) during somitogenesis causes vertebral deformities. To determine if vertebral deformities can occur due to sudden temperature changes during early development in fish, Atlantic salmon embryos were HS treated during somitogenesis. Ten months later these individuals displayed a high prevalence of caudal vertebral column condensations (27-34%). The defects were located caudally of the abdominal cavity, displaying an even distribution in this region independent of time of HS. To determine if HS disturbed vertebral development during somitogenesis, two genes coding for markers of skeletal development were identified, namely, the secreted protein Shh (Sashh) and the transcription factor Twist (Satwist). These proteins are involved in the proliferation and specification of presumptive skeletal cells (sclerotome) in vertebrates. The spatial expression pattern of sashh and satwist in salmon indicated a functional conservation of these proteins. Furthermore, HS embryos displayed expressional disturbance in both sashh and satwist, indicating an effect of HS on sclerotomal cell patterning. However, the HS-protecting ability in embryos seems to be individually regulated because reduction in gene expression was not detected at all stages; in addition, HS did not induce somitic disturbance and vertebral deformity in all embryos.

  8. Tissue morphodynamics shaping the early mouse embryo.

    PubMed

    Sutherland, Ann E

    2016-07-01

    Generation of the elongated vertebrate body plan from the initially radially symmetrical embryo requires comprehensive changes to tissue form. These shape changes are generated by specific underlying cell behaviors, coordinated in time and space. Major principles and also specifics are emerging, from studies in many model systems, of the cell and physical biology of how region-specific cell behaviors produce regional tissue morphogenesis, and how these, in turn, are integrated at the level of the embryo. New technical approaches have made it possible more recently, to examine the morphogenesis of the mouse embryo in depth, and to elucidate the underlying cellular mechanisms. This review focuses on recent advances in understanding the cellular basis for the early fundamental events that establish the basic form of the embryo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution.

    PubMed

    Johnson, Andrew D; Drum, Matthew; Bachvarova, Rosemary F; Masi, Thomas; White, Mary E; Crother, Brian I

    2003-01-01

    The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.

  10. The generation of vertebral segmental patterning in the chick embryo

    PubMed Central

    Senthinathan, Biruntha; Sousa, Cátia; Tannahill, David; Keynes, Roger

    2012-01-01

    We have carried out a series of experimental manipulations in the chick embryo to assess whether the notochord, neural tube and spinal nerves influence segmental patterning of the vertebral column. Using Pax1 expression in the somite-derived sclerotomes as a marker for segmentation of the developing intervertebral disc, our results exclude such an influence. In contrast to certain teleost species, where the notochord has been shown to generate segmentation of the vertebral bodies (chordacentra), these experiments indicate that segmental patterning of the avian vertebral column arises autonomously in the somite mesoderm. We suggest that in amniotes, the subdivision of each sclerotome into non-miscible anterior and posterior halves plays a critical role in establishing vertebral segmentation, and in maintaining left/right alignment of the developing vertebral elements at the body midline. PMID:22458512

  11. The generation of vertebral segmental patterning in the chick embryo.

    PubMed

    Senthinathan, Biruntha; Sousa, Cátia; Tannahill, David; Keynes, Roger

    2012-06-01

    We have carried out a series of experimental manipulations in the chick embryo to assess whether the notochord, neural tube and spinal nerves influence segmental patterning of the vertebral column. Using Pax1 expression in the somite-derived sclerotomes as a marker for segmentation of the developing intervertebral disc, our results exclude such an influence. In contrast to certain teleost species, where the notochord has been shown to generate segmentation of the vertebral bodies (chordacentra), these experiments indicate that segmental patterning of the avian vertebral column arises autonomously in the somite mesoderm. We suggest that in amniotes, the subdivision of each sclerotome into non-miscible anterior and posterior halves plays a critical role in establishing vertebral segmentation, and in maintaining left/right alignment of the developing vertebral elements at the body midline. © 2012 The Authors. Journal of Anatomy © 2012 Anatomical Society.

  12. Enzymatic Metabolism of Vitamin A in Developing Vertebrate Embryos

    PubMed Central

    Metzler, Melissa A.; Sandell, Lisa L.

    2016-01-01

    Embryonic development is orchestrated by a small number of signaling pathways, one of which is the retinoic acid (RA) signaling pathway. Vitamin A is essential for vertebrate embryonic development because it is the molecular precursor of the essential signaling molecule RA. The level and distribution of RA signaling within a developing embryo must be tightly regulated; too much, or too little, or abnormal distribution, all disrupt embryonic development. Precise regulation of RA signaling during embryogenesis is achieved by proteins involved in vitamin A metabolism, retinoid transport, nuclear signaling, and RA catabolism. The reversible first step in conversion of the precursor vitamin A to the active retinoid RA is mediated by retinol dehydrogenase 10 (RDH10) and dehydrogenase/reductase (SDR family) member 3 (DHRS3), two related membrane-bound proteins that functionally activate each other to mediate the interconversion of retinol and retinal. Alcohol dehydrogenase (ADH) enzymes do not contribute to RA production under normal conditions during embryogenesis. Genes involved in vitamin A metabolism and RA catabolism are expressed in tissue-specific patterns and are subject to feedback regulation. Mutations in genes encoding these proteins disrupt morphogenesis of many systems in a developing embryo. Together these observations demonstrate the importance of vitamin A metabolism in regulating RA signaling during embryonic development in vertebrates. PMID:27983671

  13. The first whole transcriptomic exploration of pre-oviposited early chicken embryos using single and bulked embryonic RNA-sequencing.

    PubMed

    Hwang, Young Sun; Seo, Minseok; Choi, Hee Jung; Kim, Sang Kyung; Kim, Heebal; Han, Jae Yong

    2018-04-01

    The chicken is a valuable model organism, especially in evolutionary and embryology research because its embryonic development occurs in the egg. However, despite its scientific importance, no transcriptome data have been generated for deciphering the early developmental stages of the chicken because of practical and technical constraints in accessing pre-oviposited embryos. Here, we determine the entire transcriptome of pre-oviposited avian embryos, including oocyte, zygote, and intrauterine embryos from Eyal-giladi and Kochav stage I (EGK.I) to EGK.X collected using a noninvasive approach for the first time. We also compare RNA-sequencing data obtained using a bulked embryo sequencing and single embryo/cell sequencing technique. The raw sequencing data were preprocessed with two genome builds, Galgal4 and Galgal5, and the expression of 17,108 and 26,102 genes was quantified in the respective builds. There were some differences between the two techniques, as well as between the two genome builds, and these were affected by the emergence of long intergenic noncoding RNA annotations. The first transcriptome datasets of pre-oviposited early chicken embryos based on bulked and single embryo sequencing techniques will serve as a valuable resource for investigating early avian embryogenesis, for comparative studies among vertebrates, and for novel gene annotation in the chicken genome.

  14. Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.

    PubMed

    Trivedi, Vikas; Choi, Harry M T; Fraser, Scott E; Pierce, Niles A

    2018-01-08

    For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization. © 2018. Published by The Company of Biologists Ltd.

  15. Confocal imaging of whole vertebrate embryos reveals novel insights into molecular and cellular mechanisms of organ development

    NASA Astrophysics Data System (ADS)

    Hadel, Diana M.; Keller, Bradley B.; Sandell, Lisa L.

    2014-03-01

    Confocal microscopy has been an invaluable tool for studying cellular or sub-cellular biological processes. The study of vertebrate embryology is based largely on examination of whole embryos and organs. The application of confocal microscopy to immunostained whole mount embryos, combined with three dimensional (3D) image reconstruction technologies, opens new avenues for synthesizing molecular, cellular and anatomical analysis of vertebrate development. Optical cropping of the region of interest enables visualization of structures that are morphologically complex or obscured, and solid surface rendering of fluorescent signal facilitates understanding of 3D structures. We have applied these technologies to whole mount immunostained mouse embryos to visualize developmental morphogenesis of the mammalian inner ear and heart. Using molecular markers of neuron development and transgenic reporters of neural crest cell lineage we have examined development of inner ear neurons that originate from the otic vesicle, along with the supporting glial cells that derive from the neural crest. The image analysis reveals a previously unrecognized coordinated spatial organization between migratory neural crest cells and neurons of the cochleovestibular nerve. The images also enable visualization of early cochlear spiral nerve morphogenesis relative to the developing cochlea, demonstrating a heretofore unknown association of neural crest cells with extending peripheral neurite projections. We performed similar analysis of embryonic hearts in mouse and chick, documenting the distribution of adhesion molecules during septation of the outflow tract and remodeling of aortic arches. Surface rendering of lumen space defines the morphology in a manner similar to resin injection casting and micro-CT.

  16. Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

    PubMed Central

    McEwen, Gayle K.; Goode, Debbie K.; Parker, Hugo J.; Woolfe, Adam; Callaway, Heather; Elgar, Greg

    2009-01-01

    Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA. PMID:20011110

  17. The evolution of early vertebrate photoreceptors.

    PubMed

    Collin, Shaun P; Davies, Wayne L; Hart, Nathan S; Hunt, David M

    2009-10-12

    Meeting the challenge of sampling an ancient aquatic landscape by the early vertebrates was crucial to their survival and would establish a retinal bauplan to be used by all subsequent vertebrate descendents. Image-forming eyes were under tremendous selection pressure and the ability to identify suitable prey and detect potential predators was thought to be one of the major drivers of speciation in the Early Cambrian. Based on the fossil record, we know that hagfishes, lampreys, holocephalans, elasmobranchs and lungfishes occupy critical stages in vertebrate evolution, having remained relatively unchanged over hundreds of millions of years. Now using extant representatives of these 'living fossils', we are able to piece together the evolution of vertebrate photoreception. While photoreception in hagfishes appears to be based on light detection and controlling circadian rhythms, rather than image formation, the photoreceptors of lampreys fall into five distinct classes and represent a critical stage in the dichotomy of rods and cones. At least four types of retinal cones sample the visual environment in lampreys mediating photopic (and potentially colour) vision, a sampling strategy retained by lungfishes, some modern teleosts, reptiles and birds. Trichromacy is retained in cartilaginous fishes (at least in batoids and holocephalans), where it is predicted that true scotopic (dim light) vision evolved in the common ancestor of all living gnathostomes. The capacity to discriminate colour and balance the tradeoff between resolution and sensitivity in the early vertebrates was an important driver of eye evolution, where many of the ocular features evolved were retained as vertebrates progressed on to land.

  18. Glassfrog embryos hatch early after parental desertion.

    PubMed

    Delia, Jesse R J; Ramírez-Bautista, Aurelio; Summers, Kyle

    2014-06-22

    Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations.

  19. Glassfrog embryos hatch early after parental desertion

    PubMed Central

    Delia, Jesse R. J.; Ramírez-Bautista, Aurelio; Summers, Kyle

    2014-01-01

    Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations. PMID:24789892

  20. Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae).

    PubMed

    González-Gutiérrez, Alejandra G; Gutiérrez-Mora, Antonia; Rodríguez-Garay, Benjamín

    2014-01-01

    Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer's-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded.

  1. Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

    PubMed

    Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

    2014-10-13

    Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (<1 hr) P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

  2. [Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

    PubMed

    Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

    2014-06-01

    To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

  3. Long-term in vivo study of vertebrate embryonic development using noninvasive harmonics optical microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Szu-Yu; Hsieh, C.-S.; Chu, S.-W.; Lin, Cheng-Yung; Ko, C.-Y.; Chen, Y.-C.; Tsai, Huai-Jen; Hu, C.-H.; Sun, Chi-Kuang

    2005-03-01

    Harmonics optical microscopy (HOM) provides a truly "noninvasive" tool for in vivo and long-term study of vertebrate embryonic development. Based on the nonlinear natures, it provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power (~1μm axial resolution) without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamages. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can be used to do functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Zebrafish embryos now have been used to study many vertebrate physiological systems. We have demonstrated an in vivo HOM study of developmental dynamics of several embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.

  4. Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos.

    PubMed

    Chandramore, Kalpana; Ito, Yuzuro; Takahashi, Shuji; Asashima, Makoto; Ghaskadbi, Surendra

    2010-01-01

    Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.

  5. Expression of renin–angiotensin system components in the early bovine embryo

    PubMed Central

    Pijacka, Wioletta; Hunter, Morag G; Broughton Pipkin, Fiona; Luck, Martin R

    2012-01-01

    The renin–angiotensin system (RAS), mainly associated with the regulation of blood pressure, has been recently investigated in female reproductive organs and the developing foetus. Angiotensin II (Ang II) influences oviductal gamete movements and foetal development, but there is no information about RAS in the early embryo. The aim of this study was to determine whether RAS components are present in the pre-implantation embryo, to determine how early they are expressed and to investigate their putative role at this stage of development. Bovine embryos produced in vitro were used for analysis of RAS transcripts (RT-PCR) and localisation of the receptors AGTR1 and AGTR2 (immunofluorescent labelling). We also investigated the effects of Ang II, Olmesartan (AGTR1 antagonist) and PD123319 (AGTR2 antagonist) on oocyte cleavage, embryo expansion and hatching. Pre-implanted embryos possessed AGTR1 and AGTR2 but not the other RAS components. Both receptors were present in the trophectoderm and in the inner cell mass of the blastocyst. AGTR1 was mainly localised in granular-like structures in the cytoplasm, suggesting its internalisation into clathrin-coated vesicles, and AGTR2 was found mainly in the nuclear membrane and in the mitotic spindle of dividing trophoblastic cells. Treating embryos with PD123319 increased the proportion of hatched embryos compared with the control. These results, the first on RAS in the early embryo, suggest that the pre-implanted embryo responds to Ang II from the mother rather than from the embryo itself. This may be a route by which the maternal RAS influences blastocyst hatching and early embryonic development. PMID:23781300

  6. What are Head Cavities? - A History of Studies on Vertebrate Head Segmentation.

    PubMed

    Kuratani, Shigeru; Adachi, Noritaka

    2016-06-01

    Motivated by the discovery of segmental epithelial coeloms, or "head cavities," in elasmobranch embryos toward the end of the 19th century, the debate over the presence of mesodermal segments in the vertebrate head became a central problem in comparative embryology. The classical segmental view assumed only one type of metamerism in the vertebrate head, in which each metamere was thought to contain one head somite and one pharyngeal arch, innervated by a set of cranial nerves serially homologous to dorsal and ventral roots of spinal nerves. The non-segmental view, on the other hand, rejected the somite-like properties of head cavities. A series of small mesodermal cysts in early Torpedo embryos, which were thought to represent true somite homologs, provided a third possible view on the nature of the vertebrate head. Recent molecular developmental data have shed new light on the vertebrate head problem, explaining that head mesoderm evolved, not by the modification of rostral somites of an amphioxus-like ancestor, but through the polarization of unspecified paraxial mesoderm into head mesoderm anteriorly and trunk somites posteriorly.

  7. Impact of PCOS on early embryo cleavage kinetics.

    PubMed

    Wissing, M L; Bjerge, M R; Olesen, A I G; Hoest, T; Mikkelsen, A L

    2014-04-01

    This study investigated whether polycystic ovary syndrome (PCOS) affected early embryo development assessed by time-lapse analysis of embryo kinetics from fertilization to the blastocyst stage. This was a prospective cohort study of two pronuclei (2PN) embryos from 25 hyperandrogenic PCOS patients (110 2PN embryos), 26 normoandrogenic PCOS patients (140 2PN embryos) and 20 healthy, regularly cycling women (controls, 97 2PN embryos). Patients underwent the same baseline evaluation and the same ovarian stimulation from April 2010 to February 2013. Oocytes were fertilized by intracytoplasmic sperm injection and incubated in an EmbryoScope with pictures taken every 20 min in seven focal planes. Time to 2PN breakdown, first cleavage and cleavage to 3, 4, 5, 6, 7 and 8 cells, morula and blastocyst (t₂, t₃, t₄, t₅, t₆, t₇, t₈, t(M), t(B)) were annotated. Differences in embryo kinetics between groups were assessed by mixed modelling. Compared with controls, embryos from hyperandrogenic PCOS patients were significantly delayed at 2PN breakdown, t₂, t₃, t₄ and t₇ but not at t₅, t₆, t₈, t(M) or t(B). Embryos from hyperandrogenic PCOS women had developed slower from fertilization to the 8-cell stage compared with embryos from controls. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  8. Approaches and species in the history of vertebrate embryology.

    PubMed

    Hopwood, Nick

    2011-01-01

    Recent debates about model organisms echo far into the past; taking a longer view adds perspective to present concerns. The major approaches in the history of research on vertebrate embryos have tended to exploit different species, though there are long-term continuities too. Early nineteenth-century embryologists worked on surrogates for humans and began to explore the range of vertebrate embryogenesis; late nineteenth-century Darwinists hunted exotic ontogenies; around 1900 experimentalists favored living embryos in which they could easily intervene; reproductive scientists tackled farm animals and human beings; after World War II developmental biologists increasingly engineered species for laboratory life; and proponents of evo-devo have recently challenged the resulting dominance of a few models. Decisions about species have depended on research questions, biological properties, supply lines, and, not least, on methods. Nor are species simply chosen; embryology has transformed them even as they have profoundly shaped the science.

  9. Evolution of vertebrate central nervous system is accompanied by novel expression changes of duplicate genes.

    PubMed

    Chen, Yuan; Ding, Yun; Zhang, Zuming; Wang, Wen; Chen, Jun-Yuan; Ueno, Naoto; Mao, Bingyu

    2011-12-20

    The evolution of the central nervous system (CNS) is one of the most striking changes during the transition from invertebrates to vertebrates. As a major source of genetic novelties, gene duplication might play an important role in the functional innovation of vertebrate CNS. In this study, we focused on a group of CNS-biased genes that duplicated during early vertebrate evolution. We investigated the tempo-spatial expression patterns of 33 duplicate gene families and their orthologs during the embryonic development of the vertebrate Xenopus laevis and the cephalochordate Brachiostoma belcheri. Almost all the identified duplicate genes are differentially expressed in the CNS in Xenopus embryos, and more than 50% and 30% duplicate genes are expressed in the telencephalon and mid-hindbrain boundary, respectively, which are mostly considered as two innovations in the vertebrate CNS. Interestingly, more than 50% of the amphioxus orthologs do not show apparent expression in the CNS in amphioxus embryos as detected by in situ hybridization, indicating that some of the vertebrate CNS-biased duplicate genes might arise from non-CNS genes in invertebrates. Our data accentuate the functional contribution of gene duplication in the CNS evolution of vertebrate and uncover an invertebrate non-CNS history for some vertebrate CNS-biased duplicate genes. Copyright © 2011. Published by Elsevier Ltd.

  10. Extra-embryonic tissue spreading directs early embryo morphogenesis in killifish

    PubMed Central

    Reig, Germán; Cerda, Mauricio; Sepúlveda, Néstor; Flores, Daniela; Castañeda, Victor; Tada, Masazumi; Härtel, Steffen; Concha, Miguel L.

    2017-01-01

    The spreading of mesenchymal-like cell layers is critical for embryo morphogenesis and tissue repair, yet we know little of this process in vivo. Here we take advantage of unique developmental features of the non-conventional annual killifish embryo to study the principles underlying tissue spreading in a simple cellular environment, devoid of patterning signals and major morphogenetic cell movements. Using in vivo experimentation and physical modelling we reveal that the extra-embryonic epithelial enveloping cell layer, thought mainly to provide protection to the embryo, directs cell migration and the spreading of embryonic tissue during early development. This function relies on the ability of embryonic cells to couple their autonomous random motility to non-autonomous signals arising from the expansion of the extra-embryonic epithelium, mediated by cell membrane adhesion and tension. Thus, we present a mechanism of extra-embryonic control of embryo morphogenesis that couples the mechanical properties of adjacent tissues in the early killifish embryo. PMID:28580937

  11. Wounded cells drive rapid epidermal repair in the early Drosophila embryo

    PubMed Central

    Fernandez-Gonzalez, Rodrigo; Zallen, Jennifer A.

    2013-01-01

    Epithelial tissues are protective barriers that display a remarkable ability to repair wounds. Wound repair is often associated with an accumulation of actin and nonmuscle myosin II around the wound, forming a purse string. The role of actomyosin networks in generating mechanical force during wound repair is not well understood. Here we investigate the mechanisms of force generation during wound repair in the epidermis of early and late Drosophila embryos. We find that wound closure is faster in early embryos, where, in addition to a purse string around the wound, actomyosin networks at the medial cortex of the wounded cells contribute to rapid wound repair. Laser ablation demonstrates that both medial and purse-string actomyosin networks generate contractile force. Quantitative analysis of protein localization dynamics during wound closure indicates that the rapid contraction of medial actomyosin structures during wound repair in early embryos involves disassembly of the actomyosin network. By contrast, actomyosin purse strings in late embryos contract more slowly in a mechanism that involves network condensation. We propose that the combined action of two force-generating structures—a medial actomyosin network and an actomyosin purse string—contributes to the increased efficiency of wound repair in the early embryo. PMID:23985320

  12. Exceptional preservation of tiny embryos documents seed dormancy in early angiosperms.

    PubMed

    Friis, Else Marie; Crane, Peter R; Pedersen, Kaj Raunsgaard; Stampanoni, Marco; Marone, Federica

    2015-12-24

    The rapid diversification of angiosperms through the Early Cretaceous period, between about 130-100 million years ago, initiated fundamental changes in the composition of terrestrial vegetation and is increasingly well understood on the basis of a wealth of palaeobotanical discoveries over the past four decades and their integration with improved knowledge of living angiosperms. Prevailing hypotheses, based on evidence both from living and from fossil plants, emphasize that the earliest angiosperms were plants of small stature with rapid life cycles that exploited disturbed habitats in open, or perhaps understorey, conditions. However, direct palaeontogical data relevant to understanding the seed biology and germination ecology of Early Cretaceous angiosperms are sparse. Here we report the discovery of embryos and their associated nutrient storage tissues in exceptionally well-preserved angiosperm seeds from the Early Cretaceous. Synchrotron radiation X-ray tomographic microscopy of the fossil embryos from many taxa reveals that all were tiny at the time of dispersal. These results support hypotheses based on extant plants that tiny embryos and seed dormancy are basic for angiosperms as a whole. The minute size of the fossil embryos, and the modest nutrient storage tissues dictated by the overall small seed size, is also consistent with the interpretation that many early angiosperms were opportunistic, early successional colonizers of disturbance-prone habitats.

  13. Budgett’s frog (Lepidobatrachus laevis): a new amphibian embryo for developmental biology

    PubMed Central

    Amin, Nirav M.; Womble, Mandy; Ledon-Rettig, Cris; Hull, Margaret; Dickinson, Amanda; Nascone-Yoder, Nanette

    2015-01-01

    The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett’s frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett’s frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett’s tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett’s frog model provides inimitable advantages for developmental studies—and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution. PMID:26169245

  14. Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in early vertebrates.

    PubMed

    Nakatani, Yoichiro; Takeda, Hiroyuki; Kohara, Yuji; Morishita, Shinichi

    2007-09-01

    Although several vertebrate genomes have been sequenced, little is known about the genome evolution of early vertebrates and how large-scale genomic changes such as the two rounds of whole-genome duplications (2R WGD) affected evolutionary complexity and novelty in vertebrates. Reconstructing the ancestral vertebrate genome is highly nontrivial because of the difficulty in identifying traces originating from the 2R WGD. To resolve this problem, we developed a novel method capable of pinning down remains of the 2R WGD in the human and medaka fish genomes using invertebrate tunicate and sea urchin genes to define ohnologs, i.e., paralogs produced by the 2R WGD. We validated the reconstruction using the chicken genome, which was not considered in the reconstruction step, and observed that many ancestral proto-chromosomes were retained in the chicken genome and had one-to-one correspondence to chicken microchromosomes, thereby confirming the reconstructed ancestral genomes. Our reconstruction revealed a contrast between the slow karyotype evolution after the second WGD and the rapid, lineage-specific genome reorganizations that occurred in the ancestral lineages of major taxonomic groups such as teleost fishes, amphibians, reptiles, and marsupials.

  15. Chronology of early embryonic development and embryo uterine migration in alpacas.

    PubMed

    Picha, Y; Tibary, A; Memon, M; Kasimanickam, R; Sumar, J

    2013-03-01

    The objectives were to: (1) describe the chronology of early embryonic development from ovulation to entry into the uterus; and (2) to determine the timing of embryo migration to the left uterine horn when ovulation occurred from the right ovary. The experiment was conducted in Peru. Females (n = 132) were randomly assigned to 15 experimental groups. All females were mated to an intact male, given 50 μg GnRH im (Cystorelin) and ovulation time determined by transrectal ultrasonography, conducted every 6 hours, starting 24 hours postmating. Animals were slaughtered at a specific intervals postovulation and reproductive tracts were recovered and subjected to oviductal and uterine flushing for females slaughtered between 1 and 6 days postovulation (dpo; Day 0 = ovulation) and uterine flushing for females slaughtered from 7 to 15 dpo for recovery of oocytes/embryos. Season of mating did not influence the interval from mating to ovulation (winter: 29 ± 6 hours vs. summer: 30 ± 6 hours; P = 0.49). Ovulation rates for females mated during winter and summer were 92% versus 100%, respectively (P = 0.05). Fertilization rates for winter and summer mated females were 72% and 82% (P = 0.29). Unfertilized ova were not retained in the uterine tube. All embryos collected were in the uterine tube ipsilateral to the side of ovulation between 1 and 5 dpo. Embryos reached the uterus on 6 dpo. Embryos began to elongate on 9 dpo; at this time, 83% of embryos derived from right-ovary ovulations were collected from the left uterine horn. Embryos occupied the entire uterine cavity by 10 dpo. In conclusion, we characterized early embryo development and location of embryo during its early developmental stages in alpaca. This was apparently the first report regarding chronology of embryo development and migration to the left horn in alpaca which merits further investigation regarding its role in maternal recognition of pregnancy. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Excess Imidacloprid Exposure Causes the Heart Tube Malformation of Chick Embryos.

    PubMed

    Gao, Lin-Rui; Li, Shuai; Zhang, Jing; Liang, Chang; Chen, En-Ni; Zhang, Shi-Yao; Chuai, Manli; Bao, Yong-Ping; Wang, Guang; Yang, Xuesong

    2016-11-30

    As a neonicotinoid pesticide, imidacloprid is widely used to control sucking insects on agricultural planting and fleas on domestic animals. However, the extent to which imidacloprid exposure has an influence on cardiogensis in early embryogenesis is still poorly understood. In vertebrates, the heart is the first organ to be formed. In this study, to address whether imidacloprid exposure affects early heart development, the early chick embryo has been used as an experimental model because of its accessibility at its early developmental stage. The results demonstrate that exposure of the early chick embryo to imidacloprid caused malformation of heart tube. Furthermore, the data reveal that down-regulation of GATA4, NKX2.5, and BMP4 and up-regulation of Wnt3a led to aberrant cardiomyocyte differentiation. In addition, imidacloprid exposure interfered with basement membrane breakdown, E-cadherin/laminin expression, and mesoderm formation during the epithelial-mesenchymal transition (EMT) in gastrula chick embryos. Finally, the DiI-labeled cell migration trajectory indicated that imidacloprid restricted the cell migration of cardiac progenitors to primary heart field in gastrula chick embryos. A similar observation was also obtained from the cell migration assay of scratch wounds in vitro. Additionally, imidacloprid exposure negatively affected the cytoskeleton structure and expression of corresponding adhesion molecules. Taken together, these results reveal that the improper EMT, cardiac progenitor migration, and differentiation are responsible for imidacloprid exposure-induced malformation of heart tube during chick embryo development.

  17. Budgett's frog (Lepidobatrachus laevis): A new amphibian embryo for developmental biology.

    PubMed

    Amin, Nirav M; Womble, Mandy; Ledon-Rettig, Cristina; Hull, Margaret; Dickinson, Amanda; Nascone-Yoder, Nanette

    2015-09-15

    The large size and rapid development of amphibian embryos has facilitated ground-breaking discoveries in developmental biology. Here, we describe the embryogenesis of the Budgett's frog (Lepidobatrachus laevis), an unusual species with eggs that are over twice the diameter of laboratory Xenopus, and embryos that can tolerate higher temperatures to develop into a tadpole four times more rapidly. In addition to detailing their early development, we demonstrate that, like Xenopus, these embryos are amenable to explant culture assays and can express exogenous transcripts in a tissue-specific manner. Moreover, the steep developmental trajectory and large scale of Lepidobatrachus make it exceptionally well-suited for morphogenesis research. For example, the developing organs of the Budgett's frog are massive compared to those of most model species, and are composed of larger individual cells, thereby affording increased subcellular resolution of early vertebrate organogenesis. Furthermore, we found that complete limb regeneration, which typically requires months to achieve in most vertebrate models, occurs in a matter of days in the Budgett's tadpole, which substantially accelerates the pace of experimentation. Thus, the unusual combination of the greater size and speed of the Budgett's frog model provides inimitable advantages for developmental studies-and a novel inroad to address the mechanisms of spatiotemporal scaling during evolution. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Culturing of avian embryos for time-lapse imaging.

    PubMed

    Rupp, Paul A; Rongish, Brenda J; Czirok, Andras; Little, Charles D

    2003-02-01

    Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h.

  19. MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

    PubMed

    Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

    2011-02-15

    At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

  20. Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

    PubMed Central

    Raissig, Michael T.; Gagliardini, Valeria; Jaenisch, Johan; Grossniklaus, Ueli; Baroux, Célia

    2013-01-01

    In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays. PMID:23770918

  1. The vertebrate phylotypic stage and an early bilaterian-related stage in mouse embryogenesis defined by genomic information.

    PubMed

    Irie, Naoki; Sehara-Fujisawa, Atsuko

    2007-01-12

    Embryos of taxonomically different vertebrates are thought to pass through a stage in which they resemble one another morphologically. This "vertebrate phylotypic stage" may represent the basic vertebrate body plan that was established in the common ancestor of vertebrates. However, much controversy remains about when the phylotypic stage appears, and whether it even exists. To overcome the limitations of studies based on morphological comparison, we explored a comprehensive quantitative method for defining the constrained stage using expressed sequence tag (EST) data, gene ontologies (GO), and available genomes of various animals. If strong developmental constraints occur during the phylotypic stage of vertebrate embryos, then genes conserved among vertebrates would be highly expressed at this stage. We established a novel method for evaluating the ancestral nature of mouse embryonic stages that does not depend on comparative morphology. The numerical "ancestor index" revealed that the mouse indeed has a highly conserved embryonic period at embryonic day 8.0-8.5, the time of appearance of the pharyngeal arch and somites. During this period, the mouse prominently expresses GO-determined developmental genes shared among vertebrates. Similar analyses revealed the existence of a bilaterian-related period, during which GO-determined developmental genes shared among bilaterians are markedly expressed at the cleavage-to-gastrulation period. The genes associated with the phylotypic stage identified by our method are essential in embryogenesis. Our results demonstrate that the mid-embryonic stage of the mouse is indeed highly constrained, supporting the existence of the phylotypic stage. Furthermore, this candidate stage is preceded by a putative bilaterian ancestor-related period. These results not only support the developmental hourglass model, but also highlight the hierarchical aspect of embryogenesis proposed by von Baer. Identification of conserved stages and tissues

  2. Early prenatal diagnosis of a lumbo-costo-vertebral syndrome.

    PubMed

    Pristavu, Anda Ioana; Furnica, Cristina; Ifrim, Mona Mihaela; Popovici, Razvan Mihai

    2018-04-01

    Lumbo-costo-vertebral syndrome (LCVS) is a rare type of lumbar hernia with associated abnormalities of the vertebral bodies, ribs, and trunk muscles. Only a few cases have been reported in the literature, all of which were diagnosed after birth. We present a case of LCVS diagnosed early in the second trimester of pregnancy using two- and three-dimensional ultrasound. In our case, the associated anomalies were: multiple costovertebral anomalies, lumbar hernia, anal imperforation, left hand supernumerary digit, and clubfoot.

  3. Zygotic Genome Activation in Vertebrates.

    PubMed

    Jukam, David; Shariati, S Ali M; Skotheim, Jan M

    2017-08-21

    The first major developmental transition in vertebrate embryos is the maternal-to-zygotic transition (MZT) when maternal mRNAs are degraded and zygotic transcription begins. During the MZT, the embryo takes charge of gene expression to control cell differentiation and further development. This spectacular organismal transition requires nuclear reprogramming and the initiation of RNAPII at thousands of promoters. Zygotic genome activation (ZGA) is mechanistically coordinated with other embryonic events, including changes in the cell cycle, chromatin state, and nuclear-to-cytoplasmic component ratios. Here, we review progress in understanding vertebrate ZGA dynamics in frogs, fish, mice, and humans to explore differences and emphasize common features. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. SNW1 Is a Critical Regulator of Spatial BMP Activity, Neural Plate Border Formation, and Neural Crest Specification in Vertebrate Embryos

    PubMed Central

    Wu, Mary Y.; Ramel, Marie-Christine; Howell, Michael; Hill, Caroline S.

    2011-01-01

    Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and

  5. Early Activation of MAPK and Apoptosis in Nutritive Embryos of Calyptraeid Gastropods.

    PubMed

    Lesoway, Maryna P; Collin, Rachel; Abouheif, Ehab

    2017-07-01

    Investigation of alternative phenotypes, different morphologies produced by a single genome, has contributed novel insights into development and evolution. Yet, the mechanisms underlying developmental switch points between alternative phenotypes remain poorly understood. The calyptraeid snails Crepidula navicella and Calyptraea lichen produce two phenotypes: viable and nutritive embryos, where nutritive embryos arrest their development after gastrulation and are ingested by their viable siblings as a form of intracapsular nutrition. Here, we investigate the activity of mitogen-activated protein kinase (MAPK, ERK1/2) and apoptosis during early cleavage. MAPK and apoptosis, found in a previous transcriptomic study, are known to be involved in organization of other spiralian embryos and nutritive embryo development, respectively. In the model Crepidula fornicata, MAPK activation begins at the 16-cell stage. In contrast, we discovered in C. navicella and C. lichen that many embryos begin MAPK activation at the one-cell stage. A subset of embryos shows a similar pattern of MAPK activation to C. fornicata at later stages. In all stages where MAPK is detected, the activation pattern is highly variable, frequently occurring in all quadrants or in multiple tiers of cells. We also detected apoptosis in cleaving embryos, while C. fornicata and Crepidula lessoni, which do not produce nutritive embryos, show no signs of apoptosis during cleavage. Our results show that MAPK and apoptosis are expressed during early development in species with nutritive embryos, and raises the possibility that these processes may play a role and even interact with one another in producing the nutritive embryo phenotype. © 2017 Wiley Periodicals, Inc.

  6. Applications of Tol2 Transposon-Mediated Gene Transfer for Stable Integration and Conditional Expression of Electroporated Genes in Chicken Embryos

    NASA Astrophysics Data System (ADS)

    Sato, Yuki; Takahashi, Yoshiko

    Because of the high accessibility to developing embryos, avian embryos (chicken and quail) have long been used as a good model animal to study embryogenesis in vertebrates, especially amniotes (reviewed in Wolpert, 2004). The techniques used for “classical” avian embryology included tissue transplantations, tissue ablations, and cell-labeling by vital dye. At the end of the last century, the in ovo electropora tion technique was developed by Nakamura and his colleagues, and this modern method opened a way to study the roles of developmental genes directly in living embryos (Funahashi et al., 1999) reviewed in (Nakamura et al., 2004; Yasuda et al., 2000; Yasugi and Nakamura, 2000). This powerful technique allows us to introduce genes (DNA, RNA, morpholino) into embryos in a tissue-specific way by targeting a restricted area of embryonic tissues. Thus, the electroporation technique using chickens has provided numerous novel insights into the understanding of early development in vertebrates, making the chicken a unique model animal.

  7. Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates.

    PubMed

    Frésard, Laure; Leroux, Sophie; Roux, Pierre-François; Klopp, Christophe; Fabre, Stéphane; Esquerré, Diane; Dehais, Patrice; Djari, Anis; Gourichon, David; Lagarrigue, Sandrine; Pitel, Frédérique

    2015-01-01

    RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome.

  8. Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates

    PubMed Central

    Frésard, Laure; Leroux, Sophie; Roux, Pierre-François; Klopp, Christophe; Fabre, Stéphane; Esquerré, Diane; Dehais, Patrice; Djari, Anis; Gourichon, David

    2015-01-01

    RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome. PMID:26024316

  9. Embryo density and medium volume effects on early murine embryo development.

    PubMed

    Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

    1992-10-01

    One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

  10. Laminarin improves developmental competence of porcine early stage embryos by inhibiting oxidative stress.

    PubMed

    Jiang, Hao; Liang, Shuang; Yao, Xue-Rui; Jin, Yong-Xun; Shen, Xing-Hui; Yuan, Bao; Zhang, Jia-Bao; Kim, Nam-Hyung

    2018-04-23

    Laminarin (LMA), a β-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20 μg/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H 2 O 2 . Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Expression of voltage-activated calcium channels in the early zebrafish embryo.

    PubMed

    Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

    2009-05-01

    Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

  12. Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention.

    PubMed

    Mitchell, Megan; Schulz, Samantha L; Armstrong, David T; Lane, Michelle

    2009-04-01

    Dietary supply of nutrients, both periconception and during pregnancy, influence the growth and development of the fetus and offspring and their health into adult life. Despite the importance of research efforts surrounding the developmental origins of health and disease hypothesis, the biological mechanisms involved remain elusive. Mitochondria are of major importance in the oocyte and early embryo, particularly as a source of ATP generation, and perturbations in their function have been related to reduced embryo quality. The present study examined embryo development following periconception exposure of females to a high-protein diet (HPD) or a low-protein diet (LPD) relative to a medium-protein diet (MPD; control), and we hypothesized that perturbed mitochondrial metabolism in the mouse embryo may be responsible for the impaired embryo and fetal development reported by others. Although the rate of development to the blastocyst stage did not differ between diets, both the HPD and LPD reduced the number of inner cell mass cells in the blastocyst-stage embryo. Furthermore, mitochondrial membrane potential was reduced and mitochondrial calcium levels increased in the 2-cell embryo. Embryos from HPD females had elevated levels of reactive oxygen species and ADP concentrations, indicative of metabolic stress and, potentially, the uncoupling of oxidative phosphorylation, whereas embryos from LPD females had reduced mitochondrial clustering around the nucleus, suggestive of an overall quietening of metabolism. Thus, although periconception dietary supply of different levels of protein is permissive of development, mitochondrial metabolism is altered in the early embryo, and the nature of the perturbation differs between HPD and LPD exposure.

  13. There is no highly conserved embryonic stage in the vertebrates: implications for current theories of evolution and development.

    PubMed

    Richardson, M K; Hanken, J; Gooneratne, M L; Pieau, C; Raynaud, A; Selwood, L; Wright, G M

    1997-08-01

    Embryos of different species of vertebrate share a common organisation and often look similar. Adult differences among species become more apparent through divergence at later stages. Some authors have suggested that members of most or all vertebrate clades pass through a virtually identical, conserved stage. This idea was promoted by Haeckel, and has recently been revived in the context of claims regarding the universality of developmental mechanisms. Thus embryonic resemblance at the tailbud stage has been linked with a conserved pattern of developmental gene expression - the zootype. Haeckel's drawings of the external morphology of various vertebrates remain the most comprehensive comparative data purporting to show a conserved stage. However, their accuracy has been questioned and only a narrow range of species was illustrated. In view of the current widespread interest in evolutionary developmental biology, and especially in the conservation of developmental mechanisms, re-examination of the extent of variation in vertebrate embryos is long overdue. We present here the first review of the external morphology of tailbud embryos, illustrated with original specimens from a wide range of vertebrate groups. We find that embryos at the tailbud stage - thought to correspond to a conserved stage - show variations in form due to allometry, heterochrony, and differences in body plan and somite number. These variations foreshadow important differences in adult body form. Contrary to recent claims that all vertebrate embryos pass through a stage when they are the same size, we find a greater than 10-fold variation in greatest length at the tailbud stage. Our survey seriously undermines the credibility of Haeckel's drawings, which depict not a conserved stage for vertebrates, but a stylised amniote embryo. In fact, the taxonomic level of greatest resemblance among vertebrate embryos is below the subphylum. The wide variation in morphology among vertebrate embryos is

  14. Microfluidic EmbryoSort technology: towards in flow analysis, sorting and dispensing of individual vertebrate embryos

    NASA Astrophysics Data System (ADS)

    Fuad, Nurul M.; Wlodkowic, Donald

    2013-12-01

    The demand to reduce the numbers of laboratory animals has facilitated the emergence of surrogate models such as tests performed on zebrafish (Danio rerio) or African clawed frog's (Xenopus levis) eggs, embryos and larvae. Those two model organisms are becoming increasingly popular replacements to current adult animal testing in toxicology, ecotoxicology and also in drug discovery. Zebrafish eggs and embryos are particularly attractive for toxicological analysis due their size (diameter 1.6 mm), optical transparency, large numbers generated per fish and very straightforward husbandry. The current bottleneck in using zebrafish embryos for screening purposes is, however, a tedious manual evaluation to confirm the fertilization status and subsequent dispensing of single developing embryos to multitier plates to perform toxicity analysis. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator's fatigue. In this work, we present a proofof- concept design of a continuous flow embryo sorter capable of analyzing, sorting and dispensing objects ranging in size from 1.5 - 2.5 mm. The prototypes were fabricated in polymethyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining. The application of additive manufacturing processes to prototype Lab-on-a-Chip sorters using both fused deposition manufacturing (FDM) and stereolithography (SLA) were also explored. The operation of the device was based on a revolving receptacle capable of receiving, holding and positioning single fish embryos for both interrogation and subsequent sorting. The actuation of the revolving receptacle was performed using a DC motor and/or microservo motor. The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers.

  15. Sensitivity of early mouse embryos to (/sup 3/H)thymidine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Spindle, A.; Wu, K.; Pedersen, R.A.

    1982-12-01

    Effects of intranuclear radiation on the developmental capacity of early mouse embryos were studied by exposing embryos to (/sup 3/H)thymidine and counting the number of embryos forming blastocysts, trophoblast outgrowths, inner cell masses (ICMs), and two-layer ICMs (differentiated into primary endoderm and ectoderm). When embryos were cultured from the 2-cell stage for 8 days in the continuous presence of (/sup 3/H)thymidine, concentrations as low as 0.2 nCi/ml reduced the number of embryos forming two-layer ICMs. At 1 nCi/ml, the number of both ICMs and two-layer ICMs were reduced, and at 10 nCi/ml the number of embryos developing to all threemore » post-blastocyst endpoints was reduced. Blastocyst formation was not affected even at the highst concentration (/sup 3/H)thymidine and then cultured further in unlabelled medium, the effects were similar to those of 8-day exposure. When embryos were exposed to (/sup 3/H)thymidine for 24 h at various developmental stages, effects were less severe than when they were exposed continuously for 3 or 8 days, and the sensitivity of embryos differed between stages. The 24-h exposure of immunosurgically isolated ICMS to (/sup 3/H)thymidine revealed that the high sensitivity of the ICM to (/sup 3/H)thymidine persists through the late blastocyst stage and declines progressively thereafter. Autoradiography indicated that the change in radiosensitivity of embryos or ICMs is generally related to their ability to incorporate (/sup 3/H)thymidine into the DNA.« less

  16. DYZ1 copy number variation, Y chromosome polymorphism and early recurrent spontaneous abortion/early embryo growth arrest.

    PubMed

    Yan, Junhao; Fan, Lingling; Zhao, Yueran; You, Li; Wang, Laicheng; Zhao, Han; Li, Yuan; Chen, Zi-Jiang

    2011-12-01

    To find the association between recurrent spontaneous abortion (RSA)/early embryo growth arrest and Y chromosome polymorphism. Peripheral blood samples of the male patients of big Y chromosome, small Y chromosome and other male patients whose partners suffered from unexplained RSA/early embryo growth arrest were collected. PCR and real-time fluorescent quantitative PCR were used to test the deletion and the copy number variation of DYZ1 region in Y chromosome of the patients. A total of 79 big Y chromosome patients (48 of whose partners suffered from RSA or early embryo growth arrest), 7 small Y chromosome patients, 106 other male patients whose partners had suffered from unexplained RSA or early embryo growth arrest, and 100 normal male controls were enrolled. There was no fraction deletion of DYZ1 detected both in big Y patients and in normal men. Of RSA patients, 1 case showed deletion of 266bp from the gene locus 25-290bp, and 2 cases showed deletion of 773bp from 1347 to 2119bp. Of only 7 small Y chromosome patients, 2 cases showed deletion of 266bp from 25 to 290bp, and 4 cases showed deletion of 773bp from 1347 to 2119bp and 275bp from 3128 to 3420bp. The mean of DYZ1 copies was 3900 in normal control men; the mean in big Y patients was 5571, in RSA patients was 2655, and in small Y patients was 1059. All of the others were significantly different (P<0.01) compared with normal control men, which meant that DYZ1 copy number in normal control men was less than that of big Y chromosome patients, and was more than that of unexplained early RSA patients and small Y patients. The integrity and copy number variation of DYZ1 are closely related to the Y chromosome length under microscope. The cause of RSA/early embryo growth arrest in some couples may be the increase (big Y patients) or decrease of DYZ1 copy number in the husbands' Y chromosome. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  17. The influence of early embryo traits on human embryonic stem cell derivation efficiency.

    PubMed

    O'Leary, Thomas; Heindryckx, Björn; Lierman, Sylvie; Van der Jeught, Margot; Menten, Björn; Deforce, Dieter; Cornelissen, Ria; de Sousa Lopes, Susana Chuva; De Sutter, Petra

    2011-05-01

    Despite its prognostic value in in vitro fertilization, early embryo morphology is not reported on in the derivation of human embryonic stem cell (hESC) lines. Standard hESC derivation does rely on blastocyst development and its efficiency is highly correlated to inner cell mass (ICM) quality. Poor-quality embryos (PQEs) donated for hESC derivation may have a range of cleavage-stage abnormalities that are known to compromise further development. This study was implemented to determine whether specific PQEs traits influence the efficiency of good-quality ICMs to derive new hESC lines. We found that although the types of PQEs investigated were all able to make blastocysts with good-quality ICMs, the ICMs were unequal in their ability to derive hESCs. Good-quality ICMs from embryos with multiple poor-quality traits were unable to generate hESC lines, in contrast to good-quality ICMs from embryos with a single poor-quality trait. In addition, our data suggest a direct correlation between the number of ICM cells present in the blastocyst and its capacity to derive new hESC lines. This study is the first to demonstrate that ICM quality alone is an incomplete indicator of hESC derivation and that application of in vitro fertilization-based early embryo scoring can help predict hESC derivation efficiency. Experiments aiming to quantify, improve upon, or compare hESC derivation efficiency should thus take into consideration early embryo morphology scoring for the comparison of groups with equal developmental competence.

  18. Precision matters for position decoding in the early fly embryo

    NASA Astrophysics Data System (ADS)

    Petkova, Mariela D.; Tkacik, Gasper; Wieschaus, Eric F.; Bialek, William; Gregor, Thomas

    Genetic networks can determine cell fates in multicellular organisms with precision that often reaches the physical limits of the system. However, it is unclear how the organism uses this precision and whether it has biological content. Here we address this question in the developing fly embryo, in which a genetic network of patterning genes reaches 1% precision in positioning cells along the embryo axis. The network consists of three interconnected layers: an input layer of maternal gradients, a processing layer of gap genes, and an output layer of pair-rule genes with seven-striped patterns. From measurements of gap gene protein expression in hundreds of wild-type embryos we construct a ``decoder'', which is a look-up table that determines cellular positions from the concentration means, variances and co-variances. When we apply the decoder to measurements in mutant embryos lacking various combinations of the maternal inputs, we predict quantitative changes in the output layer such as missing, altered or displaced stripes. We confirm these predictions by measuring pair-rule expression in the mutant embryos. Our results thereby show that the precision of the patterning network is biologically meaningful and a necessary feature for decoding cell positions in the early fly embryo.

  19. Contraction and stress-dependent growth shape the forebrain of the early chicken embryo.

    PubMed

    Garcia, Kara E; Okamoto, Ruth J; Bayly, Philip V; Taber, Larry A

    2017-01-01

    During early vertebrate development, local constrictions, or sulci, form to divide the forebrain into the diencephalon, telencephalon, and optic vesicles. These partitions are maintained and exaggerated as the brain tube inflates, grows, and bends. Combining quantitative experiments on chick embryos with computational modeling, we investigated the biophysical mechanisms that drive these changes in brain shape. Chemical perturbations of contractility indicated that actomyosin contraction plays a major role in the creation of initial constrictions (Hamburger-Hamilton stages HH11-12), and fluorescent staining revealed that F-actin is circumferentially aligned at all constrictions. A finite element model based on these findings shows that the observed shape changes are consistent with circumferential contraction in these regions. To explain why sulci continue to deepen as the forebrain expands (HH12-20), we speculate that growth depends on wall stress. This idea was examined by including stress-dependent growth in a model with cerebrospinal fluid pressure and bending (cephalic flexure). The results given by the model agree with observed morphological changes that occur in the brain tube under normal and reduced eCSF pressure, quantitative measurements of relative sulcal depth versus time, and previously published patterns of cell proliferation. Taken together, our results support a biphasic mechanism for forebrain morphogenesis consisting of differential contractility (early) and stress-dependent growth (late). Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Evolution and ecology of retinal photoreception in early vertebrates.

    PubMed

    Collin, Shaun P

    2010-01-01

    Visual ecology or the relationship between the visual system of an animal and its environment has proven to be a crucial research field for establishing general concepts of adaptation, specialization and evolution. The visual neuroscientist is indeed confronted with a plethora of different visual characteristics, each seemingly optimised for each species' ecological niche, but often without a clear understanding of the evolutionary constraints at play. However, before we are able to fully understand the influence(s) of ecology and phylogeny on visual system design in vertebrates, it is first necessary to understand the basic bauplan of key representatives of each taxa. This review examines photoreception in hagfishes, lampreys, cartilaginous fishes and lungfishes with an eye to their ecology using a range of neurobiological methods including anatomy, microspectrophotometry and molecular genetics. These early vertebrates represent critical stages in evolution and surprisingly possess a level of visual complexity that is almost unrivalled in other vertebrates. 2010 S. Karger AG, Basel.

  1. Mitotic wavefronts mediated by mechanical signaling in early Drosophila embryos

    NASA Astrophysics Data System (ADS)

    Kang, Louis; Idema, Timon; Liu, Andrea; Lubensky, Tom

    2013-03-01

    Mitosis in the early Drosophila embryo demonstrates spatial and temporal correlations in the form of wavefronts that travel across the embryo in each cell cycle. This coordinated phenomenon requires a signaling mechanism, which we suggest is mechanical in origin. We have constructed a theoretical model that supports nonlinear wavefront propagation in a mechanically-excitable medium. Previously, we have shown that this model captures quantitatively the wavefront speed as it varies with cell cycle number, for reasonable values of the elastic moduli and damping coefficient of the medium. Now we show that our model also captures the displacements of cell nuclei in the embryo in response to the traveling wavefront. This new result further supports that mechanical signaling may play an important role in mediating mitotic wavefronts.

  2. Expression profiling of the mouse early embryo: Reflections and Perspectives

    PubMed Central

    Ko, Minoru S. H.

    2008-01-01

    Laboratory mouse plays important role in our understanding of early mammalian development and provides invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. Comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over last two decades, have provided even more advantages to mouse models. Here the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells are reviewed and the future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, which include: bird’s-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks. PMID:16739220

  3. Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

    PubMed

    Liu, C; Liu, Y; Larsen, K; Hou, Y P; Callesen, H

    2018-01-01

    It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

  4. Nodal Cilia Dynamics and the Specification of the Left/Right Axis in Early Vertebrate Embryo Development

    PubMed Central

    Buceta, Javier; Ibañes, Marta; Rasskin-Gutman, Diego; Okada, Yasushi; Hirokawa, Nobutaka; Izpisúa-Belmonte, Juan Carlos

    2005-01-01

    Nodal cilia dynamics is a key factor for left/right axis determination in mouse embryos through the induction of a leftward fluid flow. So far it has not been clearly established how such dynamics is able to induce the asymmetric leftward flow within the node. Herein we propose that an asymmetric two-phase nonplanar beating cilia dynamics that involves the bending of the ciliar axoneme is responsible for the leftward fluid flow. We support our proposal with a host of hydrodynamic arguments, in silico experiments and in vivo video microscopy data in wild-type embryos and inv mutants. Our phenomenological modeling approach underscores how the asymmetry and speed of the flow depends on different relevant parameters. In addition, we discuss how the combination of internal and external mechanisms might cause the two-phase beating cilia dynamics. PMID:16040754

  5. Comparative transcriptome analysis reveals vertebrate phylotypic period during organogenesis

    PubMed Central

    Irie, Naoki; Kuratani, Shigeru

    2011-01-01

    One of the central issues in evolutionary developmental biology is how we can formulate the relationships between evolutionary and developmental processes. Two major models have been proposed: the 'funnel-like' model, in which the earliest embryo shows the most conserved morphological pattern, followed by diversifying later stages, and the 'hourglass' model, in which constraints are imposed to conserve organogenesis stages, which is called the phylotypic period. Here we perform a quantitative comparative transcriptome analysis of several model vertebrate embryos and show that the pharyngula stage is most conserved, whereas earlier and later stages are rather divergent. These results allow us to predict approximate developmental timetables between different species, and indicate that pharyngula embryos have the most conserved gene expression profiles, which may be the source of the basic body plan of vertebrates. PMID:21427719

  6. The effects of levetiracetam on neural tube development in the early stage of chick embryos.

    PubMed

    Guvenc, Yahya; Dalgic, Ali; Billur, Deniz; Karaoglu, Derya; Aydin, Sevim; Daglioglu, Ergun; Ozdol, Cagatay; Nacar, Osman Arikan; Yildirim, Ali Erdem; Belen, Deniz

    2013-01-01

    This study aimed to investigate the effects of a new generation antiepileptic agent, levetiracetam, on the neural tube development in a chick embryo model that corresponds to the first month of vertebral development in mammals. Forty-five Atabey® breed fertilized chicken eggs with no specific pathogens were randomly divided into 5 groups. All of the eggs were incubated at 37.8±2°C and 60±5 % relative humidity in an incubator. Group A was control group. The other eggs were applied physiological saline and drugs at a volume of 10 μL by the in ovo method at the 28th hour of the incubation period. Group B was given distilled water; Group C, physiological saline; Group D, Levetiracetam (L8668) at a dose equivalent to the treatment dose for humans (10 mg/ kg), and Group E, Levetiracetam (L8668) at a dose of 10 times the treatment dose. The embryos in all of the groups were removed from the shells at the 48th hour and morphologically and histologically evaluated. Of the 45 embryos incubated, neural tubes of 41 were closed and the embryos displayed normal development. Levetiracetam, at a dose equivalent to human treatment dose and 10 times the treatment dose, was shown not to cause neural tube defects in chick embryos.

  7. Reversible neuronal and muscular toxicity of caffeine in developing vertebrates.

    PubMed

    Rodriguez, Rufino S; Haugen, Rebecca; Rueber, Alexandra; Huang, Cheng-Chen

    2014-06-01

    This study utilizes zebrafish embryos to understand the cellular and molecular mechanisms of caffeine toxicity in developing vertebrate embryos. By using a high concentration of caffeine, we observed almost all the phenotypes that have been described in humans and/or in other animal models, including neural tube closure defect, jittery, touch insensitivity, and growth retardation as well as a drastic coiled body phenotype. Zebrafish embryos exposed to 5mM caffeine exhibited high frequent movement, 10 moves/min comparing with around 3 moves/min in control embryos, within half an hour post exposure (HPE). They later showed twitching, uncoordinated movement, and eventually severe body curvature by 6HPE. Exposure at later stages resulted in the same phenotypes but more posteriorly. Surprisingly, when caffeine was removed before 6HPE, the embryos were capable of recovering but still exhibited mild curvature and shorter bodies. Longer exposure caused irreversible body curvature and lethality. These results suggest that caffeine likely targets the neuro-muscular physiology in developing embryos. Immunohistochemistry revealed that the motorneurons in treated embryos developed shorter axons, abnormal branching, and excessive synaptic vesicles. Developing skeletal muscles also appeared smaller and lacked the well-defined boundaries seen in control embryos. Finally, caffeine increases the expression of genes involved in synaptic vesicle migration. In summary, our results provide molecular understanding of caffeine toxicity on developing vertebrate embryos. Published by Elsevier Inc.

  8. Epimorphin acts extracellularly to promote cell sorting and aggregation during the condensation of vertebral cartilage.

    PubMed

    Oka, Yumiko; Sato, Yuki; Tsuda, Hokari; Hanaoka, Kazunori; Hirai, Yohei; Takahashi, Yoshiko

    2006-03-01

    Formation of vertebrae occurs via endochondral ossification, a process involving condensation of precartilaginous cells. Here, we provide the first molecular evidence of mechanism that underlies initiation of this process by showing that the extracellular factor, Epimorphin, plays a role during early steps in vertebral cartilage condensation. Epimorphin mRNA is predominantly localized in the vertebral primordium. When provided exogenously in ovo, it causes precocious differentiation of chondrocytes, resulting in the formation of supernumerary vertebral cartilage in chicken embryos. To further analyze its mode of action, we used an in vitro co-culture system in which labeled 10T1/2 or sclerotomal prechondrogenic cells were co-cultured with unlabeled Epimorphin-producing cells. In the presence of Epimorphin, the labeled cells formed tightly packed aggregates, and sclerotomal cells displayed augmented accumulation of NCAM and other early markers of chondrocyte differentiation. Finally, we found that the Epimorphin expression is initiated during vertebrogenesis by Sonic hedgehog from the notochord mediated by Sox 9. We present a model in which successive action of Epimorphin in recruiting and stacking sclerotomal cells leads to a sequential elongation of a vertebral primordium.

  9. Reduced heart rate and cardiac output differentially affect angiogenesis, growth, and development in early chicken embryos (Gallus domesticus).

    PubMed

    Branum, Sylvia R; Yamada-Fisher, Miho; Burggren, Warren

    2013-01-01

    growth rate and angiogenesis in the CAM periphery. This study demonstrates for the first time that different processes in the ontogeny of the early vertebrate embryo (i.e., hypertrophic growth vs. development) have differential sensitivities to altered convective blood flow.

  10. Frodo proteins: modulators of Wnt signaling in vertebrate development.

    PubMed

    Brott, Barbara K; Sokol, Sergei Y

    2005-09-01

    The Frodo/dapper (Frd) proteins are recently discovered signaling adaptors, which functionally and physically interact with Wnt and Nodal signaling pathways during vertebrate development. The Frd1 and Frd2 genes are expressed in dynamic patterns in early embryos, frequently in cells undergoing epithelial-mesenchymal transition. The Frd proteins function in multiple developmental processes, including mesoderm and neural tissue specification, early morphogenetic cell movements, and organogenesis. Loss-of-function studies using morpholino antisense oligonucleotides demonstrate that the Frd proteins regulate Wnt signal transduction in a context-dependent manner and may be involved in Nodal signaling. The identification of Frd-associated factors and cellular targets of the Frd proteins should shed light on the molecular mechanisms underlying Frd functions in embryonic development and in cancer.

  11. Teratogenicity of Ochratoxin A and the Degradation Product, Ochratoxin α, in the Zebrafish (Danio rerio) Embryo Model of Vertebrate Development

    PubMed Central

    Haq, Mehreen; Gonzalez, Nelson; Mintz, Keenan; Jaja-Chimedza, Asha; De Jesus, Christopher Lawrence; Lydon, Christina; Welch, Aaron Z.; Berry, John P.

    2016-01-01

    Ochratoxins, and particularly ochratoxin A (OTA), are toxic fungal-derived contaminants of food and other agricultural products. Growing evidence supports the degradation of OTA by chemical, enzymatic and/or microbial means as a potential approach to remove this mycotoxin from food products. In particular, hydrolysis of OTA to ochratoxin α (OTα) and phenylalanine is the presumptive product of degradation in most cases. In the current study, we employed the zebrafish (Danio rerio) embryo, as a model of vertebrate development to evaluate, the teratogenicity of OTA and OTα. These studies show that OTA is potently active in the zebrafish embryo toxicity assay (ZETA), and that toxicity is both concentration- and time-dependent with discernible and quantifiable developmental toxicity observed at nanomolar concentrations. On the other hand, OTα had no significant effect on embryo development at all concentrations tested supporting a decreased toxicity of this degradation product. Taken together, these results suggest that ZETA is a useful, and highly sensitive, tool for evaluating OTA toxicity, as well as its degradation products, toward development of effective detoxification strategies. Specifically, the results obtained with ZETA, in the present study, further demonstrate the toxicity of OTA, and support its degradation via hydrolysis to OTα as an effective means of detoxification. PMID:26861395

  12. Teratogenicity of Ochratoxin A and the Degradation Product, Ochratoxin α, in the Zebrafish (Danio rerio) Embryo Model of Vertebrate Development.

    PubMed

    Haq, Mehreen; Gonzalez, Nelson; Mintz, Keenan; Jaja-Chimedza, Asha; De Jesus, Christopher Lawrence; Lydon, Christina; Welch, Aaron; Berry, John P

    2016-02-05

    Ochratoxins, and particularly ochratoxin A (OTA), are toxic fungal-derived contaminants of food and other agricultural products. Growing evidence supports the degradation of OTA by chemical, enzymatic and/or microbial means as a potential approach to remove this mycotoxin from food products. In particular, hydrolysis of OTA to ochratoxin α (OTα) and phenylalanine is the presumptive product of degradation in most cases. In the current study, we employed the zebrafish (Danio rerio) embryo, as a model of vertebrate development to evaluate, the teratogenicity of OTA and OTα. These studies show that OTA is potently active in the zebrafish embryo toxicity assay (ZETA), and that toxicity is both concentration- and time-dependent with discernible and quantifiable developmental toxicity observed at nanomolar concentrations. On the other hand, OTα had no significant effect on embryo development at all concentrations tested supporting a decreased toxicity of this degradation product. Taken together, these results suggest that ZETA is a useful, and highly sensitive, tool for evaluating OTA toxicity, as well as its degradation products, toward development of effective detoxification strategies. Specifically, the results obtained with ZETA, in the present study, further demonstrate the toxicity of OTA, and support its degradation via hydrolysis to OTα as an effective means of detoxification.

  13. The early-stage diagnosis of albinic embryos by applying optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Yang, Bor-Wen; Wang, Shih-Yuan; Wang, Yu-Yen; Cai, Jyun-Jhang; Chang, Chung-Hao

    2013-09-01

    Albinism is a kind of congenital disease of abnormal metabolism. Poecilia reticulata (guppy fish) is chosen as the model to study the development of albinic embryos as it is albinic, ovoviviparous and with short life period. This study proposed an imaging method for penetrative embryo investigation using optical coherence tomography. By imaging through guppy mother’s reproduction purse, we found the embryo’s eyes were the early-developed albinism features. As human’s ocular albinism typically appear at about four weeks old, it is the time to determine if an embryo will grow into an albino.

  14. A multicenter prospective study to assess the effect of early cleavage on embryo quality, implantation, and live-birth rate.

    PubMed

    de los Santos, Maria José; Arroyo, Gemma; Busquet, Ana; Calderón, Gloria; Cuadros, Jorge; Hurtado de Mendoza, Maria Victoria; Moragas, Marta; Herrer, Raquel; Ortiz, Agueda; Pons, Carme; Ten, Jorge; Vilches, Miguel Angel; Figueroa, Maria José

    2014-04-01

    To investigate the impact of early cleavage (EC) on embryo quality, implantation, and live-birth rates. Prospective cross-sectional study. Multicenter study. Seven hundred embryo transfers and 1,028 early-stage human embryos. None. Implantation according to the presence of EC and embryo quality. The presence of EC is associated with embryo quality, especially in cycles with autologous oocytes. However, the use of EC as an additional criterion for selecting an embryo for transfer does not appear to significantly improve likelihood of implantation. Furthermore, embryos that presented EC had live-birth rates per implanted embryo similar to those that did not show any sign of cleavage. At least for conventional embryo culture and morphologic evaluations, the additional evaluation of EC in embryos may not be valuable to improve embryo implantation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  15. A Standard System to Study Vertebrate Embryos

    PubMed Central

    Werneburg, Ingmar

    2009-01-01

    Staged embryonic series are important as reference for different kinds of biological studies. I summarise problems that occur when using ‘staging tables’ of ‘model organisms’. Investigations of developmental processes in a broad scope of taxa are becoming commonplace. Beginning in the 1990s, methods were developed to quantify and analyse developmental events in a phylogenetic framework. The algorithms associated with these methods are still under development, mainly due to difficulties of using non-independent characters. Nevertheless, the principle of comparing clearly defined newly occurring morphological features in development (events) in quantifying analyses was a key innovation for comparative embryonic research. Up to date no standard was set for how to define such events in a comparative approach. As a case study I compared the external development of 23 land vertebrate species with a focus on turtles, mainly based on reference staging tables. I excluded all the characters that are only identical for a particular species or general features that were only analysed in a few species. Based on these comparisons I defined 104 developmental characters that are common either for all vertebrates (61 characters), gnathostomes (26), tetrapods (3), amniotes (7), or only for sauropsids (7). Characters concern the neural tube, somite, ear, eye, limb, maxillary and mandibular process, pharyngeal arch, eyelid or carapace development. I present an illustrated guide listing all the defined events. This guide can be used for describing developmental series of any vertebrate species or for documenting specimen variability of a particular species. The guide incorporates drawings and photographs as well as consideration of species identifying developmental features such as colouration. The simple character-code of the guide is extendable to further characters pertaining to external and internal morphological, physiological, genetic or molecular development, and also

  16. Effect of exogenous transforming growth factor β1 (TGF-β1) on early bovine embryo development.

    PubMed

    Barrera, Antonio D; García, Elina V; Miceli, Dora C

    2018-06-08

    SummaryDuring preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor β1 (TGF-β1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-β1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-β receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-β1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-β1 treated embryos. These results suggest an early action of exogenous TGF-β1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-β signalling during early embryogenesis.

  17. Vertebrate assemblages from the early Late Cretaceous of southeastern Morocco: An overview

    NASA Astrophysics Data System (ADS)

    Cavin, L.; Tong, H.; Boudad, L.; Meister, C.; Piuz, A.; Tabouelle, J.; Aarab, M.; Amiot, R.; Buffetaut, E.; Dyke, G.; Hua, S.; Le Loeuff, J.

    2010-07-01

    Fossils of vertebrates have been found in great abundance in the continental and marine early Late Cretaceous sediments of Southeastern Morocco for more than 50 years. About 80 vertebrate taxa have so far been recorded from this region, many of which were recognised and diagnosed for the first time based on specimens recovered from these sediments. In this paper, we use published data together with new field data to present an updated overview of Moroccan early Late Cretaceous vertebrate assemblages. The Cretaceous series we have studied encompasses three Formations, the Ifezouane and Aoufous Formations, which are continental and deltaic in origin and are often grouped under the name "Kem Kem beds", and the Akrabou Formation which is marine in origin. New field observations allow us to place four recognised vertebrate clusters, corresponding to one compound assemblage and three assemblages, within a general temporal framework. In particular, two ammonite bioevents characterise the lower part of the Upper Cenomanian ( Calycoceras guerangeri Zone) at the base of the Akrabou Formation and the upper part of the Lower Turonian ( Mammites nodosoides Zone), that may extend into the Middle Turonian within the Akrabou Formation, and allow for more accurate dating of the marine sequence in the study area. We are not yet able to distinguish a specific assemblage that characterises the Ifezouane Formation when compared to the similar Aoufous Formation, and as a result we regard the oldest of the four vertebrate "assemblages" in this region to be the compound assemblage of the "Kem Kem beds". This well-known vertebrate assemblage comprises a mixture of terrestrial (and aerial), freshwater and brackish vertebrates. The archosaur component of this fauna appears to show an intriguingly high proportion of large-bodied carnivorous taxa, which may indicate a peculiar trophic chain, although collecting biases alter this palaeontological signal. A small and restricted assemblage, the

  18. Gene regulation in amphioxus: An insight from transgenic studies in amphioxus and vertebrates.

    PubMed

    Kozmikova, Iryna; Kozmik, Zbynek

    2015-12-01

    Cephalochordates, commonly known as amphioxus or lancelets, are the most basal subphylum of chordates. Cephalochordates are thus key to understanding the origin of vertebrates and molecular mechanisms underlying vertebrate evolution. The evolution of developmental control mechanisms during invertebrate-to-vertebrate transition involved not only gene duplication events, but also specific changes in spatial and temporal expression of many genes. To get insight into the spatiotemporal regulation of gene expression during invertebrate-to-vertebrate transition, functional studies of amphioxus gene regulatory elements are highly warranted. Here, we review transgenic studies performed in amphioxus and vertebrates using promoters and enhancers derived from the genome of Branchiostoma floridae. We describe the current methods of transgenesis in amphioxus, provide evidence of Tol2 transposon-generated transgenic embryos of Branchiostoma lanceolatum and discuss possible future directions. We envision that comparative transgenic analysis of gene regulatory sequences in the context of amphioxus and vertebrate embryos will likely provide an important mechanistic insight into the evolution of vertebrate body plan. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Central cell-derived peptides regulate early embryo patterning in flowering plants.

    PubMed

    Costa, Liliana M; Marshall, Eleanor; Tesfaye, Mesfin; Silverstein, Kevin A T; Mori, Masashi; Umetsu, Yoshitaka; Otterbach, Sophie L; Papareddy, Ranjith; Dickinson, Hugh G; Boutiller, Kim; VandenBosch, Kathryn A; Ohki, Shinya; Gutierrez-Marcos, José F

    2014-04-11

    Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non-cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

  20. Early embryonic survival and embryo development in two lines of rabbits divergently selected for uterine capacity.

    PubMed

    Peiró, R; Santacreu, M A; Climent, A; Blasco, A

    2007-07-01

    The aim of this work is to study early embryo survival and development in 2 lines divergently selected for high and low uterine capacity throughout 10 generations. A total of 162 female rabbits from the high line and 133 from the low line were slaughtered at 25, 48, or 62 h of gestation. There were no differences in ovulation rate and fertilization rate between lines in any of the 3 stages of gestation. Embryo survival, estimated as the number of normal embryos recovered at a constant ovulation rate, was similar in both lines at 25 and 48 h. Embryo survival was greater in the high line [D (posterior mean of the difference between the high and low lines) = 0.57 embryos] at 62 h of gestation. There was no difference in embryonic stage of development at 25 h, but at 48 and 62 h of gestation, the high line, compared with the low line, had a greater percentage of early morulae (83 vs. 72%) and compacted morulae (55 vs. 38%). Divergent selection for uterine capacity appeared to modify embryo development, at least from 48 h of gestation, and embryo survival from 62 h.

  1. Growth/differentiation factor-11: an evolutionary conserved growth factor in vertebrates.

    PubMed

    Funkenstein, Bruria; Olekh, Elena

    2010-11-01

    Growth and differentiation factor-11 (GDF-11) is a member of the transforming growth factor-β superfamily and is thought to be derived together with myostatin (known also as GDF-8) from an ancestral gene. In the present study, we report the isolation and characterization of GDF-11 homolog from a marine teleost, the gilthead sea bream Sparus aurata, and show that this growth factor is highly conserved throughout vertebrates. Using bioinformatics, we identified GDF-11 in Tetraodon, Takifugu, medaka, and stickleback and found that they are highly conserved at the amino acid sequence as well as gene organization. Moreover, we found conservation of syntenic relationships among vertebrates in the GDF-11 locus. Transcripts for GDF-11 can be found in eggs and early embryos, albeit at low levels, while in post-hatching larvae expression levels are high and decreases as development progresses, suggesting that GDF-11 might have a role during early development of fish as found in tetrapods and zebrafish. Finally, GDF-11 is expressed in various tissues in the adult fish including muscle, brain, and eye.

  2. [Specification of cell destiny in early Caenorhabditis elegans embryo].

    PubMed

    Schierenberg, E

    1997-02-01

    Embryogenesis of the nematode Caenorhabditis elegans has been described completely on a cell-by-cell basis and found to be essentially invariant. With this knowledge in hands, micromanipulated embryos and mutants have been analyzed for cell lineage defects and the distribution of specific gene products. The results challenge the classical view of cell-autonomous development in nematodes and indicate that the early embryo of C. elegans is a highly dynamic system. A network of inductive events between neighboring cells is being revealed, which is necessary to assign different developmental programs to blastomeres. In those cases where molecules involved in these cell-cell interactions have been identified, homologies to cell surface receptors, ligands and transcription factors found in other systems have become obvious.

  3. Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

    PubMed

    Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

    2014-10-01

    In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Sildenafil citrate (Viagra) impairs fertilization and early embryo development in mice.

    PubMed

    Glenn, David R J; McClure, Neil; Cosby, S Louise; Stevenson, Michael; Lewis, Sheena E M

    2009-03-01

    To determine the effects of sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase inhibitor known to affect sperm function, on fertilization and early embryo cleavage. This acute mammal study included male and female mice assigned randomly, the females sacrificed after mating and their oocytes/embryos evaluated at four time periods after treatment. Academic research environment. Male and female CBAB(6) mice. Female mice were injected intraperitoneally with 5 IU gonadotropin (hCG) to stimulate follicular growth and induce ovulation. They were each caged with a male that had been gavaged with sildenafil citrate (0.06 mg/0.05 mL) and allowed to mate. After 12, 36, 60, and 84 h, females were killed, their oviducts were dissected out, and retrieved embryos were assessed for blastomere number and quality. Fertilization rates and numbers of embryos were evaluated after treatment. Fertilization rates (day 1) were markedly reduced (-33%) in matings where the male had taken sildenafil citrate. Over days 2-4, the numbers of embryos developing in the treated group were significantly fewer than in the control group. There was also a trend for impaired cleavage rates within those embryos, although this did not reach significance. The impairments to fertility caused by sildenafil citrate have important implications for infertility centers and for couples who are using this drug precoitally while attempting to conceive.

  5. Node and midline defects are associated with left-right development in Delta1 mutant embryos.

    PubMed

    Przemeck, Gerhard K H; Heinzmann, Ulrich; Beckers, Johannes; Hrabé de Angelis, Martin

    2003-01-01

    Axes formation is a fundamental process of early embryonic development. In addition to the anteroposterior and dorsoventral axes, the determination of the left-right axis is crucial for the proper morphogenesis of internal organs and is evolutionarily conserved in vertebrates. Genes known to be required for the normal establishment and/or maintenance of left-right asymmetry in vertebrates include, for example, components of the TGF-beta family of intercellular signalling molecules and genes required for node and midline function. We report that Notch signalling, which previously had not been implicated in this morphogenetic process, is required for normal left-right determination in mice. We show, that the loss-of-function of the delta 1 (Dll1) gene causes a situs ambiguous phenotype, including randomisation of the direction of heart looping and embryonic turning. The most probable cause for this left-right defect in Dll1 mutant embryos is a failure in the development of proper midline structures. These originate from the node, which is disrupted and deformed in Dll1 mutant embryos. Based on expression analysis in wild-type and mutant embryos, we suggest a model, in which Notch signalling is required for the proper differentiation of node cells and node morphology.

  6. Learning about Vertebrate Limb Development

    ERIC Educational Resources Information Center

    Liang, Jennifer O.; Noll, Matthew; Olsen, Shayna

    2014-01-01

    We have developed an upper-level undergraduate laboratory exercise that enables students to replicate a key experiment in developmental biology. In this exercise, students have the opportunity to observe live chick embryos and stain the apical ectodermal ridge, a key tissue required for development of the vertebrate limb. Impressively, every…

  7. Early embryo mortality in natural human reproduction: What the data say

    PubMed Central

    Jarvis, Gavin E.

    2017-01-01

    How many human embryos die between fertilisation and birth under natural conditions? It is widely accepted that natural human embryo mortality is high, particularly during the first weeks after fertilisation, with total prenatal losses of 70% and higher frequently claimed. However, the first external sign of pregnancy occurs two weeks after fertilisation with a missed menstrual period, and establishing the fate of embryos before this is challenging. Calculations are additionally hampered by a lack of data on the efficiency of fertilisation under natural conditions. Four distinct sources are used to justify quantitative claims regarding embryo loss: (i) a hypothesis published by Roberts & Lowe in The Lancet  is widely cited but has no practical quantitative value; (ii) life table analyses give consistent assessments of clinical pregnancy loss, but cannot illuminate losses at earlier stages of development; (iii) studies that measure human chorionic gonadotrophin (hCG) reveal losses in the second week of development and beyond, but not before; and (iv) the classic studies of Hertig and Rock offer the only direct insight into the fate of human embryos from fertilisation under natural conditions. Re-examination of Hertig’s data demonstrates that his estimates for fertilisation rate and early embryo loss are highly imprecise and casts doubt on the validity of his numerical analysis. A recent re-analysis of hCG study data concluded that approximately 40-60% of embryos may be lost between fertilisation and birth, although this will vary substantially between individual women. In conclusion, natural human embryo mortality is lower than often claimed and widely accepted. Estimates for total prenatal mortality of 70% or higher are exaggerated and not supported by the available data. PMID:28580126

  8. Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Samardzija, Dragana; Stanic, Bojana; Pogrmic-Majkic, Kristina; Fa, Svetlana; Andric, Nebojsa

    2016-12-01

    Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Embryo selection using time-lapse analysis (Early Embryo Viability Assessment) in conjunction with standard morphology: a prospective two-center pilot study.

    PubMed

    Kieslinger, Dorit C; De Gheselle, Stefanie; Lambalk, Cornelis B; De Sutter, Petra; Kostelijk, E Hanna; Twisk, Jos W R; van Rijswijk, Joukje; Van den Abbeel, Etienne; Vergouw, Carlijn G

    2016-11-01

    Does prospective embryo selection using the results from the Eava Test (Early Embryo Viability Assessment) in combination with standard morphology increase the pregnancy rate of IVF and ICSI patients compared to embryo selection based on morphology only? Embryo selection using the Eeva Test plus standard morphology on Day 3 results in comparable pregnancy rates as conventional morphological embryo selection. Time-lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. The Eeva Test records the development of each embryo with a cell-tracking system and predicts the likelihood (High, Medium or Low) that an embryo will form a blastocyst based on an automated analysis of early cell division timings. This trial was designed as a prospective, observational, two-center pilot study with a propensity matched control group. The analysis involved 280 of 302 enrolled patients who were included in the Eeva Test group in 2013 and 560 control patients who were treated in the years 2011-2013. The majority of transfers (98%) were single embryo transfers. Two academic hospitals (VUmc Amsterdam and UZ Gent) enrolled patients <41 years old, with <3 previous attempts and ≥5 normally fertilized eggs. Propensity matching was used to identify a propensity matched control group from a cohort of 1777 patients based on age, cycle number, oocyte number and number of fertilized oocytes. There was no difference in patient baseline characteristics between the two groups. The ongoing pregnancy rate (OPR) of patients enrolled in the Eeva Test group (34.3%; 96/280) did not differ significantly from the OPR in the propensity matched control group (34.6%, 194/560; P = 0.92). However, significantly less top quality embryos (eight-cell embryos with ≤25% fragmentation) were transferred in the Eeva Test group compared to the propensity matched control group (70.4% vs. 82.3%; P < 0.001). The transfer of Eeva High and Medium embryos resulted in a

  10. Vestibular blueprint in early vertebrates.

    PubMed

    Straka, Hans; Baker, Robert

    2013-11-19

    Central vestibular neurons form identifiable subgroups within the boundaries of classically outlined octavolateral nuclei in primitive vertebrates that are distinct from those processing lateral line, electrosensory, and auditory signals. Each vestibular subgroup exhibits a particular morpho-physiological property that receives origin-specific sensory inputs from semicircular canal and otolith organs. Behaviorally characterized phenotypes send discrete axonal projections to extraocular, spinal, and cerebellar targets including other ipsi- and contralateral vestibular nuclei. The anatomical locations of vestibuloocular and vestibulospinal neurons correlate with genetically defined hindbrain compartments that are well conserved throughout vertebrate evolution though some variability exists in fossil and extant vertebrate species. The different vestibular subgroups exhibit a robust sensorimotor signal processing complemented with a high degree of vestibular and visual adaptive plasticity.

  11. Paternal Diet-Induced Obesity Retards Early Mouse Embryo Development, Mitochondrial Activity and Pregnancy Health

    PubMed Central

    Binder, Natalie K.; Hannan, Natalie J.; Gardner, David K.

    2012-01-01

    Worldwide, 48% of adult males are overweight or obese. An association between infertility and excessive body weight is now accepted, although focus remains primarily on females. It has been shown that parental obesity results in compromised embryo development, disproportionate changes in embryo metabolism and reduced blastocyst cell number. The aim of this study was to determine whether paternal obesity has negative effects on the resultant embryo. Specifically, using in vitro fertilisation (IVF), we wanted to isolate the functional effects of obesity on sperm by examining the subsequent embryo both pre- and post-implantation. Epididymal sperm was collected from age matched normal and obese C57BL/6 mice and cryopreserved for subsequent IVF with oocytes collected from Swiss females (normal diet/weight). Obesity was induced in male mice by feeding a high fat diet of 22% fat for 10 weeks. Resultant embryos were cultured individually and development monitored using time-lapse microscopy. Paternal obesity resulted in a significant delay in preimplantation embryo development as early as syngamy (P<0.05). Metabolic parameters were measured across key developmental stages, demonstrating significant reduction in mitochondrial membrane potential (P<0.01). Blastocysts were stained to determine trophectoderm (TE) and inner cell mass (ICM) cell numbers, revealing significant differences in the ratio of cell allocation to TE and ICM lineages (P<0.01). Functional studies examining blastocyst attachment, growth and implantation demonstrated that blastocysts derived from sperm of obese males displayed significantly reduced outgrowth on fibronectin in vitro (P<0.05) and retarded fetal development in vivo following embryo transfer (P<0.05). Taken together, these data clearly demonstrate that paternal obesity has significant negative effects on the embryo at a variety of key early developmental stages, resulting in delayed development, reduced placental size and smaller offspring

  12. Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

    PubMed Central

    Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

    2014-01-01

    During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

  13. The effect of flurbiprofen on the development of anencephaly in early stage chicken embryos.

    PubMed

    Özeren, Ersin; Er, Uygur; Güvenç, Yahya; Demirci, Adnan; Arıkök, Ata Türker; Şenveli, Engin; Ergün, Rüçhan Behzat

    2015-04-01

    The study investigated the effect of flurbiprofen on the development of anencephaly in early stage chicken embryos. We looked at four groups with a total of 36 embryos. There was a control group, a normal saline group, a normal-dose group and a high-dose group with ten, ten, eight and eight eggs with embryo respectively. Two embryos in the control group, studied with light microscopy at 48 h, were consistent with 28-29 hours' incubation in the Hamburger-Hamilton System. They had open neural tubes. The other embryos in this group were considered normal. One embryo in the normal saline group was on the occlusion stage at 48 h. One embryo showed an open neural tube. They were compatible with 28-29 hours' incubation in the Hamburger-Hamilton system. The remaining eight embryos showed normal development. In the normal dose group, one embryo showed underdevelopment of the embryonic disc and the embryo was dead. In four embryos, the neural tubes were open. One cranial malformation was found that was complicated with anencephaly in one embryo. In two embryos the neural tubes were closed, as they showed normal development, and they reached their expected stages according to the Hamburger-Hamilton classification. There was no malformation or growth retardation. Four experimental embryos were anencephalic in the high dose group, and three embryos had open neural tubes. One embryo exhibited both anencephaly and a neural tube closure defect. None of the embryos in this group showed normal development. Even the usual therapeutic doses of flurbiprofen increased the risk of neural tube defect. Flurbiprofen was found to significantly increase the risk of anencephaly. The provision of improved technical materials and studies with larger sample sizes will reveal the stage of morphological disruption during the development of embryos.

  14. Metameric pattern of intervertebral disc/vertebral body is generated independently of Mesp2/Ripply-mediated rostro-caudal patterning of somites in the mouse embryo.

    PubMed

    Takahashi, Yu; Yasuhiko, Yukuto; Takahashi, Jun; Takada, Shinji; Johnson, Randy L; Saga, Yumiko; Kanno, Jun

    2013-08-15

    The vertebrae are derived from the sclerotome of somites. Formation of the vertebral body involves a process called resegmentation, by which the caudal half of a sclerotome is combined with the rostral half of the next sclerotome. To elucidate the relationship between resegmentation and rostro-caudal patterning of somite, we used the Uncx4.1-LacZ transgene to characterize the resegmentation process. Our observations suggested that in the thoracic and lumbar vertebrae, the Uncx4.1-expressing caudal sclerotome gave rise to the intervertebral disc (IVD) and rostral portion of the vertebral body (VB). In the cervical vertebrae, the Uncx4.1-expressing caudal sclerotome appeared to contribute to the IVD and both caudal and rostral ends of the VB. This finding suggests that the rostro-caudal gene expression boundary does not necessarily coincide with the resegmentation boundary. This conclusion was supported by analyses of Mesp2 KO and Ripply1/2 double KO embryos lacking rostral and caudal properties, respectively. Resegmentation was not observed in Mesp2 KO embryos, but both the IVD and whole VB were formed from the caudalized sclerotome. Expression analysis of IVD marker genes including Pax1 in the wild-type, Mesp2 KO, and Ripply1/2 DKO embryos also supported the idea that a metameric pattern of IVD/VB is generated independently of Mesp2/Ripply-mediated rostro-caudal patterning of somite. However, in the lumbar region, IVD differentiation appeared to be stimulated by the caudal property and suppressed by the rostral property. Therefore, we propose that rostro-caudal patterning of somites is not a prerequisite for metameric patterning of the IVD and VB, but instead required to stimulate IVD differentiation in the caudal half of the sclerotome. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. A resegmentation-shift model for vertebral patterning.

    PubMed

    Ward, Lizzy; Evans, Susan E; Stern, Claudio D

    2017-02-01

    Segmentation of the vertebrate body axis is established in the embryo by formation of somites, which give rise to the axial muscles (myotome) and vertebrae (sclerotome). To allow a muscle to attach to two successive vertebrae, the myotome and sclerotome must be repositioned by half a segment with respect to each other. Two main models have been put forward: 'resegmentation' proposes that each half-sclerotome joins with the half-sclerotome from the next adjacent somite to form a vertebra containing cells from two successive somites on each side of the midline. The second model postulates that a single vertebra is made from a single somite and that the sclerotome shifts with respect to the myotome. There is conflicting evidence for these models, and the possibility that the mechanism may vary along the vertebral column has not been considered. Here we use DiI and DiO to trace somite contributions to the vertebrae in different axial regions in the chick embryo. We demonstrate that vertebral bodies and neural arches form by resegmentation but that sclerotome cells shift in a region-specific manner according to their dorsoventral position within a segment. We propose a 'resegmentation-shift' model as the mechanism for amniote vertebral patterning. © 2016 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.

  16. Discriminating signal from noise in the fossil record of early vertebrates reveals cryptic evolutionary history

    PubMed Central

    Sansom, Robert S.; Randle, Emma; Donoghue, Philip C. J.

    2015-01-01

    The fossil record of early vertebrates has been influential in elucidating the evolutionary assembly of the gnathostome bodyplan. Understanding of the timing and tempo of vertebrate innovations remains, however, mired in a literal reading of the fossil record. Early jawless vertebrates (ostracoderms) exhibit restriction to shallow-water environments. The distribution of their stratigraphic occurrences therefore reflects not only flux in diversity, but also secular variation in facies representation of the rock record. Using stratigraphic, phylogenetic and palaeoenvironmental data, we assessed the veracity of the fossil records of the jawless relatives of jawed vertebrates (Osteostraci, Galeaspida, Thelodonti, Heterostraci). Non-random models of fossil recovery potential using Palaeozoic sea-level changes were used to calculate confidence intervals of clade origins. These intervals extend the timescale for possible origins into the Upper Ordovician; these estimates ameliorate the long ghost lineages inferred for Osteostraci, Galeaspida and Heterostraci, given their known stratigraphic occurrences and stem–gnathostome phylogeny. Diversity changes through the Silurian and Devonian were found to lie within the expected limits predicted from estimates of fossil record quality indicating that it is geological, rather than biological factors, that are responsible for shifts in diversity. Environmental restriction also appears to belie ostracoderm extinction and demise rather than competition with jawed vertebrates. PMID:25520359

  17. The mRNA-bound proteome of the early fly embryo

    PubMed Central

    Wessels, Hans-Hermann; Imami, Koshi; Baltz, Alexander G.; Kolinski, Marcin; Beldovskaya, Anastasia; Selbach, Matthias; Small, Stephen; Ohler, Uwe; Landthaler, Markus

    2016-01-01

    Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation. PMID:27197210

  18. Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors.

    PubMed

    Kimber, S J; Sneddon, S F; Bloor, D J; El-Bareg, A M; Hawkhead, J A; Metcalfe, A D; Houghton, F D; Leese, H J; Rutherford, A; Lieberman, B A; Brison, D R

    2008-05-01

    Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

  19. Learning to Fish with Genetics: A Primer on the Vertebrate Model Danio rerio

    PubMed Central

    Holtzman, Nathalia G.; Iovine, M. Kathryn; Liang, Jennifer O.; Morris, Jacqueline

    2016-01-01

    In the last 30 years, the zebrafish has become a widely used model organism for research on vertebrate development and disease. Through a powerful combination of genetics and experimental embryology, significant inroads have been made into the regulation of embryonic axis formation, organogenesis, and the development of neural networks. Research with this model has also expanded into other areas, including the genetic regulation of aging, regeneration, and animal behavior. Zebrafish are a popular model because of the ease with which they can be maintained, their small size and low cost, the ability to obtain hundreds of embryos on a daily basis, and the accessibility, translucency, and rapidity of early developmental stages. This primer describes the swift progress of genetic approaches in zebrafish and highlights recent advances that have led to new insights into vertebrate biology. PMID:27384027

  20. PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

    PubMed Central

    2011-01-01

    Background PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo. Methods Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy. Results PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control). Conclusions PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF

  1. Stickleback embryos use ATP-binding cassette transporters as a buffer against exposure to maternally derived cortisol

    PubMed Central

    Bukhari, Syed Abbas; Bell, Alison M.

    2016-01-01

    Offspring from females that experience stressful conditions during reproduction often exhibit altered phenotypes and many of these effects are thought to arise owing to increased exposure to maternal glucocorticoids. While embryos of placental vertebrates are known to regulate exposure to maternal glucocorticoids via placental steroid metabolism, much less is known about how and whether egg-laying vertebrates can control their steroid environment during embryonic development. We tested the hypothesis that threespine stickleback (Gasterosteus aculeatus) embryos can regulate exposure to maternal steroids via active efflux of maternal steroids from the egg. Embryos rapidly (within 72 h) cleared intact steroids, but blocking ATP-binding cassette (ABC) transporters inhibited cortisol clearance. Remarkably, this efflux of cortisol was sufficient to prevent a transcriptional response of embryos to exogenous cortisol. Taken together, these findings suggest that, much like their placental counterparts, developing fish embryos can actively regulate their exposure to maternal cortisol. These findings highlight the fact that even in egg-laying vertebrates, the realized exposure to maternal steroids is mediated by both maternal and embryonic processes and this has important implications for understanding how maternal stress influences offspring development. PMID:26984623

  2. Method of Electroporation for the Early Chick Embryo

    NASA Astrophysics Data System (ADS)

    Hatakeyama, Jun; Shimamura, Kenji

    Chick embryos have long been one of the favored model systems in the field of embryology and developmental biology. Recent advances in the gene manipulation technologies (Muramatsu et al., 1997; Nakamura et al., 2004) make this model system even more attractive for the developmental biologists (see review by Stern, 2005). Thanks to its two dimensional geometry, easiness in accessibility and observation, and well-established fate maps (e.g. Couly and Le Douarin, 1988; Garcia-Martinez et al., 1993; Hatada and Stern, 1994; Psychoyos and Stern, 1996; Sawada and Aoyama, 1999; Cobos et al., 2001; Lopez-Sanchez et al., 2001; Redkar et al., 2001; Fernandez-Garre et al., 2002; Kimura et al., 2006; Matsushita et al., 2008), it has great advantages especially for studies at the early embryonic stages, such as the processes of gastrulation, neural induction, left-right patterning, etc. For such purposes, a whole embryo culture system, originally invented by Dennis A. T. New (New, 1955), and its derivatives (Flamme, 1987; Sundin and Eichele, 1992; Stern, 1993; Chapman et al., 2001) have been widely used.

  3. Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

    PubMed Central

    Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

    2017-01-01

    Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

  4. Early evolution of multifocal optics for well-focused colour vision in vertebrates.

    PubMed

    Gustafsson, O S E; Collin, S P; Kröger, R H H

    2008-05-01

    Jawless fishes (Agnatha; lampreys and hagfishes) most closely resemble the earliest stage in vertebrate evolution and lamprey-like animals already existed in the Lower Cambrian [about 540 million years ago (MYA)]. Agnathans are thought to have separated from the main vertebrate lineage at least 500 MYA. Hagfishes have primitive eyes, but the eyes of adult lampreys are well-developed. The southern hemisphere lamprey, Geotria australis, possesses five types of opsin genes, three of which are clearly orthologous to the opsin genes of jawed vertebrates. This suggests that the last common ancestor of all vertebrate lineages possessed a complex colour vision system. In the eyes of many bony fishes and tetrapods, well-focused colour images are created by multifocal crystalline lenses that compensate for longitudinal chromatic aberration. To trace the evolutionary origins of multifocal lenses, we studied the optical properties of the lenses in four species of lamprey (Geotria australis, Mordacia praecox, Lampetra fluviatilis and Petromyzon marinus), with representatives from all three of the extant lamprey families. Multifocal lenses are present in all lampreys studied. This suggests that the ability to create well-focused colour images with multifocal optical systems also evolved very early.

  5. Effects of embryo size at transfer (whole versus demi) and early pregnancy progesterone supplementation on embryo growth and pregnancy-specific protein bovine concentrations in recipient dairy heifers.

    PubMed

    Lopes-da-Costa, L; Chagas e Silva, J; Deloche, M C; Jeanguyot, N; Humblot, P; Horta, A E M

    2011-08-01

    The objectives of this study were to evaluate embryonic size and survival, plasma progesterone (P4) and pregnancy-specific protein bovine (PSPB) concentrations in early pregnancies (n = 99) following the transfer of one whole (n = 66) or one demi (n = 33) embryo to recipient virgin dairy heifers. The experiment was designed to evaluate the fixed effects of embryo size at transfer (whole or demi embryo) on Day 7 of the estrous cycle (Day 0 = estrus) and P4 supplementation between Days 7 to 19 through an intravaginal device (yes or no) on plasma P4 and PSPB concentrations and on embryo measurements. Plasma P4 concentrations were measured by RIA on Days 0, 7, 14, 19, 21, 25, 35, 42, 49, 56 and 63 of pregnancy and, PSPB concentrations were measured by ELISA on Days 7, 21, 25, 35, 42, 49, 56 and 63. The presence of an embryonic vesicle was detected on Day 25, embryonic/fetal movements and heartbeat were evaluated on Days 42 and 63 and embryo measurements [crown-rump length (CRL) and width at mid body] were obtained on Day 42 through ultrasonography. In non-supplemented pregnancies, Day 42 whole embryos had higher (P < 0.05) CRL and width than demi embryos, but the difference averaged only 1 to 2 mm. In P4 supplemented pregnancies, whole and demi embryos attained a similar size on Day 42 of pregnancy. Embryo size at transfer, early exogenous P4 supplementation and their interactions had no effects (P > 0.05) on plasma P4 concentrations. However, the post-hoc LSD evaluation showed that plasma P4 concentrations on Day 25 were higher (P < 0.001) in whole than in demi embryo derived pregnancies and, that exogenous P4 supplementation increased (P < 0.05) plasma P4 concentrations on Day 19 of pregnancy. The plasma PSPB detection rate on Days 7 to 63 of pregnancy was similar in pregnancies resulting from the transfer of whole and demi embryos. From a total of 93 recipients remaining pregnant until Day 63, plasma PSPB was constantly undetectable on Day 7, was detected in 4% of

  6. Timing Embryo Segmentation: Dynamics and Regulatory Mechanisms of the Vertebrate Segmentation Clock

    PubMed Central

    Resende, Tatiana P.; Andrade, Raquel P.; Palmeirim, Isabel

    2014-01-01

    All vertebrate species present a segmented body, easily observed in the vertebrate column and its associated components, which provides a high degree of motility to the adult body and efficient protection of the internal organs. The sequential formation of the segmented precursors of the vertebral column during embryonic development, the somites, is governed by an oscillating genetic network, the somitogenesis molecular clock. Herein, we provide an overview of the molecular clock operating during somite formation and its underlying molecular regulatory mechanisms. Human congenital vertebral malformations have been associated with perturbations in these oscillatory mechanisms. Thus, a better comprehension of the molecular mechanisms regulating somite formation is required in order to fully understand the origin of human skeletal malformations. PMID:24895605

  7. β-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo

    PubMed Central

    Wikramanayake, Athula H.; Huang, Ling; Klein, William H.

    1998-01-01

    In sea urchin embryos, the animal-vegetal axis is specified during oogenesis. After fertilization, this axis is patterned to produce five distinct territories by the 60-cell stage. Territorial specification is thought to occur by a signal transduction cascade that is initiated by the large micromeres located at the vegetal pole. The molecular mechanisms that mediate the specification events along the animal–vegetal axis in sea urchin embryos are largely unknown. Nuclear β-catenin is seen in vegetal cells of the early embryo, suggesting that this protein plays a role in specifying vegetal cell fates. Here, we test this hypothesis and show that β-catenin is necessary for vegetal plate specification and is also sufficient for endoderm formation. In addition, we show that β-catenin has pronounced effects on animal blastomeres and is critical for specification of aboral ectoderm and for ectoderm patterning, presumably via a noncell-autonomous mechanism. These results support a model in which a Wnt-like signal released by vegetal cells patterns the early embryo along the animal–vegetal axis. Our results also reveal similarities between the sea urchin animal–vegetal axis and the vertebrate dorsal–ventral axis, suggesting that these axes share a common evolutionary origin. PMID:9689082

  8. Egg accumulation with 3D embryos provides insight into the life history of a pterosaur.

    PubMed

    Wang, Xiaolin; Kellner, Alexander W A; Jiang, Shunxing; Cheng, Xin; Wang, Qiang; Ma, Yingxia; Paidoula, Yahefujiang; Rodrigues, Taissa; Chen, He; Sayão, Juliana M; Li, Ning; Zhang, Jialiang; Bantim, Renan A M; Meng, Xi; Zhang, Xinjun; Qiu, Rui; Zhou, Zhonghe

    2017-12-01

    Fossil eggs and embryos that provide unique information about the reproduction and early growth of vertebrates are exceedingly rare, particularly for pterosaurs. Here we report on hundreds of three-dimensional (3D) eggs of the species Hamipterus tianshanensis from a Lower Cretaceous site in China, 16 of which contain embryonic remains. Computed tomography scanning, osteohistology, and micropreparation reveal that some bones lack extensive ossification in potentially late-term embryos, suggesting that hatchlings might have been flightless and less precocious than previously assumed. The geological context, including at least four levels with embryos and eggs, indicates that this deposit was formed by a rare combination of events, with storms acting on a nesting ground. This discovery supports colonial nesting behavior and potential nesting site fidelity in the Pterosauria. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  9. Regulation of bone morphogenetic proteins in early embryonic development

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  10. Building the Vertebrate Spine

    NASA Astrophysics Data System (ADS)

    Pourquié, Olivier

    2008-03-01

    The vertebrate body can be subdivided along the antero-posterior (AP) axis into repeated structures called segments. This periodic pattern is established during embryogenesis by the somitogenesis process. Somites are generated in a rhythmic fashion from the paraxial mesoderm and subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somite formation involves an oscillator-the segmentation clock-whose periodic signal is converted into the periodic array of somite boundaries. This clock drives the dynamic expression of cyclic genes in the presomitic mesoderm and requires Notch and Wnt signaling. Microarray studies of the mouse presomitic mesoderm transcriptome reveal that the segmentation clock drives the periodic expression of a large network of cyclic genes involved in cell signaling. Mutually exclusive activation of the Notch/FGF and Wnt pathways during each cycle suggests that coordinated regulation of these three pathways underlies the clock oscillator. In humans, mutations in the genes associated to the function of this oscillator such as Dll3 or Lunatic Fringe result in abnormal segmentation of the vertebral column such as those seen in congenital scoliosis. Whereas the segmentation clock is thought to set the pace of vertebrate segmentation, the translation of this pulsation into the reiterated arrangement of segment boundaries along the AP axis involves dynamic gradients of FGF and Wnt signaling. The FGF signaling gradient is established based on an unusual mechanism involving mRNA decay which provides an efficient means to couple the spatio-temporal activation of segmentation to the posterior elongation of the embryo. Another striking aspect of somite production is the strict bilateral symmetry of the process. Retinoic acid was shown to control aspects of this coordination by buffering destabilizing effects from the embryonic left-right machinery. Defects in this embryonic program controlling vertebral symmetry might lead

  11. The Effect of Levetiracetam on Closure of the Midline in Early Chicken Embryos.

    PubMed

    Ozgural, Onur; Armagan, Ercan; Bozkurt, Melih; Eroglu, Umit; Kahilogullari, Gokmen; Unlu, Agahan

    2015-01-01

    Genetic predisposition and some environmental factors play an important role in the development of neural tube defects. Levetiracetam is a new drug that has been approved in the treatment of partial seizures. We aimed in this study to determine the effect of levetiracetam on chick embryos. One hundred and sixty fertile non-pathogenic Super Nick eggs were incubated for 24 hours and were divided into four groups of 40 eggs each. Levetiracetam was administered via the sub-blastodermic route. The eggs were incubated for another 24 hours. All eggs were opened at the 48th hour, and the embryos were evaluated morphologically and histopathologically. The effects of levetiracetam on the embryo were correlated with the dose of levetiracetam. In the light of the results, it was determined that the use of increasing doses of levetiracetam led to defects of midline closure in early chicken embryos. Levetiracetam, a new antiepileptic drug that is effective especially on calcium ion concentration, leads to defects in midline closure in embryos in a dose-dependent manner. Further studies are needed to show the mechanism of embryonic damage and the mechanisms of its teratogenous effects associated with genetic and environmental factors.

  12. A new heart for a new head in vertebrate cardiopharyngeal evolution.

    PubMed

    Diogo, Rui; Kelly, Robert G; Christiaen, Lionel; Levine, Michael; Ziermann, Janine M; Molnar, Julia L; Noden, Drew M; Tzahor, Eldad

    2015-04-23

    It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts - both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates.

  13. Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

    NASA Astrophysics Data System (ADS)

    Lau, Jeffrey M. C.; Muslin, Anthony J.

    The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

  14. Haeckel's Embryos and Evolution: Setting the Record Straight.

    ERIC Educational Resources Information Center

    Wells, Jonathan

    1999-01-01

    Argues that Ernst Haeckel's drawings of vertebrate embryos (1891), which have been widely used in biology textbooks to illustrate his "Biogenetic Law", are factually flawed. Discusses the problems with Haeckel's drawings and his theory. Contains 14 references. (WRM)

  15. Ontogeny of the vertebral column of Eleutherodactylus johnstonei (Anura: Eleutherodactylidae) reveals heterochronies relative to metamorphic frogs.

    PubMed

    Meza-Joya, Fabio Leonardo; Ramos-Pallares, Eliana Patricia; Ramírez-Pinilla, Martha Patricia

    2013-07-01

    Over the last century, the morphogenesis of the vertebral column has been considered as a highly conserved process among anurans. This statement is based on the study of few metamorphic taxa, ignoring the role of developmental mechanisms underlying the evolution of specialized life-histories. Direct development in anurans has been regarded as evolutionarily derived and involves developmental recapitulation and repatterning at different levels in all amphibian taxa studied so far. Herein, we analyze the vertebral column morphogenesis of the direct-developing frog Eleutherodactylus johnstonei, describing the sequence of chondrification and ossification, based on cleared and double-stained specimens from early stage embryos to adults. In general, our results show that the morphogenesis of the vertebral column in E. johnstonei recapitulates the ancestral tadpole-like pattern of development. However, the analysis of the sequence of events using heterochrony plots shows important heterocronies relative to metamorphic species, such as a delay in the chondrification of the vertebral centra and in osteogenesis. These ontogenetic peculiarities may represent derived traits in direct-developing frogs and are possibly correlated with its unusual life history. In addition, several features of the vertebral column of E. johnstonei are highly variable from its typical morphology. We report some malformations and small deviations, which do not seem to affect the survival of individuals. These anomalies have also been found in other frogs, and include many vertebral defects, such as vertebral fusion, and vertebral preclusion and/or induction. Copyright © 2013 Wiley Periodicals, Inc.

  16. Bovine oocytes and early embryos express Staufen and ELAVL RNA-binding proteins.

    PubMed

    Calder, M D; Madan, P; Watson, A J

    2008-05-01

    RNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to the blastocyst stage. ELAVL2 mRNAs were detectable from the GV to the morula stage, whereas ELAVL2 protein was in all stages examined and localized to both cytoplasm and nuclei. The findings provide a foundation for investigating the role of RBPs during mammalian oocyte maturation and early embryogenesis.

  17. Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport.

    PubMed

    Pirkmajer, Sergej; Kirchner, Henriette; Lundell, Leonidas S; Zelenin, Pavel V; Zierath, Juleen R; Makarova, Kira S; Wolf, Yuri I; Chibalin, Alexander V

    2017-07-15

    Small transmembrane proteins such as FXYDs, which interact with Na + ,K + -ATPase, and the micropeptides that interact with sarco/endoplasmic reticulum Ca 2+ -ATPase play fundamental roles in regulation of ion transport in vertebrates. Uncertain evolutionary origins and phylogenetic relationships among these regulators of ion transport have led to inconsistencies in their classification across vertebrate species, thus hampering comparative studies of their functions. We discovered the first FXYD homologue in sea lamprey, a basal jawless vertebrate, which suggests small transmembrane regulators of ion transport emerged early in the vertebrate lineage. We also identified 13 gene subfamilies of FXYDs and propose a revised, phylogeny-based FXYD classification that is consistent across vertebrate species. These findings provide an improved framework for investigating physiological and pathophysiological functions of small transmembrane regulators of ion transport. Small transmembrane proteins are important for regulation of cellular ion transport. The most prominent among these are members of the FXYD family (FXYD1-12), which regulate Na + ,K + -ATPase, and phospholamban, sarcolipin, myoregulin and DWORF, which regulate the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). FXYDs and regulators of SERCA are present in fishes, as well as terrestrial vertebrates; however, their evolutionary origins and phylogenetic relationships are obscure, thus hampering comparative physiological studies. Here we discovered that sea lamprey (Petromyzon marinus), a representative of extant jawless vertebrates (Cyclostomata), expresses an FXYD homologue, which strongly suggests that FXYDs predate the emergence of fishes and other jawed vertebrates (Gnathostomata). Using a combination of sequence-based phylogenetic analysis and conservation of local chromosome context, we determined that FXYDs markedly diversified in the lineages leading to cartilaginous fishes (Chondrichthyes) and bony

  18. The effect of adriamycin exposure on the notochord of mouse embryos.

    PubMed

    Hajduk, Piotr; May, Alison; Puri, Prem; Murphy, Paula

    2012-04-01

    The notochord has important structural and signaling properties during vertebrate development with key roles in patterning surrounding tissues, including the foregut. The adriamycin mouse model is an established model of foregut anomalies where exposure of embryos in utero to the drug adriamycin leads to malformations including oesophageal atresia and tracheoesophageal fistula. In addition to foregut abnormalities, treatment also causes branching, displacement, and hypertrophy of the notochord. Here, we explore the hypothesis that the notochord may be a primary target of disruption leading to abnormal patterning of the foregut by examining notochord position and structure in early embryos following adriamycin exposure. Treated (n = 46) and control (n = 30) embryos were examined during the crucial period when the notochord normally delaminates away from the foregut endoderm (6-28 somite pairs). Transverse sections were derived from the anterior foregut and analyzed by confocal microscopy following immunodetection of extracellular matrix markers E-cadherin and Laminin. In adriamycin-treated embryos across all stages, the notochord was abnormally displaced ventrally with prolonged attachment to the foregut endoderm. While E-cadherin was normally detected in the foregut endoderm with no expression in the notochord of control embryos, treated embryos up to 24 somites showed ectopic notochordal expression indicating a change in characteristics of the tissue; specifically an increase in intracellular adhesiveness, which may be instrumental in structural changes, affecting mechanical and signaling properties. This is consistent with disruption of the notochord leading to altered signaling to the foregut causing abnormal patterning and congenital foregut malformations. © 2012 Wiley Periodicals, Inc.

  19. Temporal Control of Gene Expression by Combining Electroporation and the Tetracycline Inducible Systems in Vertebrate Embryos

    NASA Astrophysics Data System (ADS)

    Dubrulle, Julien; Pourquié, Olivier

    The electroporation technique has revolutionized vertebrate embryology. It has greatly contributed to our understanding of how genes and proteins can interact and regulate various aspects of vertebrate development in the last decade. This technique provides an efficient way to transfect embryonic cells in vivo with exogenous DNA by cre ating transient holes in the plasma membrane with short, squared electric pulses of low voltage (Itasaki et al., 1999; Momose et al., 1999; Muramatsu et al., 1997; Nakamura et al., 2004; Ogura, 2002). It has been particularly well-developed in the chick model since the large size of the embryo and its easy accessibility enables to target specific tissues with great precision. With the electroporation, it is possible to precisely choose which type of cells to transfect by performing a local injection of DNA close to the cells of interest, followed by the application of a small current through the targeted area. To date, all three germ layers — endoderm, mesoderm and ectoderm — as well as an increasing number of differentiated structures have been efficiently transfected (Dubrulle et al., 2001; Grapin-Botton et al., 2001; Itasaki et al., 1999; Luo and Redies, 2005; Scaal et al., 2004) and the continuous improvement in electrode design makes it even possible to aim at sub-populations of cells within a given tissue. In addition to this spatial precision, the technique also allows great temporal precision; any stage of development, ranging from pre-gastrulation stage to adulthood can be reached as long as the cells or structures are accessible for local DNA injection and electrode placement (Bigey et al., 2002; Iimura and Pourquie, 2006).

  20. Supplementation with CTGF, SDF1, NGF, and HGF promotes ovine in vitro oocyte maturation and early embryo development.

    PubMed

    Wang, D H; Ren, J; Zhou, C J; Han, Z; Wang, L; Liang, C G

    2018-05-17

    The strategies for improving the in vitro maturation (IVM) of domestic animal oocytes focus on promoting nuclear and cytoplasmic maturation. The identification of paracrine factors and their supplementation in the culture medium represent effective approaches for oocyte maturation and embryo development. This study investigated the effects of paracrine factor supplementation including connective tissue growth factor (CTGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), and stromal derived factor 1 (SDF1) on ovine oocytes and early parthenogenetic embryos using an in vitro culture system. First, we identified the optimal concentrations of CTGF (30 ng/mL), SDF1 (10 ng/mL), NGF (3 ng/mL), and HGF (100 ng/mL) for promoting oocyte maturation, which combined, induced nuclear maturation in 94.19% of oocytes. This combination also promoted cumulus cell expansion and inhibited oocyte/cumulus apoptosis, while enabling a larger proportion (33.04%) of embryos to develop into blastocysts than in the controls and prevented embryo apoptosis. These novel findings demonstrate that the paracrine factors CTGF, SDF1, NGF, and HGF facilitate ovine oocyte and early parthenogenetic embryo development in vitro. Thus, supplementation with these factors may help optimize the IVM of ovine oocytes and early parthenogenetic embryo development strategies. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Automated image-based phenotypic analysis in zebrafish embryos

    PubMed Central

    Vogt, Andreas; Cholewinski, Andrzej; Shen, Xiaoqiang; Nelson, Scott; Lazo, John S.; Tsang, Michael; Hukriede, Neil A.

    2009-01-01

    Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to utilizing the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes. PMID:19235725

  2. Cilia are required for asymmetric nodal induction in the sea urchin embryo.

    PubMed

    Tisler, Matthias; Wetzel, Franziska; Mantino, Sabrina; Kremnyov, Stanislav; Thumberger, Thomas; Schweickert, Axel; Blum, Martin; Vick, Philipp

    2016-08-23

    Left-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet. Here we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos. Based on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.

  3. Mouse Embryo Compaction.

    PubMed

    White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

    2016-01-01

    Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. © 2016 Elsevier Inc. All rights reserved.

  4. Effect of early addition of bone morphogenetic protein 5 (BMP5) to embryo culture medium on in vitro development and expression of developmentally important genes in bovine preimplantation embryos.

    PubMed

    García, Elina V; Miceli, Dora C; Rizo, Gabriela; Valdecantos, Pablo A; Barrera, Antonio D

    2015-09-01

    Previous studies have reported that bone morphogenetic protein 5 (BMP5) is differentially expressed in the isthmus of bovine oviducts and it is present in the oviductal fluid. However, the specific action of this factor is unknown. To evaluate whether BMP5 exerts some effect during early bovine embryo development, gene expression of BMP5, BMP receptors, and the effect of exogenous BMP5 on in vitro development and expression of developmentally important genes were assessed. In experiment 1, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from in vitro fertilization, were collected for analysis of BMP5 and BMP receptors (BMPR1A, BMPR1B, and BMPR2) messenger RNA (mRNA) expression. On the basis of previous results, in experiment 2, presumptive zygotes were cultured for the first 48 hours after insemination in CR1aa medium assaying three different treatments: (1) control (CR1aa); (2) vehicle control (CR1aa + 0.04 mM HCl), and (3) BMP5 treatment (CR1aa + 100 ng/mL of BMP5). The cleavage rate was evaluated 48 hours after insemination (Day 2), and then, embryos were transferred to CR1aa + 10% fetal bovine serum. The blastocyst rate was determined on Day 7. In experiment 3, pools of embryos at two-cell, four-cell, eight-cell, and blastocyst stages, derived from control and BMP5-treated groups, were collected for analysis of ID2 (BMP target gene), OCT4, NANOG, and SOX2 (pluripotency genes) mRNA expression. BMP5 transcripts were not detectable in any of the embryonic stages examined, whereas the relative mRNA abundance of the three BMP receptors analyzed was greater in early embryo development stages before maternal-embryonic transition, raising the possibility of a direct effect of exogenous BMPs on the embryo during the first developmental period. Although early addition of 100 ng/mL of BMP5 to the embryo culture medium had no effect on the cleavage rate, a significantly higher proportion of cleaved embryos developed to the

  5. Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.

    PubMed

    Flores, Luis E; Hildebrandt, Thomas B; Kühl, Anja A; Drews, Barbara

    2014-05-10

    Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9-11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert's membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart

  6. Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy

    PubMed Central

    2014-01-01

    Background Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. Methods In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. Results The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9–11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert’s membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and

  7. Facultative parthenogenesis in vertebrates: reproductive error or chance?

    PubMed

    Lampert, K P

    2008-01-01

    Parthenogenesis, the development of an embryo from a female gamete without any contribution of a male gamete, is very rare in vertebrates. Parthenogenetically reproducing species have, so far, only been found in the Squamate reptiles (lizards and snakes). Facultative parthenogenesis, switching between sexual and clonal reproduction, although quite common in invertebrates, e.g. Daphnia and aphids, seems to be even rarer in vertebrates. However, isolated cases of parthenogenetic development have been reported in all vertebrate groups. Facultative parthenogenesis in vertebrates has only been found in captive animals but might simply have been overlooked in natural populations. Even though its evolutionary impact is hard to determine and very likely varies depending on the ploidy restoration mechanisms and sex-determining mechanisms involved, facultative parthenogenesis is already discussed in conservation biology and medical research. To raise interest for facultative parthenogenesis especially in evolutionary biology, I summarize the current knowledge about facultative parthenogenesis in the different vertebrate groups, introduce mechanisms of diploid oocyte formation and discuss the genetic consequences and potential evolutionary impact of facultative parthenogenesis in vertebrates.

  8. A new heart for a new head in vertebrate cardiopharyngeal evolution

    PubMed Central

    Diogo, Rui; Kelly, Robert G.; Christiaen, Lionel; Levine, Michael; Ziermann, Janine M.; Molnar, Julia L.; Noden, Drew M.; Tzahor, Eldad

    2015-01-01

    It has been more than 30 years since the publication of the new head hypothesis, which proposed that the vertebrate head is an evolutionary novelty resulting from the emergence of neural crest and cranial placodes. Neural crest generates the skull and associated connective tissues, whereas placodes produce sensory organs. However, neither crest nor placodes produce head muscles, which are a crucial component of the complex vertebrate head. We discuss emerging evidence for a surprising link between the evolution of head muscles and chambered hearts — both systems arise from a common pool of mesoderm progenitor cells within the cardiopharyngeal field of vertebrate embryos. We consider the origin of this field in non-vertebrate chordates and its evolution in vertebrates. PMID:25903628

  9. Tissue-specific expression of FoxD reporter constructs in amphioxus embryos.

    PubMed

    Yu, Jr-Kai; Holland, Nicholas D; Holland, Linda Z

    2004-10-15

    Cephalochordates (amphioxus), the closest living invertebrate relatives of the vertebrates, are key to understanding the evolution of developmental mechanisms during the invertebrate-to-vertebrate transition. However, a major impediment to amphioxus as a model organism for developmental biology has been the inability to introduce transgenes or other macromolecules into the embryos. Here, we report the development of a reproducible method for microinjection of amphioxus eggs. Specifically, we show that expression of a LacZ reporter construct including 6.3 kb of AmphiFoxD upstream regulatory DNA recapitulates expression of the endogenous gene in the nerve cord, somites, and notochord. We have also identified the 1.6 kb at the 5' end of this region as essential for expression in the first two of these domains and the 4.7 kb at the 3' end as sufficient for expression in the notochord. This study, which is the first report of a method for introduction of large molecules such as DNA into amphioxus embryos, opens the way for studies of gene regulation and function in amphioxus and for comparative studies with vertebrates to understand the relationship between the extensive gene duplications that occurred within the vertebrate lineage and the evolution of vertebrate innovations such as neural crest.

  10. Moral uncertainty in bioethical argumentation: a new understanding of the pro-life view on early human embryos.

    PubMed

    Żuradzki, Tomasz

    2014-12-01

    In this article, I present a new interpretation of the pro-life view on the status of early human embryos. In my understanding, this position is based not on presumptions about the ontological status of embryos and their developmental capabilities but on the specific criteria of rational decisions under uncertainty and on a cautious response to the ambiguous status of embryos. This view, which uses the decision theory model of moral reasoning, promises to reconcile the uncertainty about the ontological status of embryos with the certainty about normative obligations. I will demonstrate that my interpretation of the pro-life view, although seeming to be stronger than the standard one, has limited scope and cannot be used to limit destructive research on human embryos.

  11. TALE transcription factors during early development of the vertebrate brain and eye.

    PubMed

    Schulte, Dorothea; Frank, Dale

    2014-01-01

    Our brain's cognitive performance arises from the coordinated activities of billions of nerve cells. Despite a high degree of morphological and functional differences, all neurons of the vertebrate central nervous system (CNS) arise from a common field of multipotent progenitors. Cell fate specification and differentiation are directed by multistep processes that include inductive/external cues, such as the extracellular matrix or growth factors, and cell-intrinsic determinants, such as transcription factors and epigenetic modulators of proteins and DNA. Here we review recent findings implicating TALE-homeodomain proteins in these processes. Although originally identified as HOX-cofactors, TALE proteins also contribute to many physiological processes that do not require HOX-activity. Particular focus is, therefore, given to HOX-dependent and -independent functions of TALE proteins during early vertebrate brain development. Additionally, we provide an overview about known upstream and downstream factors of TALE proteins in the developing vertebrate brain and discuss general concepts of how TALE proteins function to modulate neuronal cell fate specification. Copyright © 2013 Wiley Periodicals, Inc.

  12. Maternal hCG concentrations in early IVF pregnancies: associations with number of cells in the Day 2 embryo and oocytes retrieved.

    PubMed

    Tanbo, T G; Eskild, A

    2015-12-01

    Do number of cells in the transferred cleavage stage embryo and number of oocytes retrieved for IVF influence maternal hCG concentrations in early pregnancies? Compared with transfer of a 2-cell embryo, transfer of a 4-cell embryo results in higher hCG concentrations on Day 12 after transfer, and more than 20 oocytes retrieved were associated with low hCG concentrations. Maternal hCG concentration in very early pregnancy varies considerably among women, but is likely to be an indicator of time since implantation of the embryo into the endometrium, in addition to number and function of trophoblast cells. We followed 1047 pregnancies after IVF/ICSI from oocyte retrieval until Day 12 after embryo transfer. Women were recruited in Norway during the years 2005-2013. Successful pregnancies after transfer of one single embryo that had been cultured for 2 days were included. Maternal hCG was quantified on Day 12 after embryo transfer by chemiluminescence immunoassay, which measures intact hCG and the free β-hCG chain. Information on a successful pregnancy, defined as birth after >16 weeks, was obtained by linkage to the Medical Birth Registry of Norway. Transfer of a 4-cell embryo resulted in higher maternal hCG concentrations compared with transfer of a 2-cell embryo (134.8 versus 87.8 IU/l, P < 0.05). A high number of oocytes retrieved (>20) was associated with low hCG concentrations (P < 0.05). The factors studied explain a limited part of the total variation of hCG concentrations in early pregnancy. Although embryo transfer was performed at the same time after fertilization, we do not know the exact time of implantation. A further limitation to our study is that the number of pregnancies after transfer of a 2-cell embryo was small (27 cases). Number of cells in the transferred embryo and number of oocytes retrieved may influence the conditions and timing for embryo implantation in different ways and thereby influence maternal hCG concentrations. Such knowledge may be

  13. vEmbryo In Silico Models: Predicting Vascular Developmental Toxicity

    EPA Science Inventory

    The cardiovascular system is the first to function in the vertebrate embryo, reflecting the critical need for nutrient delivery and waste removal during organogenesis. Blood vessel development occurs by complex interacting signaling networks, including extra-cellular matrix remod...

  14. Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation

    PubMed Central

    Liu, Yunlong; Balaraman, Yokesh; Wang, Guohua; Nephew, Kenneth P.; Zhou, Feng C.

    2009-01-01

    Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88 mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10 and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p < 0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes

  15. Alcohol exposure alters DNA methylation profiles in mouse embryos at early neurulation.

    PubMed

    Liu, Yunlong; Balaraman, Yokesh; Wang, Guohua; Nephew, Kenneth P; Zhou, Feng C

    2009-10-01

    Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10, and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p<0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in

  16. Early maternal serum ß-human chorionic gonadotropin (ß-hCG) levels and sex-related growth difference of IVF embryos.

    PubMed

    Esh-Broder, Efrat; Oron, Galia; Son, Weon-Young; Holzer, Hananel; Tulandi, Togas

    2015-10-01

    Maternal serum ß-human chorionic gonadotropin (ß-hCG) represents the trophoblastic cell mass and is an indirect measurement of embryo development at early implantation stage. Studies in animals and human embryos detected sex-related growth differences (SRGD) in favour of male embryos during the pre-implantation period. The purpose of our study was to correlate SRGD and maternal serum ß-hCG at 16 days after embryo transfer. We retrospectively analysed all (fresh and frozen) non-donor, single embryo transfers (SET), elective and not elective, that were performed between December 2008 and December 2013. We included ß-hCG values from day 16 after oocyte collection of pregnancies resulting in live birth. Neonatal gender was retrieved from patient files. Male and female embryos were further grouped to cleavage and blastocyst stage transfers. Regression analysis for confounding variables included maternal age, maternal body mass index (BMI), use of micromanipulation (ICSI), embryo quality (grade), assisted hatching, day of transfer and fresh or frozen embryo transfer. Seven hundred eighty-six non-donor SETs resulted in live birth. After including only day 16 serum ß-hCG results, 525 SETs were analysed. Neonatal gender was available for 522 cases. Mean maternal serum ß-hCG levels were similar, 347 ± 191 IU/L in the male newborn group and 371 ± 200 IU/L in the female group. The difference between ß-hCG levels remained insignificant after adjusting for confounding variables. Early maternal ß-hCG levels after embryo transfers did not represent SRGD in our study.

  17. Chicken Embryos as a Potential New Model for Early Onset Type I Diabetes

    PubMed Central

    Shi, Liheng; Ko, Michael L.; Huang, Cathy Chia-Yu; Park, So-Young; Hong, Min-Pyo; Ko, Gladys Y.-P.

    2014-01-01

    Diabetic retinopathy (DR) is the leading cause of blindness among the American working population. The purpose of this study is to establish a new diabetic animal model using a cone-dominant avian species to address the distorted color vision and altered cone pathway responses in prediabetic and early diabetic patients. Chicken embryos were injected with either streptozotocin (STZ), high concentration of glucose (high-glucose), or vehicle at embryonic day 11. Cataracts occurred in varying degrees in both STZ- and high glucose-induced diabetic chick embryos at E18. Streptozotocin-diabetic chicken embryos had decreased levels of blood insulin, glucose transporter 4 (Glut4), and phosphorylated protein kinase B (pAKT). In STZ-injected E20 embryos, the ERG amplitudes of both a- and b-waves were significantly decreased, the implicit time of the a-wave was delayed, while that of the b-wave was significantly increased. Photoreceptors cultured from STZ-injected E18 embryos had a significant decrease in L-type voltage-gated calcium channel (L-VGCC) currents, which was reflected in the decreased level of L-VGCCα1D subunit in the STZ-diabetic retinas. Through these independent lines of evidence, STZ-injection was able to induce pathological conditions in the chicken embryonic retina, and it is promising to use chickens as a potential new animal model for type I diabetes. PMID:25133191

  18. Three neuropeptide Y receptor genes in the spiny dogfish, Squalus acanthias, support en bloc duplications in early vertebrate evolution.

    PubMed

    Salaneck, Erik; Ardell, David H; Larson, Earl T; Larhammar, Dan

    2003-08-01

    It has been debated whether the increase in gene number during early vertebrate evolution was due to multiple independent gene duplications or synchronous duplications of many genes. We describe here the cloning of three neuropeptide Y (NPY) receptor genes belonging to the Y1 subfamily in the spiny dogfish, Squalus acanthias, a cartilaginous fish. The three genes are orthologs of the mammalian subtypes Y1, Y4, and Y6, which are located in paralogous gene regions on different chromosomes in mammals. Thus, these genes arose by duplications of a chromosome region before the radiation of gnathostomes (jawed vertebrates). Estimates of duplication times from linearized trees together with evidence from other gene families supports two rounds of chromosome duplications or tetraploidizations early in vertebrate evolution. The anatomical distribution of mRNA was determined by reverse-transcriptase PCR and was found to differ from mammals, suggesting differential functional diversification of the new gene copies during the radiation of the vertebrate classes.

  19. Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

    PubMed Central

    Murray, Jessica R.; Stanciauskas, Monika E.; Aralere, Tejas S.; Saha, Margaret S.

    2014-01-01

    The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology1,2, behavior3, and memory and learning4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access. PMID:24999108

  20. Early genetic consequences of defaunation in a large-seeded vertebrate-dispersed palm (Syagrus romanzoffiana)

    PubMed Central

    Giombini, M I; Bravo, S P; Sica, Y V; Tosto, D S

    2017-01-01

    Plant populations are seriously threatened by anthropogenic habitat disturbance. In particular, defaunation may disrupt plant-disperser mutualisms, thus reducing levels of seed-mediated gene flow and genetic variation in animal-dispersed plants. This may ultimately limit their adaptive potential and ability to cope with environmental change. Tropical forest remnants are typically deprived of medium to large vertebrates upon which many large-seeded plants rely for accomplishing effective seed dispersal. Our main goal was to examine the potential early genetic consequences of the loss of large vertebrates for large-seeded vertebrate-dispersed plants. We compared the genetic variation in early-stage individuals of the large-seeded palm Syagrus romanzoffiana between continuous protected forest and nearby partially defaunated fragments in the Atlantic Forest of South America. Using nine microsatellites, we found lower allelic richness and stronger fine-scale spatial genetic structure in the disturbed area. In addition, the percentage of dispersed recruits around conspecific adults was lower, although not significantly, in the disturbed area (median values: 0.0 vs 14.4%). On the other hand, no evidence of increased inbreeding or reduced pollen-mediated gene flow (selfing rate and diversity of pollen donors) was found in the disturbed area. Our findings are strongly suggestive of some early genetic consequences resulting from the limitation in contemporary gene flow via seeds, but not pollen, in defaunated areas. Plant-disperser mutualisms involving medium–large frugivores, which are seriously threatened in tropical systems, should therefore be protected to warrant the maintenance of seed-mediated gene flow and genetic diversity in large-seeded plants. PMID:28121308

  1. CRISPR/Cas9-mediated mutagenesis in the sea lamprey Petromyzon marinus: a powerful tool for understanding ancestral gene functions in vertebrates

    PubMed Central

    Square, Tyler; Romášek, Marek; Jandzik, David; Cattell, Maria V.; Klymkowsky, Michael; Medeiros, Daniel M.

    2015-01-01

    Lamprey is one of only two living jawless vertebrates, a group that includes the first vertebrates. Comparisons between lamprey and jawed vertebrates have yielded important insights into the origin and evolution of vertebrate physiology, morphology and development. Despite its key phylogenetic position, studies of lamprey have been limited by their complex life history, which makes traditional genetic approaches impossible. The CRISPR/Cas9 system is a bacterial defense mechanism that was recently adapted to achieve high-efficiency targeted mutagenesis in eukaryotes. Here we report CRISPR/Cas9-mediated disruption of the genes Tyrosinase and FGF8/17/18 in the sea lamprey Petromyzon marinus, and detail optimized parameters for producing mutant F0 embryos. Using phenotype and genotype analyses, we show that CRISPR/Cas9 is highly effective in the sea lamprey, with a majority of injected embryos developing into complete or partial mutants. The ability to create large numbers of mutant embryos without inbred lines opens exciting new possibilities for studying development in lamprey and other non-traditional model organisms with life histories that prohibit the generation of mutant lines. PMID:26511928

  2. Disparities in reproductive outcomes according to the endometrial preparation protocol in frozen embryo transfer : The risk of early pregnancy loss in frozen embryo transfer cycles.

    PubMed

    Hatoum, I; Bellon, L; Swierkowski, N; Ouazana, M; Bouba, S; Fathallah, K; Paillusson, B; Bailly, M; Boitrelle, F; Alter, L; Bergère, M; Selva, J; Wainer, R

    2018-03-01

    The purpose of this study was to determine the effect of stimulated and artificial endometrial preparation protocols on reproductive outcomes in frozen embryo transfer (FET) cycles. We performed a retrospective study of 1926 FET cycles over a 3.5-year period in the Fertility Unit at a University Hospital. Stimulated and artificial protocols were used for endometrial preparation. The embryos for FET were obtained from either in vitro fertilization or intracytoplasmic sperm injection cycles. Live birth rate and early pregnancy loss rates were retrospectively compared. In artificial protocols, oral or vaginal administration of oestradiol 2 mg two or three times a day was followed by vaginal supplementation with progesterone 200 mg two or three times a day. In stimulated protocols, recombinant follicle-stimulating hormone was administered from day 4 onward. Vaginal ultrasound was used for endometrial and ovarian monitoring. A pregnancy test was performed 14 days after FET. If it was positive, oestradiol and progesterone were administered up until the 12th week of gestation in artificial cycles. We defined early pregnancy losses as biochemical pregnancies (preclinical losses) and miscarriages. Data on 865 artificial cycles (45% of the total) and 1061 stimulated cycles (55%) were collected. Early pregnancy loss rate was significantly lower for stimulated cycles (34.2%) than for artificial cycles (56.9%), and the live birth rate was significantly higher for stimulated cycles (59.7%) than for artificial cycles (29.1%). In frozen embryo transfer, artificial cycles were associated with more early pregnancy loss and lower live birth rate than stimulated cycles.

  3. Chlorpyrifos exposure affects fgf8, sox9, and bmp4 expression required for cranial neural crest morphogenesis and chondrogenesis in Xenopus laevis embryos.

    PubMed

    Tussellino, Margherita; Ronca, Raffaele; Carotenuto, Rosa; Pallotta, Maria M; Furia, Maria; Capriglione, Teresa

    2016-10-01

    Chlorpyrifos (CPF) is an organophosphate insecticide used primarily to control foliage and soil-borne insect pests on a variety of food and feed crops. In mammals, maternal exposure to CPF has been reported to induce dose-related abnormalities such as slower brain growth and cerebral cortex thinning. In lower vertebrates, for example, fish and amphibians, teratogenic activity of this compound is correlated with several anatomical alterations. Little is known about the effects of CPF on mRNA expression of genes involved in early development of the anatomical structures appearing abnormal in embryos. This study investigated the effects of exposure to different CPF concentrations (10, 15 and 20 mg/L) on Xenopus laevis embryos from stage 4/8 to stage 46. Some of the morphological changes we detected in CPF-exposed embryos included cranial neural crest cell (NCC)-derived structures. For this reason, we analyzed the expression of select genes involved in hindbrain patterning (egr2), cranial neural crest chondrogenesis, and craniofacial development (fgf8, bmp4, sox9, hoxa2 and hoxb2). We found that CPF exposure induced a reduction in transcription of all the genes involved in NCC-dependent chondrogenesis, with largest reductions in fgf8 and sox9; whereas, in hindbrain, we did not find any alterations in egr2 expression. Changes in the expression of fgf8, bmp4, and sox9, which are master regulators of several developmental pathways, have important implications. If these changes are confirmed to belong to a general pattern of alterations in vertebrates prenatally exposed to OP, they might be useful to assess damage during vertebrate embryo development. Environ. Mol. Mutagen. 57:589-604, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Detection of botulinum toxin types A, B, E, and F activity using the quail embryo

    USDA-ARS?s Scientific Manuscript database

    We recently demonstrated an effective new model for the detection of botulinum toxin type A using quail embryos in place of the mouse model. These experiments demonstrated that the Japanese quail embryo at 15 days of incubation was an effective vertebrate animal model to detect the activity of botu...

  5. The early Cambrian fossil embryo Pseudooides is a direct-developing cnidarian, not an early ecdysozoan.

    PubMed

    Duan, Baichuan; Dong, Xi-Ping; Porras, Luis; Vargas, Kelly; Cunningham, John A; Donoghue, Philip C J

    2017-12-20

    Early Cambrian Pseudooides prima has been described from embryonic and post-embryonic stages of development, exhibiting long germ-band development. There has been some debate about the pattern of segmentation, but this interpretation, as among the earliest records of ecdysozoans, has been generally accepted. Here, we show that the 'germ band' of P. prima embryos separates along its mid axis during development, with the transverse furrows between the 'somites' unfolding into the polar aperture of the ten-sided theca of Hexaconularia sichuanensis , conventionally interpreted as a scyphozoan cnidarian; co-occurring post-embryonic remains of ecdysozoans are unrelated. We recognize H. sichuanensis as a junior synonym of P. prima as a consequence of identifying these two form-taxa as distinct developmental stages of the same organism. Direct development in P. prima parallels the co-occuring olivooids Olivooides, and Quadrapyrgites and Bayesian phylogenetic analysis of a novel phenotype dataset indicates that, despite differences in their tetra-, penta- and pseudo-hexa-radial symmetry, these hexangulaconulariids comprise a clade of scyphozoan medusozoans, with Arthrochites and conulariids, that all exhibit direct development from embryo to thecate polyp. The affinity of hexangulaconulariids and olivooids to extant scyphozoan medusozoans indicates that the prevalence of tetraradial symmetry and indirect development are a vestige of a broader spectrum of body-plan symmetries and developmental modes that was manifest in their early Phanerozoic counterparts. © 2017 The Authors.

  6. The early Cambrian fossil embryo Pseudooides is a direct-developing cnidarian, not an early ecdysozoan

    PubMed Central

    2017-01-01

    Early Cambrian Pseudooides prima has been described from embryonic and post-embryonic stages of development, exhibiting long germ-band development. There has been some debate about the pattern of segmentation, but this interpretation, as among the earliest records of ecdysozoans, has been generally accepted. Here, we show that the ‘germ band’ of P. prima embryos separates along its mid axis during development, with the transverse furrows between the ‘somites’ unfolding into the polar aperture of the ten-sided theca of Hexaconularia sichuanensis, conventionally interpreted as a scyphozoan cnidarian; co-occurring post-embryonic remains of ecdysozoans are unrelated. We recognize H. sichuanensis as a junior synonym of P. prima as a consequence of identifying these two form-taxa as distinct developmental stages of the same organism. Direct development in P. prima parallels the co-occuring olivooids Olivooides, and Quadrapyrgites and Bayesian phylogenetic analysis of a novel phenotype dataset indicates that, despite differences in their tetra-, penta- and pseudo-hexa-radial symmetry, these hexangulaconulariids comprise a clade of scyphozoan medusozoans, with Arthrochites and conulariids, that all exhibit direct development from embryo to thecate polyp. The affinity of hexangulaconulariids and olivooids to extant scyphozoan medusozoans indicates that the prevalence of tetraradial symmetry and indirect development are a vestige of a broader spectrum of body-plan symmetries and developmental modes that was manifest in their early Phanerozoic counterparts. PMID:29237861

  7. Insulin-like growth factor (IGF) signalling is required for early dorso-anterior development of the zebrafish embryo.

    PubMed

    Eivers, Edward; McCarthy, Karena; Glynn, Catherine; Nolan, Catherine M; Byrnes, Lucy

    2004-12-01

    The insulin-like growth factor (IGF) signalling pathway has been highly conserved in animal evolution and, in mammals and Xenopus, plays a key role in embryonic growth and development, with the IGF-1 receptor (IGF-1R) being a crucial regulator of the signalling cascade. Here we report the first functional role for the IGF pathway in zebrafish. Expression of mRNA coding for a dominant negative IGF-1R resulted in embryos that were small in size compared to controls and had disrupted head and CNS development. At its most extreme, this phenotype was characterized by a complete loss of head and eye structures, an absence of notochord and the presence of abnormal somites. In contrast, up-regulation of IGF signalling following injection of IGF-1 mRNA, resulted in a greatly expanded development of anterior structures at the expense of trunk and tail. IGF-1R knockdown caused a significant decrease in the expression of Otx2, Rx3, FGF8, Pax6.2 and Ntl, while excess IGF signalling expanded Otx2 expression in presumptive forebrain tissue and widened the Ntl expression domain in the developing notochord. The observation that IGF-1R knockdown reduced expression of two key organizer genes (chordin and goosecoid) suggests that IGF signalling plays a role in regulating zebrafish organizer activity. This is supported by the expression of IGF-1, IGF-2 and IGF-1R in shield-stage zebrafish embryos and the demonstration that IGF signalling influences expression of BMP2b, a gene that plays an important role in zebrafish pattern formation. Our data is consistent with a common pathway for integration of IGF, FGF8 and anti-BMPs in early vertebrate development.

  8. Early developmental gene regulation in Strongylocentrotus purpuratus embryos in response to elevated CO₂ seawater conditions.

    PubMed

    Hammond, LaTisha M; Hofmann, Gretchen E

    2012-07-15

    Ocean acidification, or the increased uptake of CO(2) by the ocean due to elevated atmospheric CO(2) concentrations, may variably impact marine early life history stages, as they may be especially susceptible to changes in ocean chemistry. Investigating the regulatory mechanisms of early development in an environmental context, or ecological development, will contribute to increased understanding of potential organismal responses to such rapid, large-scale environmental changes. We examined transcript-level responses to elevated seawater CO(2) during gastrulation and the initiation of spiculogenesis, two crucial developmental processes in the purple sea urchin, Strongylocentrotus purpuratus. Embryos were reared at the current, accepted oceanic CO(2) concentration of 380 microatmospheres (μatm), and at the elevated levels of 1000 and 1350 μatm, simulating predictions for oceans and upwelling regions, respectively. The seven genes of interest comprised a subset of pathways in the primary mesenchyme cell gene regulatory network (PMC GRN) shown to be necessary for the regulation and execution of gastrulation and spiculogenesis. Of the seven genes, qPCR analysis indicated that elevated CO(2) concentrations only had a significant but subtle effect on two genes, one important for early embryo patterning, Wnt8, and the other an integral component in spiculogenesis and biomineralization, SM30b. Protein levels of another spicule matrix component, SM50, demonstrated significant variable responses to elevated CO(2). These data link the regulation of crucial early developmental processes with the environment that these embryos would be developing within, situating the study of organismal responses to ocean acidification in a developmental context.

  9. Cell cycle in ascidian eggs and embryos.

    PubMed

    McDougall, Alex; Chenevert, Janet; Lee, Karen W; Hebras, Celine; Dumollard, Remi

    2011-01-01

    In ascidians the cell cycle machinery has been studied mainly in oocytes while ascidian embryos have been used to dissect the mechanism that controls asymmetric cell division (ACD). Here we overview the most specific and often exceptional points and events in cell cycle control in ascidian oocytes and early embryos. Mature stage IV eggs are arrested at metaphase I due to cytostatic factor (CSF). In vertebrates, unfertilized eggs are arrested at metaphase II by CSF. Meta II-CSF is mediated by the Mos/MEK/MAPK/Erp1 pathway, which inhibits the ubiquitin ligase APC/C(cdc20) preventing cyclin B destruction thus stabilizing MPF activity. CSF is inactivated by the fertilization Ca(2+) transient that stimulates the destruction of Erp1 thus releasing APC/C(cdc20) from inhibition. Although many of the components of CSF are conserved between the ascidian and the vertebrates, the lack of Erp1 in the ascidians (and indeed other invertebrates) is notable since the Mos/MAPK pathway nonetheless mediates Meta I-CSF. Moreover, since the fertilization Ca(2+) transient targets Erp1, it is not clear how the sperm-triggered Ca(2+) transient in ascidians (and again other invertebrates) stimulates cyclin B destruction in the absence of Erp1. Nonetheless, like mammalian eggs, sperm trigger a series of Ca(2+) oscillations that increases the rate of cyclin B destruction and the subsequent loss of MAPK activity leading to meiotic exit in ascidians. Positive feedback from MPF maintains the Ca(2+) oscillations in fertilized ascidian eggs ensuring the eventual loss of MPF stimulating the egg-to-embryo transition. Embryonic cell cycles in the ascidian are highly stereotyped where both the rate of cell division and the orientation of cell division planes are precisely controlled. Three successive rounds of ACD generate two small posterior germ cell precursors at the 64 cell stage. The centrosome-attracting body (CAB) is a macroscopic cortical structure visible by light microscopy that causes these

  10. No significant regulation of bicoid mRNA by Pumilio or Nanos in the early Drosophila embryo.

    PubMed

    Wharton, Tammy H; Nomie, Krystle J; Wharton, Robin P

    2018-01-01

    Drosophila Pumilio (Pum) is a founding member of the conserved Puf domain class of RNA-binding translational regulators. Pum binds with high specificity, contacting eight nucleotides, one with each of the repeats in its RNA-binding domain. In general, Pum is thought to block translation in collaboration with Nanos (Nos), which exhibits no binding specificity in isolation but is recruited jointly to regulatory sequences containing a Pum binding site in the 3'-UTRs of target mRNAs. Unlike Pum, which is ubiquitous in the early embryo, Nos is tightly restricted to the posterior, ensuring that repression of its best-characterized target, maternal hunchback (hb) mRNA, takes place exclusively in the posterior. An exceptional case of Nos-independent regulation by Pum has been described-repression of maternal bicoid (bcd) mRNA at the anterior pole of the early embryo, dependent on both Pum and conserved Pum binding sites in the 3'-UTR of the mRNA. We have re-investigated regulation of bcd in the early embryo; our experiments reveal no evidence of a role for Pum or its conserved binding sites in regulation of the perdurance of bcd mRNA or protein. Instead, we find that Pum and Nos control the accumulation of bcd mRNA in testes.

  11. Late development of hagfish vertebral elements.

    PubMed

    Ota, Kinya G; Fujimoto, Satoko; Oisi, Yasuhiro; Kuratani, Shigeru

    2013-05-01

    It has been demonstrated recently that hagfishes, one of two groups of extant jawless vertebrates, have cartilaginous vertebral elements. Embryological and gene expression analyses have also shown that this group of animals develops a sclerotome, the potential primordium of the axial skeleton. However, it has not been shown unequivocally that the hagfish sclerotome truly differentiates into cartilage, because access to late-stage embryos and information about the cartilaginous extracellular matrix (ECM) are lacking for these animals. Here we investigated the expression patterns of the biglycan/decorin (BGN/DCN) gene in the inshore hagfish, Eptatretus burgeri. The homologue of this gene encodes the major noncollagenous component of the cartilaginous ECM among gnathostomes. We clearly identified the expression of this gene in adult vertebral tissues and in embryonic mesenchymal cells on the ventral aspect of the notochord. Taking into account that the sclerotome in the gnathostomes expresses BGN/DCN gene during the chondrogenesis, it is highly expected the hagfish BGN/DCN-positive mesenchymal cells are derived from the sclerotomes. We propose that hagfishes and gnathostomes share conserved developmental mechanisms not only in their somite differentiation, but also in chondrogenesis of their vertebral elements. Copyright © 2013 Wiley Periodicals, Inc.

  12. Studies on trypsin-like enzymes in sperm and early embryos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Penn, A.

    1975-12-09

    Results are reported from a study of acrosomal proteinase, a trypsin-like enzyme (TLE), found in the acrosome of all eutherian mammals studied to date. It has been implicated in the dissolution of a passage for the sperm through the zona pellucida of the egg, a step necessary for in vivo fertilization. A cytochemical procedure employing autoradiographic film as a gelatin substrate is described for in situ detection and localization of acrosomal proteolytic activity. A role for TLE in the early development of embryos is suggested. (CH)

  13. Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription.

    PubMed

    Balbi, Teresa; Franzellitti, Silvia; Fabbri, Rita; Montagna, Michele; Fabbri, Elena; Canesi, Laura

    2016-11-01

    Bisphenol A (BPA), a monomer used in plastic manufacturing, is weakly estrogenic and a potential endocrine disruptor in mammals. Although it degrades quickly, it is pseudo-persistent in the environment because of continual inputs, with reported concentrations in aquatic environments between 0.0005 and 12 μg/L. BPA represents a potential concern for aquatic ecosystems, as shown by its reproductive and developmental effects in aquatic vertebrates. In invertebrates, endocrine-related effects of BPA were observed in different species and experimental conditions, with often conflicting results, indicating that the sensitivity to this compound can vary considerably among related taxa. In the marine mussel Mytilus galloprovincialis BPA was recently shown to affect early development at environmental concentrations. In this work, the possible effects of BPA on mussel embryos were investigated at the molecular level by evaluating transcription of 13 genes, selected on the basis of their biological functions in adult mussels. Gene expression was first evaluated in trocophorae and D-veligers (24 and 48 h post fertilization) grown in physiological conditions, in comparison with unfertilized eggs. Basal expressions showed a general up-regulation during development, with distinct transcript levels in trocophorae and D-veligers. Exposure of fertilized eggs to BPA (10 μg/L) induced a general upregulation at 24 h pf, followed by down regulation at 48 h pf. Mytilus Estrogen Receptors, serotonin receptor and genes involved in biomineralization (Carbonic Anydrase and Extrapallial Protein) were the most affected by BPA exposure. At 48 h pf, changes in gene expression were associated with irregularities in shell formation, as shown by scanning electron microscopy (SEM), indicating that the formation of the first shelled embryo, a key step in mussel development, represents a sensitive target for BPA. Similar results were obtained with the natural estrogen 17β-estradiol. The

  14. AmphiPax3/7, an amphioxus paired box gene: insights into chordate myogenesis, neurogenesis, and the possible evolutionary precursor of definitive vertebrate neural crest.

    PubMed

    Holland, L Z; Schubert, M; Kozmik, Z; Holland, N D

    1999-01-01

    Amphioxus probably has only a single gene (AmphiPax3/7) in the Pax3/7 subfamily. Like its vertebrate homologs (Pax3 and Pax7), amphioxus AmphiPax3/7 is probably involved in specifying the axial musculature and muscularized notochord. During nervous system development, AmphiPax3/7 is first expressed in bilateral anteroposterior stripes along the edges of the neural plate. This early neural expression may be comparable to the transcription of Pax3 and Pax7 in some of the anterior neural crest cells of vertebrates. Previous studies by others and ourselves have demonstrated that several genes homologous to genetic markers for vertebrate neural crest are expressed along the neural plate-epidermis boundary in embryos of tunicates and amphioxus. Taken together, the early neural expression patterns of AmphiPax3/7 and other neural crest markers of amphioxus and tunicates suggest that cell populations that eventually gave rise to definitive vertebrate neural crest may have been present in ancestral invertebrate chordates. During later neurogenesis in amphioxus, AmphiPax3/7, like its vertebrate homologs, is expressed dorsally and dorsolaterally in the neural tube and may be involved in dorsoventral patterning. However, unlike its vertebrate homologs, AmphiPax3/7 is expressed only at the anterior end of the central nervous system instead of along much of the neuraxis; this amphioxus pattern may represent the loss of a primitive chordate character.

  15. Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

    PubMed Central

    Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

    2015-01-01

    The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene. PMID:25706953

  16. Early Evolution of Vertebrate Mybs: An Integrative Perspective Combining Synteny, Phylogenetic, and Gene Expression Analyses

    PubMed Central

    Campanini, Emeline B.; Vandewege, Michael W.; Pillai, Nisha E.; Tay, Boon-Hui; Jones, Justin L.; Venkatesh, Byrappa; Hoffmann, Federico G.

    2015-01-01

    Abstract The genes in the Myb superfamily encode for three related transcription factors in most vertebrates, A-, B-, and c-Myb, with functionally distinct roles, whereas most invertebrates have a single Myb. B-Myb plays an essential role in cell division and cell cycle progression, c-Myb is involved in hematopoiesis, and A-Myb is involved in spermatogenesis and regulating expression of pachytene PIWI interacting RNAs, a class of small RNAs involved in posttranscriptional gene regulation and the maintenance of reproductive tissues. Comparisons between teleost fish and tetrapods suggest that the emergence and functional divergence of the Myb genes were linked to the two rounds of whole-genome duplication early in vertebrate evolution. We combined phylogenetic, synteny, structural, and gene expression analyses of the Myb paralogs from elephant shark and lampreys with data from 12 bony vertebrates to reconstruct the early evolution of vertebrate Mybs. Phylogenetic and synteny analyses suggest that the elephant shark and Japanese lamprey have copies of the A-, B-, and c-Myb genes, implying their origin could be traced back to the common ancestor of lampreys and gnathostomes. However, structural and gene expression analyses suggest that their functional roles diverged between gnathostomes and cyclostomes. In particular, we did not detect A-Myb expression in testis suggesting that the involvement of A-Myb in the pachytene PIWI interacting RNA pathway is probably a gnathostome-specific innovation. We speculate that the secondary loss of a central domain in lamprey A-Myb underlies the functional differences between the cyclostome and gnathostome A-Myb proteins. PMID:26475318

  17. Computerized detection of vertebral compression fractures on lateral chest radiographs: Preliminary results with a tool for early detection of osteoporosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kasai, Satoshi; Li Feng; Shiraishi, Junji

    Vertebral fracture (or vertebral deformity) is a very common outcome of osteoporosis, which is one of the major public health concerns in the world. Early detection of vertebral fractures is important because timely pharmacologic intervention can reduce the risk of subsequent additional fractures. Chest radiographs are used routinely for detection of lung and heart diseases, and vertebral fractures can be visible on lateral chest radiographs. However, investigators noted that about 50% of vertebral fractures visible on lateral chest radiographs were underdiagnosed or under-reported, even when the fractures were severe. Therefore, our goal was to develop a computerized method for detectionmore » of vertebral fractures on lateral chest radiographs in order to assist radiologists' image interpretation and thus allow the early diagnosis of osteoporosis. The cases used in this study were 20 patients with severe vertebral fractures and 118 patients without fractures, as confirmed by the consensus of two radiologists. Radiologists identified the locations of fractured vertebrae, and they provided morphometric data on the vertebral shape for evaluation of the accuracy of detecting vertebral end plates by computer. In our computerized method, a curved search area, which included a number of vertebral end plates, was first extracted automatically, and was straightened so that vertebral end plates became oriented horizontally. Edge candidates were enhanced by use of a horizontal line-enhancement filter in the straightened image, and a multiple thresholding technique, followed by feature analysis, was used for identification of the vertebral end plates. The height of each vertebra was determined from locations of identified vertebral end plates, and fractured vertebrae were detected by comparison of the measured vertebral height with the expected height. The sensitivity of our computerized method for detection of fracture cases was 95% (19/20), with 1.03 (139/135) false

  18. The Late Devonian Gogo Formation Lägerstatte of Western Australia: Exceptional Early Vertebrate Preservation and Diversity

    NASA Astrophysics Data System (ADS)

    Long, John A.; Trinajstic, Kate

    2010-05-01

    The Gogo Formation of Western Australia preserves a unique Late Devonian (Frasnian) reef fauna. The exceptional three-dimensional preservation of macrofossils combined with unprecedented soft-tissue preservation (including muscle bundles, nerve cells, and umbilical structures) has yielded a particularly rich assemblage with almost 50 species of fishes described. The most significant discoveries have contributed to resolving placoderm phylogeny and elucidating their reproductive physiology. Specifically, these discoveries have produced data on the oldest known vertebrate embryos; the anatomy of the primitive actinopterygian neurocranium and phylogeny of the earliest actinopterygians; the histology, radiation, and plasticity of dipnoan (lungfish) dental and cranial structures; the anatomy and functional morphology of the extinct onychodonts; and the anatomy of the primitive tetrapodomorph head and pectoral fin.

  19. The First Human Cloned Embryo.

    ERIC Educational Resources Information Center

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  20. Notch and Delta mRNAs in early-stage and mid-stage Drosophila embryos exhibit complementary patterns of protein producing potentials

    PubMed Central

    Shepherd, Andrew; Wesley, Uma; Wesley, Cedric

    2010-01-01

    Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3′ processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early-stage and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3′ UTR and mRNA 3′ processing factors. PMID:20201103

  1. Early embryo development in Fucus distichus is auxin sensitive

    NASA Technical Reports Server (NTRS)

    Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

    2002-01-01

    Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.

  2. Redundant roles of Sox17 and Sox18 in early cardiovascular development of mouse embryos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sakamoto, Youhei; Hara, Kenshiro; Kanai-Azuma, Masami

    Sox7, -17 and -18 constitute the Sox subgroup F (SoxF) of HMG box transcription factor genes, which all are co-expressed in developing vascular endothelial cells in mice. Here we characterized cardiovascular phenotypes of Sox17/Sox18-double and Sox17-single null embryos during early-somite stages. Whole-mount PECAM staining demonstrated the aberrant heart looping, enlarged cardinal vein and mild defects in anterior dorsal aorta formation in Sox17 single-null embryos. The Sox17/Sox18 double-null embryos showed more severe defects in formation of anterior dorsal aorta and head/cervical microvasculature, and in some cases, aberrant differentiation of endocardial cells and defective fusion of the endocardial tube. However, the posteriormore » dorsal aorta and allantoic microvasculature was properly formed in all of the Sox17/Sox18 double-null embryos. The anomalies in both anterior dorsal aorta and head/cervical vasculature corresponded with the weak Sox7 expression sites. This suggests the region-specific redundant activities of three SoxF members along the anteroposterior axis of embryonic vascular network.« less

  3. The behavior of larval zebrafish reveals stressor-mediated anorexia during early vertebrate development.

    PubMed

    De Marco, Rodrigo J; Groneberg, Antonia H; Yeh, Chen-Min; Treviño, Mario; Ryu, Soojin

    2014-01-01

    The relationship between stress and food consumption has been well documented in adults but less so in developing vertebrates. Here we demonstrate that an encounter with a stressor can suppress food consumption in larval zebrafish. Furthermore, we provide indication that food intake suppression cannot be accounted for by changes in locomotion, oxygen consumption and visual responses, as they remain unaffected after exposure to a potent stressor. We also show that feeding reoccurs when basal levels of cortisol (stress hormone in humans and teleosts) are re-established. The results present evidence that the onset of stress can switch off the drive for feeding very early in vertebrate development, and add a novel endpoint for analyses of metabolic and behavioral disorders in an organism suitable for high-throughput genetics and non-invasive brain imaging.

  4. The behavior of larval zebrafish reveals stressor-mediated anorexia during early vertebrate development

    PubMed Central

    De Marco, Rodrigo J.; Groneberg, Antonia H.; Yeh, Chen-Min; Treviño, Mario; Ryu, Soojin

    2014-01-01

    The relationship between stress and food consumption has been well documented in adults but less so in developing vertebrates. Here we demonstrate that an encounter with a stressor can suppress food consumption in larval zebrafish. Furthermore, we provide indication that food intake suppression cannot be accounted for by changes in locomotion, oxygen consumption and visual responses, as they remain unaffected after exposure to a potent stressor. We also show that feeding reoccurs when basal levels of cortisol (stress hormone in humans and teleosts) are re-established. The results present evidence that the onset of stress can switch off the drive for feeding very early in vertebrate development, and add a novel endpoint for analyses of metabolic and behavioral disorders in an organism suitable for high-throughput genetics and non-invasive brain imaging. PMID:25368561

  5. Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos.

    PubMed

    Rothschild, Sarah C; Francescatto, Ludmila; Tombes, Robert M

    2016-02-19

    The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr. Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol. This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

  6. Light adaptation and the evolution of vertebrate photoreceptors.

    PubMed

    Morshedian, Ala; Fain, Gordon L

    2017-07-15

    Lamprey are cyclostomes, a group of vertebrates that diverged from lines leading to jawed vertebrates (including mammals) in the late Cambrian, 500 million years ago. It may therefore be possible to infer properties of photoreceptors in early vertebrate progenitors by comparing lamprey to other vertebrates. We show that lamprey rods and cones respond to light much like rods and cones in amphibians and mammals. They operate over a similar range of light intensities and adapt to backgrounds and bleaches nearly identically. These correspondences are pervasive and detailed; they argue for the presence of rods and cones very early in the evolution of vertebrates with properties much like those of rods and cones in existing vertebrate species. The earliest vertebrates were agnathans - fish-like organisms without jaws, which first appeared near the end of the Cambrian radiation. One group of agnathans became cyclostomes, which include lamprey and hagfish. Other agnathans gave rise to jawed vertebrates or gnathostomes, the group including all other existing vertebrate species. Because cyclostomes diverged from other vertebrates 500 million years ago, it may be possible to infer some of the properties of the retina of early vertebrate progenitors by comparing lamprey to other vertebrates. We have previously shown that rods and cones in lamprey respond to light much like photoreceptors in other vertebrates and have a similar sensitivity. We now show that these affinities are even closer. Both rods and cones adapt to background light and to bleaches in a manner almost identical to other vertebrate photoreceptors. The operating range in darkness is nearly the same in lamprey and in amphibian or mammalian rods and cones; moreover background light shifts response-intensity curves downward and to the right over a similar range of ambient intensities. Rods show increment saturation at about the same intensity as mammalian rods, and cones never saturate. Bleaches decrease

  7. The lamprey: a jawless vertebrate model system for examining origin of the neural crest and other vertebrate traits.

    PubMed

    Green, Stephen A; Bronner, Marianne E

    2014-01-01

    Lampreys are a group of jawless fishes that serve as an important point of comparison for studies of vertebrate evolution. Lampreys and hagfishes are agnathan fishes, the cyclostomes, which sit at a crucial phylogenetic position as the only living sister group of the jawed vertebrates. Comparisons between cyclostomes and jawed vertebrates can help identify shared derived (i.e. synapomorphic) traits that might have been inherited from ancestral early vertebrates, if unlikely to have arisen convergently by chance. One example of a uniquely vertebrate trait is the neural crest, an embryonic tissue that produces many cell types crucial to vertebrate features, such as the craniofacial skeleton, pigmentation of the skin, and much of the peripheral nervous system (Gans and Northcutt, 1983). Invertebrate chordates arguably lack unambiguous neural crest homologs, yet have cells with some similarities, making comparisons with lampreys and jawed vertebrates essential for inferring characteristics of development in early vertebrates, and how they may have evolved from nonvertebrate chordates. Here we review recent research on cyclostome neural crest development, including research on lamprey gene regulatory networks and differentiated neural crest fates. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  8. Metamerism in cephalochordates and the problem of the vertebrate head.

    PubMed

    Onai, Takayuki; Adachi, Noritaka; Kuratani, Shigeru

    2017-01-01

    The vertebrate head characteristically exhibits a complex pattern with sense organs, brain, paired eyes and jaw muscles, and the brain case is not found in other chordates. How the extant vertebrate head has evolved remains enigmatic. Historically, there have been two conflicting views on the origin of the vertebrate head, segmental and non-segmental views. According to the segmentalists, the vertebrate head is organized as a metameric structure composed of segments equivalent to those in the trunk; a metamere in the vertebrate head was assumed to consist of a somite, a branchial arch and a set of cranial nerves, considering that the head evolved from rostral segments of amphioxus-like ancestral vertebrates. Non-segmentalists, however, considered that the vertebrate head was not segmental. In that case, the ancestral state of the vertebrate head may be non-segmented, and rostral segments in amphioxus might have been secondarily gained, or extant vertebrates might have evolved through radical modifications of amphioxus-like ancestral vertebrate head. Comparative studies of mesodermal development in amphioxus and vertebrate gastrula embryos have revealed that mesodermal gene expressions become segregated into two domains anteroposteriorly to specify the head mesoderm and trunk mesoderm only in vertebrates; in this segregation, key genes such as delta and hairy, involved in segment formation, are expressed in the trunk mesoderm, but not in the head mesoderm, strongly suggesting that the head mesoderm of extant vertebrates is not segmented. Taken together, the above finding possibly adds a new insight into the origin of the vertebrate head; the vertebrate head mesoderm would have evolved through an anteroposterior polarization of the paraxial mesoderm if the ancestral vertebrate had been amphioxus-like.

  9. Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

    PubMed

    Chen, Linan; Dumelie, Jason G; Li, Xiao; Cheng, Matthew Hk; Yang, Zhiyong; Laver, John D; Siddiqui, Najeeb U; Westwood, J Timothy; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

    2014-01-07

    Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

  10. Characterization of the altered gene expression profile in early porcine embryos generated from parthenogenesis and somatic cell chromatin transfer.

    PubMed

    Zhou, Chi; Dobrinsky, John; Tsoi, Stephen; Foxcroft, George R; Dixon, Walter T; Stothard, Paul; Verstegen, John; Dyck, Michael K

    2014-01-01

    The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo-derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The "eIF2 signalling", "mitochondrial dysfunction", "regulation of eIF4 and p70S6K signalling", "protein ubiquitination", and "mTOR signalling" pathways were down-regulated in PA HB. Dysregulation of notch signalling-associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.

  11. Coordination of cellular differentiation, polarity, mitosis and meiosis - New findings from early vertebrate oogenesis.

    PubMed

    Elkouby, Yaniv M; Mullins, Mary C

    2017-10-15

    A mechanistic dissection of early oocyte differentiation in vertebrates is key to advancing our knowledge of germline development, reproductive biology, the regulation of meiosis, and all of their associated disorders. Recent advances in the field include breakthroughs in the identification of germline stem cells in Medaka, in the cellular architecture of the germline cyst in mice, in a mechanistic dissection of chromosomal pairing and bouquet formation in meiosis in mice, in tracing oocyte symmetry breaking to the chromosomal bouquet of meiosis in zebrafish, and in the biology of the Balbiani body, a universal oocyte granule. Many of the major events in early oogenesis are universally conserved, and some are co-opted for species-specific needs. The chromosomal events of meiosis are of tremendous consequence to gamete formation and have been extensively studied. New light is now being shed on other aspects of early oocyte differentiation, which were traditionally considered outside the scope of meiosis, and their coordination with meiotic events. The emerging theme is of meiosis as a common groundwork for coordinating multifaceted processes of oocyte differentiation. In an accompanying manuscript we describe methods that allowed for investigations in the zebrafish ovary to contribute to these breakthroughs. Here, we review these advances mostly from the zebrafish and mouse. We discuss oogenesis concepts across established model organisms, and construct an inclusive paradigm for early oocyte differentiation in vertebrates. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

    PubMed

    Wang, Chao-Jie; Wang, Guang; Wang, Xiao-Yu; Liu, Meng; Chuai, Manli; Lee, Kenneth Ka Ho; He, Xiao-Song; Lu, Da-Xiang; Yang, Xuesong

    2016-06-15

    Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

  13. γ-BUTYROBETAINE AS A SPECIFIC ANTAGONIST FOR CARNITINE IN THE DEVELOPMENT OF THE EARLY CHICK EMBRYO

    PubMed Central

    Ito, Toshio; Fraenkel, G.

    1957-01-01

    The effect of γ-butyrobetaine alone and with the addition of carnitine on the development of the early excised chick embryo has been studied. γ-Butyrobetaine in appropriate amounts exerts an inhibitory effect which can be relieved or annulled by the inclusion of appropriate amounts of carnitine. This has been interpreted as a metabolite-antimetabolite relationship, in which the normal metabolite, carnitine, is antagonized by the structurally closely related γ-butyrobetaine, and is regarded as evidence of an important role of carnitine in the metabolism of the developing chick embryo. PMID:13475691

  14. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    PubMed Central

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  15. A Zebrafish Embryo Culture System Defines Factors that Promote Vertebrate Myogenesis across Species

    PubMed Central

    Ciarlo, Christie; Liu, Jingxia; Castiglioni, Alessandra; Price, Emily; Liu, Min; Barton, Elisabeth R.; Kahn, C. Ronald; Wagers, Amy J.; Zon, Leonard I.

    2013-01-01

    SUMMARY Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle. PMID:24209627

  16. Refrigeration of rainbow trout gametes and embryos.

    PubMed

    Babiak, Igor; Dabrowski, Konrad

    2003-12-01

    Prolonged access to early embryos composed of undifferentiated, totipotent blastomeres is desirable in situations when multiple collections of gametes are not possible. The objective of the present study is to examine whether the refrigeration of rainbow trout Oncorhynchus mykiss gametes and early embryos would be a suitable, reliable, and efficient tool for prolonging the availability of early developmental stages up to the advanced blastula stage. The study was conducted continuously during fall, winter, and spring spawning seasons. In all, more than 500 experimental variants were performed involving individual samples from 26 females and 33 males derived from three strains. These strains represented three possible circumstances. In optimal one, gametes from good quality donors were obtained soon after ovulation. In the two non-optimal sources, either donors were of poor genetic quality or gametes were collected from a distant location and transported as unfertilized gametes. A highly significant effect of variability of individual sample quality on efficiency of gamete and embryo refrigeration was revealed. The source of gametes significantly affected viability of refrigerated oocytes and embryos, but not spermatozoa. On average, oocytes from optimal source retained full fertilization viability for seven days of chilled storage, significantly longer than from non-optimal sources. Spermatozoa, regardless of storage method, retained full fertilization ability for the first week of storage. Refrigeration of embryos at 1.4+/-0.4 degrees C significantly slowed the development. Two- week-old embryos were still in blastula stage. Average survival rate of embryos refrigerated for 10 days and then transferred to regular incubation temperatures of 9-14 degrees C was 92% in optimal and 51 and 71% in non-optimal source variants. No effect of gamete and embryo refrigeration on the occurrence of developmental abnormalities was observed. Cumulative refrigeration of oocytes and

  17. Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

    PubMed

    Shiokawa, K; Kai, M; Higo, T; Kaito, C; Yokoska, J; Yasuhiko, Y; Kajita, E; Nagano, M; Yamada, Y; Shibata, M; Muto, T; Shinga, J; Hara, H; Takayama, E; Fukamachi, H; Yaoita, Y; Igarashi, K

    2000-06-01

    When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

  18. Pleurodeles waltl, amphibian, Urodele, is a suitable biological model for embryological and physiological space experiments on a vertebrate

    NASA Astrophysics Data System (ADS)

    Gualandris-Parisot, L.; Husson, D.; Foulquier, F.; Kan, P.; Davet, J.; Aimar, C.; Dournon, C.; Duprat, A. M.

    2001-01-01

    Pleurodeles waltl (amphibian, Urodele) is an appropriate biological model for space experiments on a vertebrate. One reason for interest in this animal concerns the study of the effects of absence of gravity on embryonic development. First, after mating (on Earth) the females retain live, functional sperm in their cloacum for up to 5 months, allowing normal in vivo fertilisation after hormonal stimulation. Second, their development is slow, which allows analyses of all the key stages of ontogenesis from the oocyte to swimming tailbud embryos or larvae. We have performed detailed studies and analyses of the effects of weightlessness on amphibian Pleurodeles embryos, fertilised and allowed to develop until the swimming larvae stage. These experiments were performed in space during three missions on the MIR-station: FERTILE I, FERTILE II and NEUROGENESIS respectively in 1996, 1998 and 1999. We show that in microgravity abnormalities appeared at specific stages of development compared to 1g-centrifuge control embryos and 1g-ground control embryos. In this report we describe abnormalities occurring in the central nervous system. These modifications occur during the neurulation process (delay in the closure of the neural tube and failure of closure of this tube in the cephalic area) and at the early tailbud stage (microcephaly observed in 40% of the microgravity-embryos). However, if acephalic and microcephalic embryos are not taken into account, these abnormalities did not disturb further morphological, biochemical and functional development and the embryos were able to regulate and a majority of normal hatching and swimming larvae were obtained in weightlessness with a developmental time-course equivalent to that of 1g-centrifuge control embryos (on the MIR station) and 1g-ground control embryos.

  19. Convergent evolution of hemoglobin switching in jawed and jawless vertebrates.

    PubMed

    Rohlfing, Kim; Stuhlmann, Friederike; Docker, Margaret F; Burmester, Thorsten

    2016-02-01

    During development, humans and other jawed vertebrates (Gnathostomata) express distinct hemoglobin genes, resulting in different hemoglobin tetramers. Embryonic and fetal hemoglobin have higher oxygen affinities than the adult hemoglobin, sustaining the oxygen demand of the developing organism. Little is known about the expression of hemoglobins during development of jawless vertebrates (Agnatha). We identified three hemoglobin switches in the life cycle of the sea lamprey. Three hemoglobin genes are specifically expressed in the embryo, four genes in the filter feeding larva (ammocoete), and nine genes correspond to the adult hemoglobin chains. During the development from the parasitic to the reproductive adult, the composition of hemoglobin changes again, with a massive increase of chain aHb1. A single hemoglobin chain is expressed constitutively in all stages. We further showed the differential expression of other globin genes: Myoglobin 1 is most highly expressed in the reproductive adult, myoglobin 2 expression peaks in the larva. Globin X1 is restricted to the embryo; globin X2 was only found in the reproductive adult. Cytoglobin is expressed at low levels throughout the life cycle. Because the hemoglobins of jawed and jawless vertebrates evolved independently from a common globin ancestor, hemoglobin switching must also have evolved convergently in these taxa. Notably, the ontogeny of sea lamprey hemoglobins essentially recapitulates their phylogeny, with the embryonic hemoglobins emerging first, followed by the evolution of larval and adult hemoglobins.

  20. Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in early Xenopus embryos induces cell dissociation and inhibits transition from the blastula to gastrula stage.

    PubMed

    Shibata, M; Shinga, J; Yasuhiko, Y; Kai, M; Miura, K; Shimogori, T; Kashiwagi, K; Igarashi, K; Shiokawa, K

    1998-07-01

    Xenopus early embryos contain relatively low levels of S-adenosyl-methionine decarboxylase (SAMDC) and its mRNA. When SAMDC mRNA was injected into Xenopus embryos, it was preserved until the blastula stage and induced a large increase in SAMDC activity. The SAMDC-overexpressed embryos developed normally until the blastula stage but at the early gastrula stage cells which received the mRNA, dissociated autonomously and stopped synthesizing protein. In a hypotonic medium, the dissociated cells, and hence whole embryos, autolyzed. However, in isotonic media dissociated cells did not autolyze, although they did not divide and their DNA and RNA synthesis activity was greatly inhibited. The effects of SAMDC overexpression were abolished by coinjection of ethylglyoxal-bis(guanylhydrazone) (EGBG), a specific inhibitor of SAMDC. In SAMDC-overexpressed embryos the level of putrescine decreased and that of spermidine increased, though to limited extents, resulting in a considerable decrease in the putrescine/spermidine ratio. However, direct injection of spermidine did not mimic the effect of SAMDC overexpression, and putrescine coinjected with SAMDC mRNA to maintain the normal putrescine/spermidine ratio did not rescue the embryos. Conversely, the level of S-adenosylmethionine (SAM) greatly decreased and coinjection of SAM, which restored the level of SAM, rescued the embryos. We concluded that in SAMDC-overexpressed embryos a SAM-deficient state was induced and this caused cell dissociation and inhibition of transition from the blastula to gastrula stage. We suggest that the SAM-deficient embryos obtained in the present study provide a unique system for studying the cellular control mechanism underlying the blastula-gastrula transition.

  1. The effect of repeated light-dark shifts on uterine receptivity and early gestation in mice undergoing embryo transfer.

    PubMed

    Goldstein, Cathy A; O'Brien, Louise M; Bergin, Ingrid L; Saunders, Thomas L

    2018-04-01

    Female shift workers are at increased risk for negative reproductive outcomes, and animal evidence suggests that manipulation of the light-dark cycle is detrimental to early gestation in female mice. Specifically, failure of implantation may be responsible for these findings. The objective of this study was to better delineate which reproductive processes are vulnerable to detrimental effects of maternal circadian disturbance. We exposed mice undergoing embryo transfer to repetitive phase advances of the photoperiod. Embryos were derived from donor sperm and eggs from mice living in normal light-dark conditions to isolate the effects of photoperiod disruption on uterine receptivity and early gestation. Twenty-eight mice receiving embryo transfer underwent an experimental light-dark condition (advance of lights on and lights off by 6 hours every 4 days). Twenty-eight mice remained in a normal light-dark condition. Animals lived in their assigned light-dark condition beginning 2 weeks prior to embryo transfer and ending the day of uterine necropsy (post-coitus day 14.5). Wilcoxon-Mann-Whitney test demonstrated no significant differences between control and experimental light-dark conditions in pups (Z=0.10, p=.92), resorptions (Z=0.20, p=.84), or implantations (Z=-0.34, p=.73). Pup and placental weights were similar between groups. In this investigation, uterine receptivity and maintenance of early gestation were preserved despite recurrent phase advances in photoperiod. This finding, in the context of the current literature, suggests that the negative effects of circadian disruption are mediated by reproductive processes upstream of implantation.

  2. Bayesian Morphological Clock Methods Resurrect Placoderm Monophyly and Reveal Rapid Early Evolution in Jawed Vertebrates.

    PubMed

    King, Benedict; Qiao, Tuo; Lee, Michael S Y; Zhu, Min; Long, John A

    2017-07-01

    The phylogeny of early gnathostomes provides an important framework for understanding one of the most significant evolutionary events, the origin and diversification of jawed vertebrates. A series of recent cladistic analyses have suggested that the placoderms, an extinct group of armoured fish, form a paraphyletic group basal to all other jawed vertebrates. We revised and expanded this morphological data set, most notably by sampling autapomorphies in a similar way to parsimony-informative traits, thus ensuring this data (unlike most existing morphological data sets) satisfied an important assumption of Bayesian tip-dated morphological clock approaches. We also found problems with characters supporting placoderm paraphyly, including character correlation and incorrect codings. Analysis of this data set reveals that paraphyly and monophyly of core placoderms (excluding maxillate forms) are essentially equally parsimonious. The two alternative topologies have different root positions for the jawed vertebrates but are otherwise similar. However, analysis using tip-dated clock methods reveals strong support for placoderm monophyly, due to this analysis favoring trees with more balanced rates of evolution. Furthermore, enforcing placoderm paraphyly results in higher levels and unusual patterns of rate heterogeneity among branches, similar to that generated from simulated trees reconstructed with incorrect root positions. These simulations also show that Bayesian tip-dated clock methods outperform parsimony when the outgroup is largely uninformative (e.g., due to inapplicable characters), as might be the case here. The analysis also reveals that gnathostomes underwent a rapid burst of evolution during the Silurian period which declined during the Early Devonian. This rapid evolution during a period with few articulated fossils might partly explain the difficulty in ascertaining the root position of jawed vertebrates. © The Author(s) 2016. Published by Oxford University

  3. Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

    PubMed Central

    Timme-Laragy, Alicia R.; Goldstone, Jared V.; Imhoff, Barry R.; Stegeman, John J.; Hahn, Mark E.; Hansen, Jason M.

    2013-01-01

    Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous non-protein antioxidant defense molecule is the tri-peptide glutathione (γ-glutamyl-cysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days post-fertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione (GSH, GSSG) using HPLC, and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0–120 hours of zebrafish development (including mature oocytes, fertilization, mid-blastula transition, gastrulation, somitogenesis, pharyngula, pre-hatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12 hours post fertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12 h, and then oscillated around −190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (−220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study

  4. Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein

    PubMed Central

    2014-01-01

    Background Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. Results To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Conclusions Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism. PMID:24393533

  5. [Effect of human oviductal embryotrophic factors on gene expression of mouse preimplantation embryos].

    PubMed

    Yao, Yuan-Qing; Lee, Kai-Fai; Xu, Jia-Seng; Ho, Pak-Chung; Yeung, Shu-Biu

    2007-09-01

    To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.

  6. Expression and evolution of Tiki1 and Tiki2 genes in vertebrates

    PubMed Central

    FEISTEL, KERSTIN; BRITO, JOSE M.; AMADO, NATHALIA G.; XU, CHIWEI; ABREU, JOSE G.; HE, XI

    2015-01-01

    Tiki1 is a Wnt protease and antagonist specifically expressed in the Spemann-Mangold Organizer and is required for head formation in Xenopus embryos. Here we report neighbor-joining phylogenetic analysis of vertebrate Tiki genes and their mRNA expression patterns in chick, mouse, and rabbit embryos. Tiki1 and Tiki2 orthologues are highly conserved, and exhibit similar but also different developmental expression patterns among the vertebrate/mammalian species analyzed. The Tiki1 gene is noticeably absent in the rodent lineage, but is present in lagomorphs and all other vertebrate/mammalian species examined. Expression in Hensen’s node, the equivalent of the Xenopus Organizer, was observed for Chick Tiki2 and Rabbit Tiki1 and Tiki2. Mouse Tiki2 was detected at low levels at gastrulation and head fold stages, but not in the node. Mouse Tiki2 and chick Tiki1 display similar expression in the dorsal spinal cord. Chick Tiki1 expression was also detected in the surface ectoderm and maxillary bud, while chick Tiki2 was found in the anterior intestinal portal, head mensenchyme and primitive atrium. Our expression analyses provide evidence that Tiki1 and Tiki2 are evolutionary conserved among vertebrate species and their expression in the Organizer and other regions suggests contributions of these Wnt inhibitors to embryonic patterning as well as organogenesis. Our analyses further reveal mis-regulation of TIKI1 and TIKI2 in human cancer and diseases. PMID:25354456

  7. Nucleolus Precursor Bodies and Ribosome Biogenesis in Early Mammalian Embryos: Old Theories and New Discoveries.

    PubMed

    Fulka, Helena; Aoki, Fugaku

    2016-06-01

    In mammals, mature oocytes and early preimplantation embryos contain transcriptionally inactive structures termed nucleolus precursor bodies instead of the typical fibrillo-granular nucleoli. These nuclear organelles are essential and strictly of maternal origin. If they are removed from oocytes, the resulting embryos are unable to replace them and consequently fail to develop. Historically, nucleolus precursor bodies have been perceived as a passive repository site of nucleolar proteins that are required for embryos to form fully functional nucleoli. Recent results, however, contradict this long-standing dogma and show that these organelles are dispensable for nucleologenesis and ribosome biogenesis. In this article, we discuss the possible roles of nucleolus precursor bodies and propose how they might be involved in embryogenesis. Furthermore, we argue that these organelles are essential only shortly after fertilization and suggest that they might actively participate in centromeric chromatin establishment. © 2016 by the Society for the Study of Reproduction, Inc.

  8. Ecomorphological inferences in early vertebrates: reconstructing Dunkleosteus terrelli (Arthrodira, Placodermi) caudal fin from palaeoecological data.

    PubMed

    Ferrón, Humberto G; Martínez-Pérez, Carlos; Botella, Héctor

    2017-01-01

    Our knowledge about the body morphology of many extinct early vertebrates is very limited, especially in regard to their post-thoracic region. The prompt disarticulation of the dermo-skeletal elements due to taphonomic processes and the lack of a well-ossified endoskeleton in a large number of groups hinder the preservation of complete specimens. Previous reconstructions of most early vertebrates known from partial remains have been wholly based on phylogenetically closely related taxa. However, body design of fishes is determined, to a large extent, by their swimming mode and feeding niche, making it possible to recognise different morphological traits that have evolved several times in non-closely related groups with similar lifestyles. Based on this well-known ecomorphological correlation, here we propose a useful comparative framework established on extant taxa for predicting some anatomical aspects in extinct aquatic vertebrates from palaeoecological data and vice versa. For this, we have assessed the relationship between the locomotory patterns and the morphological variability of the caudal region in extant sharks by means of geometric morphometrics and allometric regression analysis. Multivariate analyses reveal a strong morphological convergence in non-closely related shark species that share similar modes of life, enabling the characterization of the caudal fin morphology of different ecological subgroups. In addition, interspecific positive allometry, affecting mainly the caudal fin span, has been detected. This phenomenon seems to be stronger in sharks with more pelagic habits, supporting its role as a compensation mechanism for the loss of hydrodynamic lift associated with the increase in body size, as previously suggested for many other living and extinct aquatic vertebrates. The quantification of shape change per unit size in each ecological subgroup has allowed us to establish a basis for inferring not only qualitative aspects of the caudal fin

  9. Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice.

    PubMed

    Wu, Yibo; Lv, Zhuo; Yang, Yang; Dong, Guoying; Yu, Yang; Cui, Yiqiang; Tong, Man; Wang, Liu; Zhou, Zuomin; Zhu, Hui; Zhou, Qi; Sha, Jiahao

    2014-05-01

    Blastomere biopsy is used in preimplantation genetic diagnosis; however, the long-term implications on the offspring are poorly characterized. We previously reported a high risk of memory defects in adult biopsied mice. Here, we assessed nervous function of aged biopsied mice and further investigated the mechanism of neural impairment after biopsy. We found that aged biopsied mice had poorer spatial learning ability, increased neuron degeneration, and altered expression of proteins involved in neural degeneration or dysfunction in the brain compared to aged control mice. Furthermore, the MeDIP assay indicated a genome-wide low methylation in the brains of adult biopsied mice when compared to the controls, and most of the genes containing differentially methylated loci in promoter regions were associated with neural disorders. When we further compared the genomic DNA methylation profiles of 7.5-days postconception (dpc) embryos between the biopsy and control group, we found the whole genome low methylation in the biopsied group, suggesting that blastomere biopsy was an obstacle to de novo methylation during early embryo development. Further analysis on mRNA profiles of 4.5-dpc embryos indicated that reduced expression of de novo methylation genes in biopsied embryos may impact de novo methylation. In conclusion, we demonstrate an abnormal neural development and function in mice generated after blastomere biopsy. The impaired epigenetic reprogramming during early embryo development may be the latent mechanism contributing to the impairment of the nervous system in the biopsied mice, which results in a hypomethylation status in their brains.

  10. Early Embryo Development in Fucus distichus Is Auxin Sensitive1

    PubMed Central

    Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

    2002-01-01

    Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [3H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development. PMID:12226509

  11. Proteomic analysis of zebrafish embryos exposed to simulated-microgravity

    NASA Astrophysics Data System (ADS)

    Hang, Xiaoming; Ma, Wenwen; Wang, Wei; Liu, Cong; Sun, Yeqing

    Microgravity can induce a serial of physiological and pathological changes in human body, such as cardiovascular functional disorder, bone loss, muscular atrophy and impaired immune system function, etc. In this research, we focus on the influence of microgravity to vertebrate embryo development. As a powerful model for studying vertebrate development, zebrafish embryos at 8 hpf (hour past fertilization) and 24 hpf were placed into a NASA developed bioreac-tor (RCCS) to simulate microgravity for 64 and 48 hours, respectively. The same number of control embryos from the same parents were placed in a tissue culture dish at the same temper-ature of 28° C. Each experiment was repeated 3 times and analyzed by two-dimensional (2-D) gel electrophoresis. Image analysis of silver stained 2-D gels revealed that 64 from total 292 protein spots showed quantitative and qualitative variations that were significantly (P<0.05) and reproducibly different between simulate-microgravity treatment and the stationary control samples. 4 protein spots with significant expression alteration (P<0.01) were excised from 2-D gels and analyzed by MALDI-TOF/TOF mass spectra primarily. Of these proteins, 3 down-regulated proteins were identified as bectin 2, centrosomal protein of 135kDa and tropomyosin 4, while the up-regulated protein was identified as creatine kinase muscle B. Other protein spots showed significant expression alteration will be identified successively and the corresponding genes expression will also be measured by Q-PCR method at different development stages. The data presented in this study illustrate that zebrafish embryo can be significantly induced by microgravity on the expression of proteins involved in bone and muscle formation. Key Words: Danio rerio; Simulated-microgravity; Proteomics

  12. Cytoskeletal changes in oocytes and early embryos during in vitro fertilization process in mice.

    PubMed

    Gumus, E; Bulut, H E; Kaloglu, C

    2010-02-01

    The cytoskeleton plays crucial roles in the development and fertilization of germ cells and in the early embryo development. The growth, maturation and fertilization of oocytes require an active movement and a correct localization of cellular organelles. This is performed by the re-organization of microtubules and actin filaments. Therefore, the aim of the present study was to determine the changes in cytoskeleton during in vitro fertilization process using appropriate immunofluorescence techniques. While the chromatin content was found to be scattered throughout the nucleus during the oocyte maturation period, it was seen only around nucleolus following the completion of the maturation. Microtubules, during oocyte maturation, were regularly distributed throughout the ooplasm which was then localized in the subcortical region of oocytes. Similarly microfilaments were scattered throughout the ooplasm during the oocyte maturation period whereas they were seen in the subcortical region around the polar body and above the meiotic spindle throughout the late developmental stages. In conclusion, those changes occurred in microtubules and microfilaments might be closely related to the re-organization of the genetic material during the oocyte maturation and early embryo development.

  13. The vertebrate ancestral repertoire of visual opsins, transducin alpha subunits and oxytocin/vasopressin receptors was established by duplication of their shared genomic region in the two rounds of early vertebrate genome duplications.

    PubMed

    Lagman, David; Ocampo Daza, Daniel; Widmark, Jenny; Abalo, Xesús M; Sundström, Görel; Larhammar, Dan

    2013-11-02

    Vertebrate color vision is dependent on four major color opsin subtypes: RH2 (green opsin), SWS1 (ultraviolet opsin), SWS2 (blue opsin), and LWS (red opsin). Together with the dim-light receptor rhodopsin (RH1), these form the family of vertebrate visual opsins. Vertebrate genomes contain many multi-membered gene families that can largely be explained by the two rounds of whole genome duplication (WGD) in the vertebrate ancestor (2R) followed by a third round in the teleost ancestor (3R). Related chromosome regions resulting from WGD or block duplications are said to form a paralogon. We describe here a paralogon containing the genes for visual opsins, the G-protein alpha subunit families for transducin (GNAT) and adenylyl cyclase inhibition (GNAI), the oxytocin and vasopressin receptors (OT/VP-R), and the L-type voltage-gated calcium channels (CACNA1-L). Sequence-based phylogenies and analyses of conserved synteny show that the above-mentioned gene families, and many neighboring gene families, expanded in the early vertebrate WGDs. This allows us to deduce the following evolutionary scenario: The vertebrate ancestor had a chromosome containing the genes for two visual opsins, one GNAT, one GNAI, two OT/VP-Rs and one CACNA1-L gene. This chromosome was quadrupled in 2R. Subsequent gene losses resulted in a set of five visual opsin genes, three GNAT and GNAI genes, six OT/VP-R genes and four CACNA1-L genes. These regions were duplicated again in 3R resulting in additional teleost genes for some of the families. Major chromosomal rearrangements have taken place in the teleost genomes. By comparison with the corresponding chromosomal regions in the spotted gar, which diverged prior to 3R, we could time these rearrangements to post-3R. We present an extensive analysis of the paralogon housing the visual opsin, GNAT and GNAI, OT/VP-R, and CACNA1-L gene families. The combined data imply that the early vertebrate WGD events contributed to the evolution of vision and the

  14. Development of the embryonic heat shock response and the impact of repeated thermal stress in early stage lake whitefish (Coregonus clupeaformis) embryos.

    PubMed

    Whitehouse, Lindy M; McDougall, Chance S; Stefanovic, Daniel I; Boreham, Douglas R; Somers, Christopher M; Wilson, Joanna Y; Manzon, Richard G

    2017-10-01

    Lake whitefish (Coregonus clupeaformis) embryos were exposed to thermal stress (TS) at different developmental stages to determine when the heat shock response (HSR) can be initiated and if it is altered by exposure to repeated TS. First, embryos were subject to one of three different TS temperatures (6, 9, or 12°C above control) at 4 points in development (21, 38, 60 and 70 days post-fertilisation (dpf)) for 2h followed by a 2h recovery to understand the ontogeny of the HSR. A second experiment explored the effects of repeated TS on the HSR in embryos from 15 to 75 dpf. Embryos were subjected to one of two TS regimes; +6°C TS for 1h every 6 days or +9°C TS for 1h every 6 days. Following a 2h recovery, a subset of embryos was sampled. Our results show that embryos could initiate a HSR via upregulation of heat shock protein 70 (hsp70) mRNA at all developmental ages studied, but that this response varied with age and was only observed with a TS of +9 or +12°C. In comparison, when embryos received multiple TS treatments, hsp70 was not induced in response to the 1h TS and 2h recovery, and a downregulation was observed at 39 dpf. Downregulation of hsp47 and hsp90α mRNA was also observed in early age embryos. Collectively, these data suggest that embryos are capable of initiating a HSR at early age and throughout embryogenesis, but that repeated TS can alter the HSR, and may result in either reduced responsiveness or a downregulation of inducible hsps. Our findings warrant further investigation into both the short- and long-term effects of repeated TS on lake whitefish development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

    DOE PAGES

    Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz; ...

    2017-03-22

    Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

  16. Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz

    Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

  17. Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ju, Yan; Liu, Chunying; Lu, Wenwen

    The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysismore » revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.« less

  18. Vascular development in the vertebrate pancreas

    PubMed Central

    Azizoglu, D. Berfin; Chong, Diana C.; Villasenor, Alethia; Magenheim, Judith; Barry, David M.; Lee, Simon; Marty-Santos, Leilani; Fu, Stephen; Dor, Yuval; Cleaver, Ondine

    2016-01-01

    The vertebrate pancreas is comprised of a highly branched tubular epithelium, which is intimately associated with an extensive and specialized vasculature. While we know a great deal about basic vascular anatomy of the adult pancreas, as well as islet capillaries, surprisingly little is known about the ontogeny of its blood vessels. Here, we analyze development of the pancreatic vasculature in the mouse embryo. We show that pancreatic epithelial branches intercalate with the fine capillary plexus of the surrounding pancreatic mesenchyme. Endothelial cells (ECs) within this mesenchyme are heterogeneous from the onset of organogenesis. Pancreatic arteries take shape before veins, in a manner analogous to early embryonic vessels. The main central artery forms during mid-gestation, as a result of vessel coalescence and remodeling of a vascular plexus. In addition, we show that vessels in the forming pancreas display a predictable architecture that is dependent on VEGF signaling. Over-expression of VEGF disrupts vascular patterning and arteriovenous differentiation within the developing pancreas. This study constitutes a first-time cellular and molecular characterization of pancreatic blood vessels, as they coordinately grow along with the pancreatic epithelium. PMID:27789228

  19. Vascular development in the vertebrate pancreas.

    PubMed

    Azizoglu, D Berfin; Chong, Diana C; Villasenor, Alethia; Magenheim, Judith; Barry, David M; Lee, Simon; Marty-Santos, Leilani; Fu, Stephen; Dor, Yuval; Cleaver, Ondine

    2016-12-01

    The vertebrate pancreas is comprised of a highly branched tubular epithelium, which is intimately associated with an extensive and specialized vasculature. While we know a great deal about basic vascular anatomy of the adult pancreas, as well as islet capillaries, surprisingly little is known about the ontogeny of its blood vessels. Here, we analyze development of the pancreatic vasculature in the mouse embryo. We show that pancreatic epithelial branches intercalate with the fine capillary plexus of the surrounding pancreatic mesenchyme. Endothelial cells (ECs) within this mesenchyme are heterogeneous from the onset of organogenesis. Pancreatic arteries take shape before veins, in a manner analogous to early embryonic vessels. The main central artery forms during mid-gestation, as a result of vessel coalescence and remodeling of a vascular plexus. In addition, we show that vessels in the forming pancreas display a predictable architecture that is dependent on VEGF signaling. Over-expression of VEGF disrupts vascular patterning and arteriovenous differentiation within the developing pancreas. This study constitutes a first-time in-depth cellular and molecular characterization of pancreatic blood vessels, as they coordinately grow along with the pancreatic epithelium. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Environmental estrogens alter early development in Xenopus laevis.

    PubMed

    Bevan, Cassandra L; Porter, Donna M; Prasad, Anita; Howard, Marthe J; Henderson, Leslie P

    2003-04-01

    A growing number of environmental toxicants found in pesticides, herbicides, and industrial solvents are believed to have deleterious effects on development by disrupting hormone-sensitive processes. We exposed Xenopus laevis embryos at early gastrula to the commonly encountered environmental estrogens nonylphenol, octylphenol, and methoxychlor, the antiandrogen, p,p-DDE, or the synthetic androgen, 17 alpha-methyltestosterone at concentrations ranging from 10 nM to 10 microM and examined them at tailbud stages (approximately 48 hr of treatment). Exposure to the three environmental estrogens, as well as to the natural estrogen 17 beta-estradiol, increased mortality, induced morphologic deformations, increased apoptosis, and altered the deposition and differentiation of neural crest-derived melanocytes in tailbud stage embryos. Although neural crest-derived melanocytes were markedly altered in embryos treated with estrogenic toxicants, expression of the early neural crest maker Xslug, a factor that regulates both the induction and subsequent migration of neural crest cells, was not affected, suggesting that the disruption induced by these compounds with respect to melanocyte development may occur at later stages of their differentiation. Co-incubation of embryos with the pure antiestrogen ICI 182,780 blocked the ability of nonylphenol to induce abnormalities in body shape and in melanocyte differentiation but did not block the effects of methoxychlor. Our data indicate not only that acute exposure to these environmental estrogens induces deleterious effects on early vertebrate development but also that different environmental estrogens may alter the fate of a specific cell type via different mechanisms. Finally, our data suggest that the differentiation of neural crest-derived melanocytes may be particularly sensitive to the disruptive actions of these ubiquitous chemical contaminants.

  1. Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.

    PubMed

    Gandhi, Shashank; Piacentino, Michael L; Vieceli, Felipe M; Bronner, Marianne E

    2017-12-01

    The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Quantitative analyses of cell behaviors underlying notochord formation and extension in mouse embryos.

    PubMed

    Sausedo, R A; Schoenwolf, G C

    1994-05-01

    Formation and extension of the notochord (i.e., notogenesis) is one of the earliest and most obvious events of axis development in vertebrate embryos. In birds and mammals, prospective notochord cells arise from Hensen's node and come to lie beneath the midline of the neural plate. Throughout the period of neurulation, the notochord retains its close spatial relationship with the developing neural tube and undergoes rapid extension in concert with the overlying neuroepithelium. In the present study, we examined notochord development quantitatively in mouse embryos. C57BL/6 mouse embryos were collected at 8, 8.5, 9, 9.5, and 10 days of gestation. They were then embedded in paraffin and sectioned transversely. Serial sections from 21 embryos were stained with Schiff's reagent according to the Feulgen-Rossenbeck procedure and used for quantitative analyses of notochord extension. Quantitative analyses revealed that extension of the notochord involves cell division within the notochord proper and cell rearrangement within the notochordal plate (the immediate precursor of the notochord). In addition, extension of the notochord involves cell accretion, that is, the addition of cells to the notochord's caudal end, a process that involves considerable cell rearrangement at the notochordal plate-node interface. Extension of the mouse notochord occurs similarly to that described previously for birds (Sausedo and Schoenwolf, 1993 Anat. Rec. 237:58-70). That is, in both birds (i.e., quail and chick) and mouse embryos, notochord extension involves cell division, cell rearrangement, and cell accretion. Thus higher vertebrates utilize similar morphogenetic movements to effect notogenesis.

  3. Radiation induced abnormalities in early in vitro mouse embryos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kirkpatrick, J.F.

    1973-08-01

    Female mice were superovulated and mated, and the two-cell embryos were collected and cultured in vitro. The embryos were exposed to x-irradiation (0 to 491 rads) during the two-cell stage before the appearance of the next cleavage plate, placed in new unirradiated culture medium and observed during subsequent development. Morphological abnormalities, which occurred as a result of irradiation, included fragmentation, disintegration, granlation, incomplete cleavage, cleavage cessation, nuclear degeneration and pycnosis and cytoplasmic vacuolization. There was no damage to the zona pellucida. The types of abnormalities indicate an agreement with the results of previous in vivo studies. A distinct correlation existedmore » between morphological abnormalities and embryo death. The greatest number of abnormalities resulted within five hours following irradiation, but increased through 20 hours post-exposure. At doses above 300 rads, the magnitude of damage was greater in the in vitro embryos than that shown in previous in vivo studies. (auth)« less

  4. Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos

    NASA Astrophysics Data System (ADS)

    Fini, Jean-Baptiste; Mughal, Bilal B.; Le Mével, Sébastien; Leemans, Michelle; Lettmann, Mélodie; Spirhanzlova, Petra; Affaticati, Pierre; Jenett, Arnim; Demeneix, Barbara A.

    2017-03-01

    Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.

  5. Early chordate origins of the vertebrate second heart field.

    PubMed

    Stolfi, Alberto; Gainous, T Blair; Young, John J; Mori, Alessandro; Levine, Michael; Christiaen, Lionel

    2010-07-30

    The vertebrate heart is formed from diverse embryonic territories, including the first and second heart fields. The second heart field (SHF) gives rise to the right ventricle and outflow tract, yet its evolutionary origins are unclear. We found that heart progenitor cells of the simple chordate Ciona intestinalis also generate precursors of the atrial siphon muscles (ASMs). These precursors express Islet and Tbx1/10, evocative of the splanchnic mesoderm that produces the lower jaw muscles and SHF of vertebrates. Evidence is presented that the transcription factor COE is a critical determinant of ASM fate. We propose that the last common ancestor of tunicates and vertebrates possessed multipotent cardiopharyngeal muscle precursors, and that their reallocation might have contributed to the emergence of the SHF.

  6. Shorter Exposures to Harder X-Rays Trigger Early Apoptotic Events in Xenopus laevis Embryos

    PubMed Central

    Dong, JiaJia; Mury, Sean P.; Drahos, Karen E.; Moscovitch, Marko

    2010-01-01

    Background A long-standing conventional view of radiation-induced apoptosis is that increased exposure results in augmented apoptosis in a biological system, with a threshold below which radiation doses do not cause any significant increase in cell death. The consequences of this belief impact the extent to which malignant diseases and non-malignant conditions are therapeutically treated and how radiation is used in combination with other therapies. Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems. Methodology/Principal Findings We explored how the energy of individual X-ray photons and exposure time, both factors that determine the total dose, influence the occurrence of cell death in early Xenopus embryo. Three different experimental scenarios were analyzed and morphological and biochemical hallmarks of apoptosis were evaluated. Initially, we examined cell death events in embryos exposed to increasing incident energies when the exposure time was preset. Then, we evaluated the embryo's response when the exposure time was augmented while the energy value remained constant. Lastly, we studied the incidence of apoptosis in embryos exposed to an equal total dose of radiation that resulted from increasing the incoming energy while lowering the exposure time. Conclusions/Significance Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system. In particular, for embryos exposed under identical conditions and delivered the same absorbed dose of radiation, the response is significantly increased when shorter bursts of more energetic photons are used. These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and utilized for therapeutic

  7. Role of nucleation-promoting factors in mouse early embryo development.

    PubMed

    Wang, Qiao-Chu; Liu, Jun; Wang, Fei; Duan, Xing; Dai, Xiao-Xin; Wang, Teng; Liu, Hong-Lin; Cui, Xiang-Shun; Sun, Shao-Chen; Kim, Nam-Hyung

    2013-06-01

    During mitosis nucleation-promoting factors (NPFs) bind to the Arp2/3 complex and activate actin assembly. JMY and WAVE2 are two critical members of the NPFs. Previous studies have demonstrated that NPFs promote multiple processes such as cell migration and cytokinesis. However, the role of NPFs in development of mammalian embryos is still unknown. Results of the present study show that the NPFs JMY and WAVE2 are critical for cytokinesis during development of mouse embryos. Both JMY and WAVE2 are expressed in mouse embryos. After injection of JMY or WAVE2 siRNA, all embryos failed to develop to the morula or blastocyst stages. Moreover, using fluorescence intensity analysis, we found that the expression of actin decreased, and multiple nuclei were observed within a single cell indicating that NPFs-induced actin reduction caused the failure of cell division. In addition, injection of JMY and WAVE2 siRNA also caused ARP2 degradation, indicating that involvement of NPFs in development of mouse embryos is mainly through regulation of ARP2/3-induced actin assembly. Taken together, these data suggested that WAVE2 and JMY are involved in development of mouse embryos, and their regulation may be through a NPFs-Arp2/3-actin pathway.

  8. The role of Mixer in patterning the early Xenopus embryo.

    PubMed

    Kofron, Matt; Wylie, Chris; Heasman, Janet

    2004-05-01

    The transcription factor VegT, is required in early Xenopus embryos for the formation of both the mesoderm and endoderm germ layers. Inherited as a maternal mRNA localized only in vegetal cells, VegT activates the transcription of a large number of transcription factors, as well as signaling ligands that induce cells in the vegetal mass to form endoderm, and the marginal zone to form mesoderm. It is important now to understand the extent to which transcription factors downstream of VegT play individual, or overlapping, roles in the specification and patterning of the endoderm and mesoderm. In addition, it is important to understand the mechanism that specifies the boundary between endoderm and mesoderm. One of the downstream targets of VegT, the homeodomain protein Mixer, is expressed at high levels at the mesoderm/endoderm boundary at the late blastula stage. We therefore examined its functions by blocking its translation using morpholino oligos. In Mixer-depleted embryos, the expression of many signaling ligands and transcription factors was affected. In particular, we found that the expression of several genes, including several normally expressed in mesoderm, was upregulated. Functional assays of Mixer-depleted vegetal cells showed that they have increased mesoderm-inducing activity. This demonstrates that Mixer plays an essential role in controlling the amount of mesoderm induction by the vegetal cells.

  9. PITX2 and NODAL expression during axis formation in the early rabbit embryo.

    PubMed

    Plöger, Ruben; Viebahn, Christoph

    2018-04-26

    Attaining molecular and morphological axial polarity during gastrulation is a fundamental early requirement for normal development of the embryo. In mammals, the first morphological sign of the anterior-posterior axis appears anteriorly in the form of the anterior marginal crescent (or anterior visceral endoderm) while in the avian the first such sign is the Koller's sickle at the posterior pole of the embryonic disc. Despite this inverse mode of axis formation many genes and molecular pathways involved in various steps of this process seem to be evolutionary conserved amongst amniotes, the nodal gene being a well-known example with its functional involvement prior and during gastrulation. The pitx2 gene, however, is a new candidate described in the chick as an early marker for anterior-posterior polarity and as regulator of axis formation including twinning. To find out whether pitx2 has retained its inductive and early marker function during the evolution of mammals, this study analyzes pitx2 and nodal expression at parallel stages during formation of the anterior-posterior polarity in the early rabbit embryo using whole-mount in situ hybridization and serial light-microscopical sections. At a late pre-gastrulation stage a localized reduction of nodal expression presages the position of the anterior pole of the embryonic disc and thus serves as the earliest molecular marker of anterior-posterior polarity known so far. pitx2 is expressed in a polarized manner in the anterior marginal crescent and in the posterior half of the embryonic disc during further development only while nodal expression in the anterior segment of the posterior pitx2 expression domain helps to define the so-called anterior streak domain (ASD), a novel progenitor region of the anterior half of the primitive streak. The expression patterns of both genes thus serve as signs of a conserved involvement in early axis formation in amniotes and, possibly, in twinning in mammals as well. Copyright

  10. The vertebral column of Australopithecus sediba.

    PubMed

    Williams, Scott A; Ostrofsky, Kelly R; Frater, Nakita; Churchill, Steven E; Schmid, Peter; Berger, Lee R

    2013-04-12

    Two partial vertebral columns of Australopithecus sediba grant insight into aspects of early hominin spinal mobility, lumbar curvature, vertebral formula, and transitional vertebra position. Au. sediba likely possessed five non-rib-bearing lumbar vertebrae and five sacral elements, the same configuration that occurs modally in modern humans. This finding contrasts with other interpretations of early hominin regional vertebral numbers. Importantly, the transitional vertebra is distinct from and above the last rib-bearing vertebra in Au. sediba, resulting in a functionally longer lower back. This configuration, along with a strongly wedged last lumbar vertebra and other indicators of lordotic posture, would have contributed to a highly flexible spine that is derived compared with earlier members of the genus Australopithecus and similar to that of the Nariokotome Homo erectus skeleton.

  11. The role of the notochord in amniote vertebral column segmentation.

    PubMed

    Ward, Lizzy; Pang, Angel S W; Evans, Susan E; Stern, Claudio D

    2018-07-01

    The vertebral column is segmented, comprising an alternating series of vertebrae and intervertebral discs along the head-tail axis. The vertebrae and outer portion (annulus fibrosus) of the disc are derived from the sclerotome part of the somites, whereas the inner nucleus pulposus of the disc is derived from the notochord. Here we investigate the role of the notochord in vertebral patterning through a series of microsurgical experiments in chick embryos. Ablation of the notochord causes loss of segmentation of vertebral bodies and discs. However, the notochord cannot segment in the absence of the surrounding sclerotome. To test whether the notochord dictates sclerotome segmentation, we grafted an ectopic notochord. We find that the intrinsic segmentation of the sclerotome is dominant over any segmental information the notochord may possess, and no evidence that the chick notochord is intrinsically segmented. We propose that the segmental pattern of vertebral bodies and discs in chick is dictated by the sclerotome, which first signals to the notochord to ensure that the nucleus pulposus develops in register with the somite-derived annulus fibrosus. Later, the notochord is required for maintenance of sclerotome segmentation as the mature vertebral bodies and intervertebral discs form. These results highlight differences in vertebral development between amniotes and teleosts including zebrafish, where the notochord dictates the segmental pattern. The relative importance of the sclerotome and notochord in vertebral patterning has changed significantly during evolution. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

    PubMed Central

    Petrek, Jiri; Zitka, Ondrej; Adam, Vojtech; Bartusek, Karel; Anjum, Naser A.; Pereira, Eduarda; Havel, Ladislav; Kizek, Rene

    2015-01-01

    Background Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. Methodology/Principal Findings The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we

  13. Early Cambrian Pentamerous Cubozoan Embryos from South China

    PubMed Central

    Han, Jian; Kubota, Shin; Li, Guoxiang; Yao, Xiaoyong; Yang, Xiaoguang; Shu, Degan; Li, Yong; Kinoshita, Shunichi; Sasaki, Osamu; Komiya, Tsuyoshi; Yan, Gang

    2013-01-01

    Background Extant cubozoans are voracious predators characterized by their square shape, four evenly spaced outstretched tentacles and well-developed eyes. A few cubozoan fossils are known from the Middle Cambrian Marjum Formation of Utah and the well-known Carboniferous Mazon Creek Formation of Illinois. Undisputed cubozoan fossils were previously unknown from the early Cambrian; by that time probably all representatives of the living marine phyla, especially those of basal animals, should have evolved. Methods Microscopic fossils were recovered from a phosphatic limestone in the Lower Cambrian Kuanchuanpu Formation of South China using traditional acetic-acid maceration. Seven of the pre-hatched pentamerous cubozoan embryos, each of which bears five pairs of subumbrellar tentacle buds, were analyzed in detail through computed microtomography (Micro-CT) and scanning electron microscopy (SEM) without coating. Results The figured microscopic fossils are unequivocal pre-hatching embryos based on their spherical fertilization envelope and the enclosed soft-tissue that has preserved key anatomical features arranged in perfect pentaradial symmetry, allowing detailed comparison with modern cnidarians, especially medusozoans. A combination of features, such as the claustrum, gonad-lamella, suspensorium and velarium suspended by the frenula, occur exclusively in the gastrovascular system of extant cubozoans, indicating a cubozoan affinity for these fossils. Additionally, the interior anatomy of these embryonic cubozoan fossils unprecedentedly exhibits the development of many new septum-derived lamellae and well-partitioned gastric pockets unknown in living cubozoans, implying that ancestral cubozoans had already evolved highly specialized structures displaying unexpected complexity at the dawn of the Cambrian. The well-developed endodermic lamellae and gastric pockets developed in the late embryonic stages of these cubozoan fossils are comparable with extant pelagic

  14. Studying the effects of genistein on gene expression of fish embryos as an alternative testing approach for endocrine disruption.

    PubMed

    Schiller, Viktoria; Wichmann, Arne; Kriehuber, Ralf; Muth-Köhne, Elke; Giesy, John P; Hecker, Markus; Fenske, Martina

    2013-01-01

    Assessment of endocrine disruption currently relies on testing strategies involving adult vertebrates. In order to minimize the use of animal tests according to the 3Rs principle of replacement, reduction and refinement, we propose a transcriptomics and fish embryo based approach as an alternative to identify and analyze an estrogenic activity of environmental chemicals. For this purpose, the suitability of 48 h and 7 days post-fertilization zebrafish and medaka embryos to test for estrogenic disruption was evaluated. The embryos were exposed to the phytoestrogen genistein and subsequently analyzed by microarrays and quantitative real-time PCR. The functional analysis showed that the genes affected related to multiple metabolic and signaling pathways in the early fish embryo, which reflect the known components of genistein's mode of actions, like apoptosis, estrogenic response, hox gene expression and steroid hormone synthesis. Moreover, the transcriptomic data also suggested a thyroidal mode of action and disruption of the nervous system development. The parallel testing of two fish species provided complementary data on the effects of genistein at gene expression level and facilitated the separation of common from species-dependent effects. Overall, the study demonstrated that combining fish embryo testing with transcriptomics can deliver abundant information about the mechanistic effects of endocrine disrupting chemicals, rendering this strategy a promising alternative approach to test for endocrine disruption in a whole organism in-vitro scale system. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

    PubMed

    Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E; Kozyraki, Renata

    2013-06-07

    Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

  16. A technique for sexing fully developed embryos and early-instar larvae of the gypsy moth

    Treesearch

    Gilbert Levesque

    1963-01-01

    Because variation in sex ratio is an important factor in the population dynamics of the gypsy moth (Porthetria dispar), it is necessary to have some means of determining the ratio of males to females in a population at the beginning of the larval period as well as in the later stages. For determining the sex of fully developed embryos and early-...

  17. Rapid onset aggressive vertebral haemangioma.

    PubMed

    Cheung, Nicholas K; Doorenbosch, Xenia; Christie, John G

    2011-03-01

    Vertebral haemangiomas are generally benign asymptomatic vascular tumours seen commonly in the adult population. Presentations in paediatric populations are extremely rare, which can result in rapid onset of neurological symptoms. We present a highly unusual case of an aggressive paediatric vertebral haemangioma causing significant cord compression. A 13-year-old boy presented with only 2 weeks duration of progressive gait disturbance, truncal ataxia and loss of bladder control. Magnetic resonance imaging (MRI) of the spine revealed a large vascular epidural mass extending between T6 and T8 vertebral bodies. Associated displacement and compression of the spinal cord was present. A highly vascular bony lesion was found during surgery. Histopathology identified this tumour to be a vertebral haemangioma. We present an extremely unusual acute presentation of a paediatric vertebral haemangioma. This study highlights the need for early diagnosis, MRI for investigation and urgent surgical management. © Springer-Verlag 2011

  18. A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis.

    PubMed

    Tassy, Olivier; Daian, Fabrice; Hudson, Clare; Bertrand, Vincent; Lemaire, Patrick

    2006-02-21

    The prospects of deciphering the genetic program underlying embryonic development were recently boosted by the generation of large sets of precisely organized quantitative molecular data. In contrast, although the precise arrangement, interactions, and shapes of cells are crucial for the fulfilment of this program, their description remains coarse and qualitative. To bridge this gap, we developed a generic software, 3D Virtual Embryo, to quantify the geometry and interactions of cells in interactive three-dimensional embryo models. We applied this approach to early ascidian embryos, chosen because of their simplicity and their phylogenetic proximity to vertebrates. We generated a collection of 19 interactive ascidian embryos between the 2- and 44-cell stages. We characterized the evolution with time, and in different cell lineages, of the volume of cells and of eight mathematical descriptors of their geometry, and we measured the surface of contact between neighboring blastomeres. These analyses first revealed that early embryonic blastomeres adopt a surprising variety of shapes, which appeared to be under strict and dynamic developmental control. Second, we found novel asymmetric cell divisions in the posterior vegetal lineages, which gave birth to sister cells with different fates. Third, during neural induction, differences in the area of contact between individual competent animal cells and inducing vegetal blastomeres appeared important to select the induced cells. In addition to novel insight into both cell-autonomous and inductive processes controlling early ascidian development, we establish a generic conceptual framework for the quantitative analysis of embryo geometry that can be applied to other model organisms.

  19. Higher growth rate and gene expression in male zebra finch embryos are independent of manipulation of maternal steroids in the eggs.

    PubMed

    Lutyk, Dorota; Tagirov, Makhsud; Drobniak, Szymon; Rutkowska, Joanna

    2017-12-01

    Sexual dimorphism in prenatal development is widespread among vertebrates, including birds. Its mechanism remains unclear, although it has been attributed to the effect of maternal steroid hormones. The aim of this study was to investigate how increased levels of steroid hormones in the eggs influence early embryonic development of male and female offspring. We also asked whether maternal hormones take part in the control of sex-specific expression of the genes involved in prenatal development. We experimentally manipulated hormones' concentrations in the egg yolk by injecting zebra finch females prior to ovulation with testosterone or corticosterone. We assessed growth rate and expression levels of CDK7, FBP1 and GHR genes in 37h-old embryos. We found faster growth and higher expression of two studied genes in male compared to female embryos. Hormonal treatment, despite clearly differentiating egg steroid levels, had no effect on the sex-specific pattern of the embryonic gene expression, even though we confirmed expression of receptors of androgens and glucocorticoids at such an early stage of development. Thus, our study shows high stability of the early sex differences in the embryonic development before the onset of sexual differentiation and indicates their independence of maternal hormones in the egg. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Essential Role of Chromatin Remodeling Protein Bptf in Early Mouse Embryos and Embryonic Stem Cells

    PubMed Central

    Landry, Joseph; Sharov, Alexei A.; Piao, Yulan; Sharova, Lioudmila V.; Xiao, Hua; Southon, Eileen; Matta, Jennifer; Tessarollo, Lino; Zhang, Ying E.; Ko, Minoru S. H.; Kuehn, Michael R.; Yamaguchi, Terry P.; Wu, Carl

    2008-01-01

    We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo. PMID:18974875

  1. Fish embryos on land: terrestrial embryo deposition lowers oxygen uptake without altering growth or survival in the amphibious fish Kryptolebias marmoratus.

    PubMed

    Wells, Michael W; Turko, Andy J; Wright, Patricia A

    2015-10-01

    Few teleost fishes incubate embryos out of water, but the oxygen-rich terrestrial environment could provide advantages for early growth and development. We tested the hypothesis that embryonic oxygen uptake is limited in aquatic environments relative to air using the self-fertilizing amphibious mangrove rivulus, Kryptolebias marmoratus, which typically inhabits hypoxic, water-filled crab burrows. We found that adult mangrove rivulus released twice as many embryos in terrestrial versus aquatic environments and that air-reared embryos had accelerated developmental rates. Surprisingly, air-reared embryos consumed 44% less oxygen and possessed larger yolk reserves, but attained the same mass, length and chorion thickness. Water-reared embryos moved their opercula ∼2.5 more times per minute compared with air-reared embryos at 7 days post-release, which probably contributed to the higher rates of oxygen uptake and yolk utilization we observed. Genetically identical air- and water-reared embryos from the same parent were raised to maturity, but the embryonic environment did not affect growth, reproduction or emersion ability in adults. Therefore, although aspects of early development were plastic, these early differences were not sustained into adulthood. Kryptolebias marmoratus embryos hatched out of water when exposed to aerial hypoxia. We conclude that exposure to a terrestrial environment reduces the energetic costs of development partly by reducing the necessity of embryonic movements to dispel stagnant boundary layers. Terrestrial incubation of young would be especially beneficial to amphibious fishes that occupy aquatic habitats of poor water quality, assuming low terrestrial predation and desiccation risks. © 2015. Published by The Company of Biologists Ltd.

  2. Identification of Ohnolog Genes Originating from Whole Genome Duplication in Early Vertebrates, Based on Synteny Comparison across Multiple Genomes.

    PubMed

    Singh, Param Priya; Arora, Jatin; Isambert, Hervé

    2015-07-01

    Whole genome duplications (WGD) have now been firmly established in all major eukaryotic kingdoms. In particular, all vertebrates descend from two rounds of WGDs, that occurred in their jawless ancestor some 500 MY ago. Paralogs retained from WGD, also coined 'ohnologs' after Susumu Ohno, have been shown to be typically associated with development, signaling and gene regulation. Ohnologs, which amount to about 20 to 35% of genes in the human genome, have also been shown to be prone to dominant deleterious mutations and frequently implicated in cancer and genetic diseases. Hence, identifying ohnologs is central to better understand the evolution of vertebrates and their susceptibility to genetic diseases. Early computational analyses to identify vertebrate ohnologs relied on content-based synteny comparisons between the human genome and a single invertebrate outgroup genome or within the human genome itself. These approaches are thus limited by lineage specific rearrangements in individual genomes. We report, in this study, the identification of vertebrate ohnologs based on the quantitative assessment and integration of synteny conservation between six amniote vertebrates and six invertebrate outgroups. Such a synteny comparison across multiple genomes is shown to enhance the statistical power of ohnolog identification in vertebrates compared to earlier approaches, by overcoming lineage specific genome rearrangements. Ohnolog gene families can be browsed and downloaded for three statistical confidence levels or recompiled for specific, user-defined, significance criteria at http://ohnologs.curie.fr/. In the light of the importance of WGD on the genetic makeup of vertebrates, our analysis provides a useful resource for researchers interested in gaining further insights on vertebrate evolution and genetic diseases.

  3. Identification of Ohnolog Genes Originating from Whole Genome Duplication in Early Vertebrates, Based on Synteny Comparison across Multiple Genomes

    PubMed Central

    Singh, Param Priya; Arora, Jatin; Isambert, Hervé

    2015-01-01

    Whole genome duplications (WGD) have now been firmly established in all major eukaryotic kingdoms. In particular, all vertebrates descend from two rounds of WGDs, that occurred in their jawless ancestor some 500 MY ago. Paralogs retained from WGD, also coined ‘ohnologs’ after Susumu Ohno, have been shown to be typically associated with development, signaling and gene regulation. Ohnologs, which amount to about 20 to 35% of genes in the human genome, have also been shown to be prone to dominant deleterious mutations and frequently implicated in cancer and genetic diseases. Hence, identifying ohnologs is central to better understand the evolution of vertebrates and their susceptibility to genetic diseases. Early computational analyses to identify vertebrate ohnologs relied on content-based synteny comparisons between the human genome and a single invertebrate outgroup genome or within the human genome itself. These approaches are thus limited by lineage specific rearrangements in individual genomes. We report, in this study, the identification of vertebrate ohnologs based on the quantitative assessment and integration of synteny conservation between six amniote vertebrates and six invertebrate outgroups. Such a synteny comparison across multiple genomes is shown to enhance the statistical power of ohnolog identification in vertebrates compared to earlier approaches, by overcoming lineage specific genome rearrangements. Ohnolog gene families can be browsed and downloaded for three statistical confidence levels or recompiled for specific, user-defined, significance criteria at http://ohnologs.curie.fr/. In the light of the importance of WGD on the genetic makeup of vertebrates, our analysis provides a useful resource for researchers interested in gaining further insights on vertebrate evolution and genetic diseases. PMID:26181593

  4. Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

    PubMed Central

    Bernut, Audrey; Dupont, Christian; Sahuquet, Alain; Herrmann, Jean-Louis; Lutfalla, Georges; Kremer, Laurent

    2015-01-01

    Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate. PMID:26382225

  5. Endogenous Nod-Factor-Like Signal Molecules Promote Early Somatic Embryo Development in Norway Spruce1

    PubMed Central

    Dyachok, Julia V.; Wiweger, Malgorzata; Kenne, Lennart; von Arnold, Sara

    2002-01-01

    Embryogenic cultures of Norway spruce (Picea abies) are composed of pro-embryogenic masses (PEMs) and somatic embryos of various developmental stages. Auxin is important for PEM formation and proliferation. In this report we show that depletion of auxin blocks PEM development and causes large-scale cell death. Extracts of the media conditioned by embryogenic cultures stimulate development of PEM aggregates in auxin-deficient cultures. Partial characterization of the conditioning factor shows that it is a lipophilic, low-molecular-weight molecule, which is sensitive to chitinase and contains GlcNAc residues. On the basis of this information, we propose that the factor is a lipophilic chitin oligosaccharide (LCO). The amount of LCO correlates to the developmental stages of PEMs and embryos, with the highest level in the media conditioned by developmentally blocked cultures. LCO is not present in nonembryogenic cultures. Cell death, induced by withdrawal of auxin, is suppressed by extra supply of endogenous LCO or Nod factor from Rhizobium sp. NGR234. The effect can be mimicked by a chitotetraose or chitinase from Streptomyces griseus. Taken together, our data suggest that endogenous LCO acts as a signal molecule stimulating PEM and early embryo development in Norway spruce. PMID:11842156

  6. Time-lapse culture with morphokinetic embryo selection improves pregnancy and live birth chances and reduces early pregnancy loss: a meta-analysis.

    PubMed

    Pribenszky, Csaba; Nilselid, Anna-Maria; Montag, Markus

    2017-11-01

    Embryo evaluation and selection is fundamental in clinical IVF. Time-lapse follow-up of embryo development comprises undisturbed culture and the application of the visual information to support embryo evaluation. A meta-analysis of randomized controlled trials was carried out to study whether time-lapse monitoring with the prospective use of a morphokinetic algorithm for selection of embryos improves overall clinical outcome (pregnancy, early pregnancy loss, stillbirth and live birth rate) compared with embryo selection based on single time-point morphology in IVF cycles. The meta-analysis of five randomized controlled trials (n = 1637) showed that the application of time-lapse monitoring was associated with a significantly higher ongoing clinical pregnancy rate (51.0% versus 39.9%), with a pooled odds ratio of 1.542 (P < 0.001), significantly lower early pregnancy loss (15.3% versus 21.3%; OR: 0.662; P = 0.019) and a significantly increased live birth rate (44.2% versus 31.3%; OR 1.668; P = 0.009). Difference in stillbirth was not significant between groups (4.7% versus 2.4%). Quality of the evidence was moderate to low owing to inconsistencies across the studies. Selective application and variability were also limitations. Although time-lapse is shown to significantly improve overall clinical outcome, further high-quality evidence is needed before universal conclusions can be drawn. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  7. Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

    PubMed Central

    Pires, Camilla Valente; Freitas, Flávia Cristina de Paula; Cristino, Alexandre S.; Dearden, Peter K.; Simões, Zilá Luz Paulino

    2016-01-01

    In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development. PMID:26751956

  8. Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth.

    PubMed

    Christiansen, Helena E; Lang, Michael R; Pace, James M; Parichy, David M

    2009-12-29

    Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.

  9. Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology

    PubMed Central

    Sandell, Lisa L.; Kurosaka, Hiroshi; Trainor, Paul A.

    2012-01-01

    Here we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional widefield fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images of similar specimens produced by Scanning Electron Microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. PMID:22930523

  10. The evolution of the dorsal thalamus of jawed vertebrates, including mammals: cladistic analysis and a new hypothesis.

    PubMed

    Butler, A B

    1994-01-01

    The evolution of the dorsal thalamus in various vertebrate lineages of jawed vertebrates has been an enigma, partly due to two prevalent misconceptions: the belief that the multitude of nuclei in the dorsal thalamus of mammals could be meaningfully compared neither with the relatively few nuclei in the dorsal thalamus of anamniotes nor with the intermediate number of dorsal thalamic nuclei of other amniotes and a definition of the dorsal thalamus that too narrowly focused on the features of the dorsal thalamus of mammals. The cladistic analysis carried out here allows us to recognize which features are plesiomorphic and which apomorphic for the dorsal thalamus of jawed vertebrates and to then reconstruct the major changes that have occurred in the dorsal thalamus over evolution. Embryological data examined in the context of Von Baerian theory (embryos of later-descendant species resemble the embryos of earlier-descendant species to the point of their divergence) supports a new 'Dual Elaboration Hypothesis' of dorsal thalamic evolution generated from this cladistic analysis. From the morphotype for an early stage in the embryological development of the dorsal thalamus of jawed vertebrates, the divergent, sequential stages of the development of the dorsal thalamus are derived for each major radiation and compared. The new hypothesis holds that the dorsal thalamus comprises two basic divisions--the collothalamus and the lemnothalamus--that receive their predominant input from the midbrain roof and (plesiomorphically) from lemniscal pathways, including the optic tract, respectively. Where present, the collothalamic, midbrain-sensory relay nuclei are homologous to each other in all vertebrate radiations as discrete nuclei. Within the lemnothalamus, the dorsal lateral geniculate nucleus of mammals and the dorsal lateral optic nucleus of non-synapsid amniotes (diapsid reptiles, birds and turtles) are homologous as discrete nuclei; most or all of the ventral nuclear group

  11. Selection and dynamics of embryonic stem cell integration into early mouse embryos

    PubMed Central

    Alexandrova, Stoyana; Kalkan, Tuzer; Humphreys, Peter; Riddell, Andrew; Scognamiglio, Roberta; Trumpp, Andreas; Nichols, Jennifer

    2016-01-01

    The process by which pluripotent cells incorporate into host embryos is of interest to investigate cell potency and cell fate decisions. Previous studies suggest that only a minority of the embryonic stem cell (ESC) inoculum contributes to the adult chimaera. How incoming cells are chosen for integration or elimination remains unclear. By comparing a heterogeneous mix of undifferentiated and differentiating ESCs (serum/LIF) with more homogeneous undifferentiated culture (2i/LIF), we examine the role of cellular heterogeneity in this process. Time-lapse ex vivo imaging revealed a drastic elimination of serum/LIF ESCs during early development in comparison with 2i/LIF ESCs. Using a fluorescent reporter for naive pluripotency (Rex1-GFP), we established that the acutely eliminated serum/LIF ESCs had started to differentiate. The rejected cells were apparently killed by apoptosis. We conclude that a selection process exists by which unwanted differentiating cells are eliminated from the embryo. However, occasional Rex1− cells were able to integrate. Upregulation of Rex1 occurred in a proportion of these cells, reflecting the potential of the embryonic environment to expedite diversion from differentiation priming to enhance the developing embryonic epiblast. PMID:26586221

  12. Live-bearing manta ray: how the embryo acquires oxygen without placenta and umbilical cord

    PubMed Central

    Tomita, Taketeru; Toda, Minoru; Ueda, Keiichi; Uchida, Senzo; Nakaya, Kazuhiro

    2012-01-01

    We conducted an ultrasonographic experiment on a pregnant manta ray, Manta alfredi (Chondrichthyes, Batoidea). This study showed how the embryo of the live-bearing elasmobranchs respires in the body of the female. In the embryonic stage, the manta ray embryo takes in uterine fluid by buccal-pumping. After birth, the manta ray shifts its respiratory mode from buccal-pumping to ram-ventilation. The rapid reduction of the spiracle size in the young manta ray may reflect this shift of respiratory mode. Unlike mammals or some carcharhinid sharks that acquire oxygen through a placenta and umbilical cord, the manta ray embryo does not have a direct connection with the mother. Thus, the manta ray embryo obtains oxygen by buccal-pumping of the uterine fluid, in the same way that the embryos of egg-laying species obtain oxygen from the water in the egg case. This finding extends our understanding of the diversity of embryonic respiratory systems in live-bearing vertebrates. PMID:22675137

  13. Microspore embryogenesis in wheat: new marker genes for early, middle and late stages of embryo development.

    PubMed

    Sánchez-Díaz, Rosa Angélica; Castillo, Ana María; Vallés, María Pilar

    2013-09-01

    Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation.

  14. Endometrium as an early sensor of in vitro embryo manipulation technologies

    PubMed Central

    Mansouri-Attia, Nadéra; Sandra, Olivier; Aubert, Julie; Degrelle, Séverine; Everts, Robin E.; Giraud-Delville, Corinne; Heyman, Yvan; Galio, Laurent; Hue, Isabelle; Yang, Xiangzhong; Tian, X. Cindy; Lewin, Harris A.; Renard, Jean-Paul

    2009-01-01

    Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy. PMID:19297625

  15. Identification and functional analysis of long non-coding RNAs in human and mouse early embryos based on single-cell transcriptome data

    PubMed Central

    Qiu, Jia-jun; Ren, Zhao-rui; Yan, Jing-bin

    2016-01-01

    Epigenetics regulations have an important role in fertilization and proper embryonic development, and several human diseases are associated with epigenetic modification disorders, such as Rett syndrome, Beckwith-Wiedemann syndrome and Angelman syndrome. However, the dynamics and functions of long non-coding RNAs (lncRNAs), one type of epigenetic regulators, in human pre-implantation development have not yet been demonstrated. In this study, a comprehensive analysis of human and mouse early-stage embryonic lncRNAs was performed based on public single-cell RNA sequencing data. Expression profile analysis revealed that lncRNAs are expressed in a developmental stage–specific manner during human early-stage embryonic development, whereas a more temporal-specific expression pattern was identified in mouse embryos. Weighted gene co-expression network analysis suggested that lncRNAs involved in human early-stage embryonic development are associated with several important functions and processes, such as oocyte maturation, zygotic genome activation and mitochondrial functions. We also found that the network of lncRNAs involved in zygotic genome activation was highly preservative between human and mouse embryos, whereas in other stages no strong correlation between human and mouse embryo was observed. This study provides insight into the molecular mechanism underlying lncRNA involvement in human pre-implantation embryonic development. PMID:27542205

  16. Single-cell transcriptome of early embryos and cultured embryonic stem cells of cynomolgus monkeys

    PubMed Central

    Nakamura, Tomonori; Yabuta, Yukihiro; Okamoto, Ikuhiro; Sasaki, Kotaro; Iwatani, Chizuru; Tsuchiya, Hideaki; Saitou, Mitinori

    2017-01-01

    In mammals, the development of pluripotency and specification of primordial germ cells (PGCs) have been studied predominantly using mice as a model organism. However, divergences among mammalian species for such processes have begun to be recognized. Between humans and mice, pre-implantation development appears relatively similar, but the manner and morphology of post-implantation development are significantly different. Nevertheless, the embryogenesis just after implantation in primates, including the specification of PGCs, has been unexplored due to the difficulties in analyzing the embryos at relevant developmental stages. Here, we present a comprehensive single-cell transcriptome dataset of pre- and early post-implantation embryo cells, PGCs and embryonic stem cells (ESCs) of cynomolgus monkeys as a model of higher primates. The identities of each transcriptome were also validated rigorously by other way such as immunofluorescent analysis. The information reported here will serve as a foundation for our understanding of a wide range of processes in the developmental biology of primates, including humans. PMID:28649393

  17. Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits.

    PubMed

    Bertrand, Stephanie; Camasses, Alain; Somorjai, Ildiko; Belgacem, Mohamed R; Chabrol, Olivier; Escande, Marie-Line; Pontarotti, Pierre; Escriva, Hector

    2011-05-31

    FGF signaling is one of the few cell-cell signaling pathways conserved among all metazoans. The diversity of FGF gene content among different phyla suggests that evolution of FGF signaling may have participated in generating the current variety of animal forms. Vertebrates possess the greatest number of FGF genes, the functional evolution of which may have been implicated in the acquisition of vertebrate-specific morphological traits. In this study, we have investigated the roles of the FGF signal during embryogenesis of the cephalochordate amphioxus, the best proxy for the chordate ancestor. We first isolate the full FGF gene complement and determine the evolutionary relationships between amphioxus and vertebrate FGFs via phylogenetic and synteny conservation analysis. Using pharmacological treatments, we inhibit the FGF signaling pathway in amphioxus embryos in different time windows. Our results show that the requirement for FGF signaling during gastrulation is a conserved character among chordates, whereas this signal is not necessary for neural induction in amphioxus, in contrast to what is known in vertebrates. We also show that FGF signal, acting through the MAPK pathway, is necessary for the formation of the most anterior somites in amphioxus, whereas more posterior somite formation is not FGF-dependent. This result leads us to propose that modification of the FGF signal function in the anterior paraxial mesoderm in an amphioxus-like vertebrate ancestor might have contributed to the loss of segmentation in the preotic paraxial mesoderm of the vertebrate head.

  18. Making lineage decisions with biological noise: Lessons from the early mouse embryo.

    PubMed

    Simon, Claire S; Hadjantonakis, Anna-Katerina; Schröter, Christian

    2018-04-30

    Understanding how individual cells make fate decisions that lead to the faithful formation and homeostatic maintenance of tissues is a fundamental goal of contemporary developmental and stem cell biology. Seemingly uniform populations of stem cells and multipotent progenitors display a surprising degree of heterogeneity, primarily originating from the inherent stochastic nature of molecular processes underlying gene expression. Despite this heterogeneity, lineage decisions result in tissues of a defined size and with consistent proportions of differentiated cell types. Using the early mouse embryo as a model we review recent developments that have allowed the quantification of molecular intercellular heterogeneity during cell differentiation. We first discuss the relationship between these heterogeneities and developmental cellular potential. We then review recent theoretical approaches that formalize the mechanisms underlying fate decisions in the inner cell mass of the blastocyst stage embryo. These models build on our extensive knowledge of the genetic control of fate decisions in this system and will become essential tools for a rigorous understanding of the connection between noisy molecular processes and reproducible outcomes at the multicellular level. We conclude by suggesting that cell-to-cell communication provides a mechanism to exploit and buffer intercellular variability in a self-organized process that culminates in the reproducible formation of the mature mammalian blastocyst stage embryo that is ready for implantation into the maternal uterus. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Gene Expression and Transcriptional Hierarchies > Quantitative Methods and Models. © 2018 Wiley Periodicals, Inc.

  19. Amphibian metamorphosis as a model for studying endocrine disruption on vertebrate development: effect of bisphenol A on thyroid hormone action.

    PubMed

    Heimeier, Rachel A; Shi, Yun-Bo

    2010-09-01

    Thyroid hormone (TH) is essential for proper development in vertebrates. TH deficiency during gestation and early postnatal development produces severe neurological, skeletal, metabolism and growth abnormalities. It is therefore important to consider environmental chemicals that may interfere with TH signaling. Exposure to environmental contaminants that disrupt TH action may underlie the increasing incidence of human developmental disorders worldwide. One contaminant of concern is the xenoestrogen bisphenol A (BPA), a chemical widely used to manufacture polycarbonate plastics and epoxy resins. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. As TH action at the cellular level is highly conserved across vertebrate species, amphibian metamorphosis serves as an important TH-dependent in vivo vertebrate model for studying potential contributions of BPA toward human developmental disorders. Using Xenopus laevis as a model, we and others have demonstrated the inhibitory effects of BPA exposure on metamorphosis. Genome-wide gene expression analysis revealed that surprisingly, BPA primarily targets the TH-signaling pathway essential for metamorphosis in Xenopus laevis. Given the importance of the genomic effects of TH during metamorphosis and the conservation in its regulation in higher vertebrates, these observations suggest that the effect of BPA in human embryogenesis is through the inhibition of the TH pathway and warrants further investigation. Our findings further argue for the critical need to use in vivo animal models coupled with systematic molecular analysis to determine the developmental effects of endocrine disrupting compounds. Published by Elsevier Inc.

  20. IN VITRO CULTURE OF POSTIMPLANTATION HAMSTER EMBRYOS

    EPA Science Inventory

    In vitro culture of intact rat and mouse embryos has been described extensively, but information on the culture of other species is sparse. The present study examined some culture requirements of early somite stage hamster embryos and assessed the embryotoxic effects of sodium sa...

  1. Patterns of protein synthesis in oocytes and early embryos of Rana esculenta complex.

    PubMed

    Chen, P S; Stumm-Zollinger, E

    1986-01-01

    We have used isotopic labelling and both one-and two-dimensional electrophoretic procedures to analyse the protien synthesis patterns in oocytes and early embryos of three phenotypes of the European green frogs. The results demonstrated that protein patterns of Rana ridibunda and R. esculenta are identical, but that they differ from those of R. lessonae. Progeny of the lethal cross R. esculenta × R. esculenta showed a distinct delay in the appearance of stage-specific proteins during early embryogenesis. The heat-shock response of R. ridibunda and R. esculenta oocytes was found to be identical, but different from that of Xenopus laevis. The implications of these findings, with respect to hybridogenesis in R. esculenta complex and variations in the regulations of heat shock genes in different amphibian species, are discussed.

  2. The phylogeny and ontogeny of autonomic control of the heart and cardiorespiratory interactions in vertebrates.

    PubMed

    Taylor, Edwin W; Leite, Cleo A C; Sartori, Marina R; Wang, Tobias; Abe, Augusto S; Crossley, Dane A

    2014-03-01

    Heart rate in vertebrates is controlled by activity in the autonomic nervous system. In spontaneously active or experimentally prepared animals, inhibitory parasympathetic control is predominant and is responsible for instantaneous changes in heart rate, such as occur at the first air breath following a period of apnoea in discontinuous breathers like inactive reptiles or species that surface to air breathe after a period of submersion. Parasympathetic control, exerted via fast-conducting, myelinated efferent fibres in the vagus nerve, is also responsible for beat-to-beat changes in heart rate such as the high frequency components observed in spectral analysis of heart rate variability. These include respiratory modulation of the heartbeat that can generate cardiorespiratory synchrony in fish and respiratory sinus arrhythmia in mammals. Both may increase the effectiveness of respiratory gas exchange. Although the central interactions generating respiratory modulation of the heartbeat seem to be highly conserved through vertebrate phylogeny, they are different in kind and location, and in most species are as yet little understood. The heart in vertebrate embryos possesses both muscarinic cholinergic and β-adrenergic receptors very early in development. Adrenergic control by circulating catecholamines seems important throughout development. However, innervation of the cardiac receptors is delayed and first evidence of a functional cholinergic tonus on the heart, exerted via the vagus nerve, is often seen shortly before or immediately after hatching or birth, suggesting that it may be coordinated with the onset of central respiratory rhythmicity and subsequent breathing.

  3. Surgical treatment of hematogenous vertebral Aspergillus osteomyelitis.

    PubMed

    Bridwell, K H; Campbell, J W; Barenkamp, S J

    1990-04-01

    Three cases of Aspergillus fumigatas vertebral osteomyelitis failed courses of medical treatment. Each was subsequently treated with anterior vertebral debridement and posterior segmental spinal instrumentation. Despite poor nutritional and immune systems, resolution of the infection and subsequent anterior ankylosis occurred in each patient, with follow-up ranging from 1 to 3 years. If patients with aspergillus vertebral osteomyelitis do not respond to medical treatment, early surgical debridement and stabilization in combination with intravenous amphotericin B can lead to resolution and bony ankylosis.

  4. Whole mount nuclear fluorescent imaging: convenient documentation of embryo morphology.

    PubMed

    Sandell, Lisa L; Kurosaka, Hiroshi; Trainor, Paul A

    2012-11-01

    Here, we describe a relatively inexpensive and easy method to produce high quality images that reveal fine topological details of vertebrate embryonic structures. The method relies on nuclear staining of whole mount embryos in combination with confocal microscopy or conventional wide field fluorescent microscopy. In cases where confocal microscopy is used in combination with whole mount nuclear staining, the resulting embryo images can rival the clarity and resolution of images produced by scanning electron microscopy (SEM). The fluorescent nuclear staining may be performed with a variety of cell permeable nuclear dyes, enabling the technique to be performed with multiple standard microscope/illumination or confocal/laser systems. The method may be used to document morphology of embryos of a variety of organisms, as well as individual organs and tissues. Nuclear stain imaging imposes minimal impact on embryonic specimens, enabling imaged specimens to be utilized for additional assays. Copyright © 2012 Wiley Periodicals, Inc.

  5. Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy.

    PubMed

    Watanabe, Tomoko; Thayil, Anisha; Jesacher, Alexander; Grieve, Kate; Debarre, Delphine; Wilson, Tony; Booth, Martin; Srinivas, Shankar

    2010-06-03

    Lipid droplets (LD) are organelles with an important role in normal metabolism and disease. The lipid content of embryos has a major impact on viability and development. LD in Drosophila embryos and cultured cell lines have been shown to move and fuse in a microtubule dependent manner. Due to limitations in current imaging technology, little is known about the behaviour of LD in the mammalian embryo. Harmonic generation microscopy (HGM) allows one to image LD without the use of exogenous labels. Adaptive optics can be used to correct aberrations that would otherwise degrade the quality and information content of images. We have built a harmonic generation microscope with adaptive optics to characterise early mouse embryogenesis. At fertilization, LD are small and uniformly distributed, but in the implanting blastocyst, LD are larger and enriched in the invading giant cells of the trophectoderm. Time-lapse studies reveal that LD move continuously and collide but do not fuse, instead forming aggregates that subsequently behave as single units. Using specific inhibitors, we show that the velocity and dynamic behaviour of LD is dependent not only on microtubules as in other systems, but also on microfilaments. We explore the limits within which HGM can be used to study living embryos without compromising viability and make the counterintuitive finding that 16 J of energy delivered continuously over a period of minutes can be less deleterious than an order of magnitude lower energy delivered dis-continuously over a period of hours. LD in pre-implantation mouse embryos show a previously unappreciated complexity of behaviour that is dependent not only on microtubules, but also microfilaments. Unlike LD in other systems, LD in the mouse embryo do not fuse but form aggregates. This study establishes HGM with adaptive optics as a powerful tool for the study of LD biology and provides insights into the photo-toxic effects of imaging embryos.

  6. Fyn/Yes and non-canonical Wnt signalling converge on RhoA in vertebrate gastrulation cell movements

    PubMed Central

    Jopling, Chris; den Hertog, Jeroen

    2005-01-01

    Convergent extension (CE) cell movements during gastrulation mediate extension of the anterior–posterior body axis of vertebrate embryos. Non-canonical Wnt5 and Wnt11 signalling is essential for normal CE movements in vertebrate gastrulation. Here, we show that morpholino (MO)-mediated double knock-down of the Fyn and Yes tyrosine kinases in zebrafish embryos impaired normal CE cell movements, resembling the silberblick and pipetail mutants, caused by mutations in wnt11 and wnt5, respectively. Co-injection of Fyn/Yes- and Wnt11- or Wnt5-MO was synergistic, but wnt11 or wnt5 RNA did not rescue the Fyn/Yes knockdown or vice versa. Remarkably, active RhoA rescued the Fyn/Yes knockdown as well as the Wnt11 knockdown, indicating that Fyn/Yes and Wnt11 signalling converged on RhoA. Our results show that Fyn and Yes act together with non-canonical Wnt signalling via RhoA in CE cell movements during gastrulation. PMID:15815683

  7. The CW domain, a structural module shared amongst vertebrates, vertebrate-infecting parasites and higher plants.

    PubMed

    Perry, Jason; Zhao, Yunde

    2003-11-01

    A previously undetected domain, named CW for its conserved cysteine and tryptophan residues, appears to be a four-cysteine zinc-finger motif found exclusively in vertebrates, vertebrate-infecting parasites and higher plants. Of the twelve distinct nuclear protein families that comprise the CW domain-containing superfamily, only the microrchida (MORC) family has begun to be characterized. However, several families contain other domains suggesting a relationship between the CW domain and either chromatin methylation status or early embryonic development.

  8. Transient expression and activity of human DNA polymerase iota in loach embryos.

    PubMed

    Makarova, Irina V; Kazakov, Andrey A; Makarova, Alena V; Khaidarova, Nella V; Kozikova, Larisa V; Nenasheva, Valentina V; Gening, Leonid V; Tarantul, Vyacheslav Z; Andreeva, Ludmila E

    2012-02-01

    Human DNA polymerase iota (Pol ι) is a Y-family DNA polymerase with unusual biochemical properties and not fully understood functions. Pol ι preferentially incorporates dGTP opposite template thymine. This property can be used to monitor Pol ι activity in the presence of other DNA polymerases, e.g. in cell extracts of tissues and tumors. We have now confirmed the specificity and sensitivity of the method of Pol ι activity detection in cell extracts using an animal model of loach Misgurnus fossilis embryos transiently expressing human Pol ι. The overexpression of Pol ι was shown to be accompanied by an increase in abnormalities in development and the frequency of pycnotic nuclei in fish embryos. Further analysis of fish embryos with constitutive or regulated Pol ι expression may provide insights into Pol ι functions in vertebrate animals.

  9. Dynamic Properties of Electrotonic Coupling between Cells of Early Xenopus Embryos

    PubMed Central

    DiCaprio, R. A.; French, A. S.; Sanders, E. J.

    1974-01-01

    Frequency response functions were measured between the cells of Xenopus laevis embryos during the first two cleavage stages. Linear systems theory was then used to produce electronic models which account for the electrical behavior of the systems. Coupling between the cells may be explained by models which have simple resistive elements joining each cell to its neighbors. The vitelline, or fertilization, membrane which surrounds the embryos has no detectable resistance to the passage of electric current. The electrical properties of the four-cell embryo can only be explained by the existence of individual junctions linking each pair of cells. This arrangement suggests that electrotonic coupling is important in the development of the embryos, at least until the four-cell stage. ImagesFIGURE 5FIGURE 14FIGURE 15 PMID:19431351

  10. The pre-vertebrate origins of neurogenic placodes.

    PubMed

    Abitua, Philip Barron; Gainous, T Blair; Kaczmarczyk, Angela N; Winchell, Christopher J; Hudson, Clare; Kamata, Kaori; Nakagawa, Masashi; Tsuda, Motoyuki; Kusakabe, Takehiro G; Levine, Michael

    2015-08-27

    The sudden appearance of the neural crest and neurogenic placodes in early branching vertebrates has puzzled biologists for over a century. These embryonic tissues contribute to the development of the cranium and associated sensory organs, which were crucial for the evolution of the vertebrate "new head". A previous study suggests that rudimentary neural crest cells existed in ancestral chordates. However, the evolutionary origins of neurogenic placodes have remained obscure owing to a paucity of embryonic data from tunicates, the closest living relatives to those early vertebrates. Here we show that the tunicate Ciona intestinalis exhibits a proto-placodal ectoderm (PPE) that requires inhibition of bone morphogenetic protein (BMP) and expresses the key regulatory determinant Six1/2 and its co-factor Eya, a developmental process conserved across vertebrates. The Ciona PPE is shown to produce ciliated neurons that express genes for gonadotropin-releasing hormone (GnRH), a G-protein-coupled receptor for relaxin-3 (RXFP3) and a functional cyclic nucleotide-gated channel (CNGA), which suggests dual chemosensory and neurosecretory activities. These observations provide evidence that Ciona has a neurogenic proto-placode, which forms neurons that appear to be related to those derived from the olfactory placode and hypothalamic neurons of vertebrates. We discuss the possibility that the PPE-derived GnRH neurons of Ciona resemble an ancestral cell type, a progenitor to the complex neuronal circuit that integrates sensory information and neuroendocrine functions in vertebrates.

  11. The Potential Role of As-sumo-1 in the Embryonic Diapause Process and Early Embryo Development of Artemia sinica

    PubMed Central

    Chu, Bing; Yao, Feng; Cheng, Cheng; Wu, Yang; Mei, Yanli; Li, Xuejie; Liu, Yan; Wang, Peisheng; Hou, Lin; Zou, Xiangyang

    2014-01-01

    During embryonic development of Artemia sinica, environmental stresses induce the embryo diapause phenomenon, required to resist apoptosis and regulate cell cycle activity. The small ubiquitin-related modifier-1 (SUMO), a reversible post-translational protein modifier, plays an important role in embryo development. SUMO regulates multiple cellular processes, including development and other biological processes. The molecular mechanism of diapause, diapause termination and the role of As-sumo-1 in this processes and in early embryo development of Artemia sinica still remains unknown. In this study, the complete cDNA sequences of the sumo-1 homolog, sumo ligase homolog, caspase-1 homolog and cyclin B homolog from Artemia sinica were cloned. The mRNA expression patterns of As-sumo-1, sumo ligase, caspase-1, cyclin B and the location of As-sumo-1 were investigated. SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E proteins were analyzed during different developmental stages of the embryo of A. sinica. Small interfering RNA (siRNA) was used to verify the function of sumo-1 in A. sinica. The full-length cDNA of As-sumo-1 was 476 bp, encoding a 92 amino acid protein. The As-caspases-1 cDNA was 966 bp, encoding a 245 amino-acid protein. The As-sumo ligase cDNA was 1556 bp encoding, a 343 amino acid protein, and the cyclin B cDNA was 739 bp, encoding a 133 amino acid protein. The expressions of As-sumo-1, As-caspase-1 and As-cyclin B were highest at the 10 h stage of embryonic development, and As-sumo ligase showed its highest expression at 0 h. The expression of As-SUMO-1 showed no tissue or organ specificity. Western blotting showed high expression of As-SUMO-1, p53, Mdm2, Caspase-1, Cyclin B and Cyclin E at the 10 h stage. The siRNA caused abnormal development of the embryo, with increased malformation and mortality. As-SUMO-1 is a crucial regulation and modification protein resumption of embryonic diapause and early embryo development of A. sinica. PMID:24404204

  12. Intracellular pH in early Xenopus embryos: its effect on current flow between blastomeres.

    PubMed Central

    Turin, L; Warner, A E

    1980-01-01

    1. Electrophysiological techniques were used to monitor the flow of electric current from one cell to the next in Xenopus laevis embryos between the 4-cell and early blastula stages of development. Intracellular pH and blastocoel pH were determined using pH-sensitive micro-electrodes. 2. The resting intracellular pH was 7.74+/-0.02 (S.E. of mean, n = 29); there were no systematic differences between developmental stages. Blastocoel cavity pH was 8.4+/-0.06 (S.E. of mean, n = 10). The intracellular buffer value was 18 m-equiv. H+/pH unit per litre. 3. In embryos treated with bicarbonate buffered Holtfreter solution equilibrated with 100% CO2 the intracellular pH fell to 6.3+/-0.17 (S.D., n = 8). The membrane potential fell and the input resistance increased. The size of the effect on membrane potential and input resistance varied. 4. From the 32-cell stage onwards current flow from one cell to the next was abolished when the intracellular pH fell to below 6.5; the effect was rapid in onset and completely reversible. At cleavage stages of development lowering intracellular pH with CO2 had no effect on current flow from cell to cell. 5. The relationship between intracellular pH and current flow from cell to cell was sigmoid and covered between 0.2 and 0.4 pH units. The pH at which current flow was completely abolished ranged from 6.85 to 6.4. 6. Alterations in extraembryonic pH over the range 5.8-7.5 had no effect on any parameter measured. 7. We conclude that lowering the intracellular pH increases the resistance of both non-junctional junctional membranes. The data do not allow us to extract the pH junctional conductance relationship. 8. Variations in intracellular pH may provide a useful tool for the study of the functional role of direct cell to cell communication in both adult organs and early embryos. PMID:6770084

  13. Ca2+ signalling and early embryonic patterning during zebrafish development.

    PubMed

    Webb, Sarah E; Miller, Andrew L

    2007-09-01

    1. It has been proposed that Ca2+ signalling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern-forming events during early vertebrate development. 2. With reference to the embryo of the zebrafish (Danio rerio), herein we review the Ca2+ transients reported from the cleavage to segmentation periods. This time-window includes most of the major pattern-forming events of early development, which transform a single-cell zygote into a complex multicellular embryo with established primary germ layers and body axes. 3. Data are presented to support our proposal that intracellular Ca2+ waves are an essential feature of embryonic cytokinesis and that propagating intercellular Ca2+ waves (both long and short range) may play a crucial role in: (i) the establishment of the embryonic periderm and the coordination of cell movements during epiboly, convergence and extension; (ii) the establishment of the basic embryonic axes and germ layers; and (iii) definition of the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen. 4. The potential downstream targets of these Ca2+ transients are also discussed, as well as how they may integrate with other pattern-forming signalling pathways known to modulate early developmental events.

  14. Building the backbone: the development and evolution of vertebral patterning.

    PubMed

    Fleming, Angeleen; Kishida, Marcia G; Kimmel, Charles B; Keynes, Roger J

    2015-05-15

    The segmented vertebral column comprises a repeat series of vertebrae, each consisting of two key components: the vertebral body (or centrum) and the vertebral arches. Despite being a defining feature of the vertebrates, much remains to be understood about vertebral development and evolution. Particular controversy surrounds whether vertebral component structures are homologous across vertebrates, how somite and vertebral patterning are connected, and the developmental origin of vertebral bone-mineralizing cells. Here, we assemble evidence from ichthyologists, palaeontologists and developmental biologists to consider these issues. Vertebral arch elements were present in early stem vertebrates, whereas centra arose later. We argue that centra are homologous among jawed vertebrates, and review evidence in teleosts that the notochord plays an instructive role in segmental patterning, alongside the somites, and contributes to mineralization. By clarifying the evolutionary relationship between centra and arches, and their varying modes of skeletal mineralization, we can better appreciate the detailed mechanisms that regulate and diversify vertebral patterning. © 2015. Published by The Company of Biologists Ltd.

  15. Role of the phagocytes on embryos: some morphological aspects.

    PubMed

    Da Silva, José Roberto Machado Cunha

    2002-06-15

    Phagocytosis in embryos was studied by Elie Metchnikoff more than a century ago and is a pillar of the Phagocytic Theory. Throughout the last three decades phagocytosis in embryos has been studied from different perspectives, which this review describes and analyzes. The following branches were identified: 1) the search for the origin and first identification of well-known adult phagocytes in embryos, including their role after induced injuries; 2) the search for the occurrence of phagocytosis in embryos and its role during their physiological development; and 3) the search for phagocytosis in embryos, as a tool to study identity and self-recognition. It is possible to verify that different cell types are able to undertake phagocytosis, under a variety of different stimuli, and that the nature of what is phagocytosed also varies widely. Although the overwhelming majority of species described among metazoarians are invertebrates, most published articles in this field relate to mammals (particularly mice and humans) and birds (particularly chicks). In order to enrich this field of knowledge, research using a wider variety of vertebrate and invertebrate species should be undertaken. Furthermore, the present knowledge of phagocytosis in embryos needs a revised paradigm capable of embracing all the above-mentioned research trends under a single, more general, biological theory. In this sense, Metchnikoff's Phagocytic Theory, which is based on a broad biological paradigm and is thus capable of dealing with all research trends mentioned herein, should be revisited in order to contribute to this edification. Copyright 2002 Wiley-Liss, Inc.

  16. Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos.

    PubMed

    Tao, T; Reichelt, B; Niemann, H

    1995-07-01

    Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos.

    PubMed

    Sussarellu, Rossana; Lebreton, Morgane; Rouxel, Julien; Akcha, Farida; Rivière, Guillaume

    2018-03-01

    Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development. For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 μg L -1 Cu 2+ ) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR. A significant increase in larval abnormalities was observed from copper concentrations of 10 μg L -1 , while significant genotoxic effects were detected at 1 μg L -1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn't show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such

  18. Macroevolutionary developmental biology: Embryos, fossils, and phylogenies.

    PubMed

    Organ, Chris L; Cooper, Lisa Noelle; Hieronymus, Tobin L

    2015-10-01

    The field of evolutionary developmental biology is broadly focused on identifying the genetic and developmental mechanisms underlying morphological diversity. Connecting the genotype with the phenotype means that evo-devo research often considers a wide range of evidence, from genetics and morphology to fossils. In this commentary, we provide an overview and framework for integrating fossil ontogenetic data with developmental data using phylogenetic comparative methods to test macroevolutionary hypotheses. We survey the vertebrate fossil record of preserved embryos and discuss how phylogenetic comparative methods can integrate data from developmental genetics and paleontology. Fossil embryos provide limited, yet critical, developmental data from deep time. They help constrain when developmental innovations first appeared during the history of life and also reveal the order in which related morphologies evolved. Phylogenetic comparative methods provide a powerful statistical approach that allows evo-devo researchers to infer the presence of nonpreserved developmental traits in fossil species and to detect discordant evolutionary patterns and processes across levels of biological organization. © 2015 Wiley Periodicals, Inc.

  19. Early patterning in a chondrichthyan model, the small spotted dogfish: towards the gnathostome ancestral state

    PubMed Central

    Godard, B G; Mazan, S

    2013-01-01

    In the past few years, the small spotted dogfish has become the primary model for analyses of early development in chondrichthyans. Its phylogenetic position makes it an ideal outgroup to reconstruct the ancestral gnathostome state by comparisons with established vertebrate model organisms. It is also a suitable model to address the molecular bases of lineage-specific diversifications such as the rise of extraembryonic tissues, as it is endowed with a distinct extraembryonic yolk sac and yolk duct ensuring exchanges between the embryo and a large undivided vitelline mass. Experimental or functional approaches such as cell marking or in ovo pharmacological treatments are emerging in this species, but recent analyses of early development in this species have primarily concentrated on molecular descriptions. These data show the dogfish embryo exhibits early polarities reflecting the dorso-ventral axis of amphibians and teleosts at early blastula stages and an atypical anamniote molecular pattern during gastrulation, independently of the presence of extraembryonic tissues. They also highlight unexpected relationships with amniotes, with a strikingly similar Nodal-dependent regional pattern in the extraembryonic endoderm. In this species, extraembryonic cell fates seem to be determined by differential cell behaviors, which lead to cell allocation in extraembryonic and embryonic tissues, rather than by cell regional identity. We suggest that this may exemplify an early evolutionary step in the rise of extraembryonic tissues, possibly related to quantitative differences in the signaling activities, which shape the early embryo. These results highlight the conservation across gnathostomes of a highly constrained core genetic program controlling early patterning. This conservation may be obscured in some lineages by taxa-specific diversifications such as specializations of extraembryonic nutritive tissues. PMID:22905913

  20. Localization of Proliferating Cells in the Inter-Vertebral Region of the Developing and Adult Vertebrae of Lizards in Relation to Growth and Regeneration.

    PubMed

    Alibardi, Lorenzo

    2016-04-01

    New cartilaginous tissues in lizards is formed during the regeneration of the tail or after vertebral damage. In order to understand the origin of new cartilaginous cells in the embryo and after injury of adult vertebrae we have studied the distribution of proliferating cartilaginous cells in the vertebral column of embryos and adults of the lizard Anolis lineatopus using autoradiography for H3-thymidine and light and ultrastructural immunocytochemistry for 5BrdU. Proliferating sclerotomal cells initially surround the notochord in a segmental pattern and give rise to the chondrocytes of the vertebral centrum that replace the original chordal cells. Qualitative observations show that proliferating sclerotomal cells dilute the labeling up to 13 days post-injection but a few maintain the labeling as long labeling retention cells and remain in the inter-centra and perichondrium after birth. These cells supply new chondroblasts for post-natal growth of vertebrae but can also proliferate in case of vertebral damage or tail amputation in lizards, a process that sustains tail regeneration. The lack of somitic organization in the regenerating tail impedes the re-formation of a segmental vertebral column that is instead replaced by a continuous cartilaginous tube. It is hypothesized that long labeling retaining cells might represent stem/primordial cells, and that their permanence in the inter-vertebral cartilages and the nearby perichondrium in adult lizards pre-adapt these reptiles to elicit a broad cartilage regeneration in case of injury of the vertebrae. © 2016 Wiley Periodicals, Inc.

  1. Axial elongation in mouse embryos involves mediolateral cell intercalation behavior in the paraxial mesoderm

    NASA Astrophysics Data System (ADS)

    Yen, WeiWei; Burdsal, Carol; Periasamy, Ammasi; Sutherland, Ann E.

    2006-02-01

    The cell mechanical and signaling pathways involved in gastrulation have been studied extensively in invertebrates and amphibians, such as Xenopus, and more recently in non-mammalian vertebrates such as zebrafish and chick. However, because culturing mouse embryos extra-utero is very difficult, this fundamental process has been least characterized in the mouse. As the primary mammalian model for genetics, biochemistry, and the study of human disease and birth defects, it is important to investigate how gastrulation proceeds in murine embryos. We have developed a method of using 4D multiphoton excitation microscopy and extra-utero culture to visualize and characterize the morphogenetic movements in mouse embryos dissected at 8.5 days of gestation. Cells are labeled by expression of an X chromosome-linked enhanced green fluorescent protein (EGFP) transgene. This method has provided a unique approach, where, for the first time, patterns of cell behavior in the notochord and surrounding paraxial mesoderm can be visualized and traced quantitatively. Our observations of mouse embryos reveal both distinct differences as well as striking similarities in patterned cell motility relative to other vertebrate models such as Xenopus, where axial extension is driven primarily by mediolateral oriented cell behaviors in the notochord and paraxial somitic mesoderm. Unlike Xenopus, the width of the mouse notochord remains the same between 4-somite stage and 8-somite stage embryos. This implies the mouse notochord plays a lesser role in driving axial extension compared to Xenopus, although intercalation may occur where the anterior region of the node becomes notochordal plate. In contrast, the width of mouse paraxial mesoderm narrows significantly during this period and cells within the paraxial mesoderm are both elongated and aligned perpendicular to the midline. In addition, these cells are observed to intercalate, consistent with a role for paraxial mesoderm in driving convergence

  2. Ethanol impedes embryo transport and impairs oviduct epithelium.

    PubMed

    Xu, Tonghui; Yang, Qiuhong; Liu, Ruoxi; Wang, Wenfu; Wang, Shuanglian; Liu, Chuanyong; Li, Jingxin

    2016-05-16

    Most studies have demonstrated that alcohol consumption is associated with decreased fertility. The aim of this study was to investigate the effects of alcohol on pre-implantation embryo transport and/or early embryo development in the oviduct. We reported here that ethanol concentration-dependently suppressed the spontaneous motility of isolated human oviduct strips (EC50 50±6mM), which was largely attenuated in the present of L-NAME, a classical nitric oxide synthase(NOS) competitive inhibitor. Notably, either acute or chronic alcohol intake delayed egg transport and retarded early development of the embryo in the mouse oviduct, which was largely rescued by co-administration of L-NAME in a acute alcohol intake group but not in chronic alcohol intake group. It is worth mentioning that the oviductal epithelium destruction was verified by scanning electron microscope (SEM) observations in chronic alcohol intake group. In conclusion, alcohol intake delayed egg transport and retarded early development of the embryo in the oviduct by suppressing the spontaneous motility of oviduct and/or impairing oviductal epithelium. These findings suggested that alcohol abuse increases the incident of ectopic pregnancy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Extracellular matrix dynamics during vertebrate axis formation.

    PubMed

    Czirók, András; Rongish, Brenda J; Little, Charles D

    2004-04-01

    The first evidence for the dynamics of in vivo extracellular matrix (ECM) pattern formation during embryogenesis is presented below. Fibrillin 2 filaments were tracked for 12 h throughout the avian intraembryonic mesoderm using automated light microscopy and algorithms of our design. The data show that these ECM filaments have a reproducible morphogenic destiny that is characterized by directed transport. Fibrillin 2 particles initially deposited in the segmental plate mesoderm are translocated along an unexpected trajectory where they eventually polymerize into an intricate scaffold of cables parallel to the anterior-posterior axis. The cables coalesce near the midline before the appearance of the next-formed somite. Moreover, the ECM filaments define global tissue movements with high precision because the filaments act as passive motion tracers. Quantification of individual and collective filament "behaviors" establish fate maps, trajectories, and velocities. These data reveal a caudally propagating traveling wave pattern in the morphogenetic movements of early axis formation. We conjecture that within vertebrate embryos, long-range mechanical tension fields are coupled to both large-scale patterning and local organization of the ECM. Thus, physical forces or stress fields are essential requirements for executing an emergent developmental pattern-in this case, paraxial fibrillin cable assembly.

  4. The effects of the early uterine environment on the subsequent development of embryo and fetus.

    PubMed

    Barnes, F L

    2000-01-15

    Synchrony between the embryo and the uterine endometrium is essential for the establishment of pregnancy and birth in people and livestock. When asynchronous conditions occur a variety of complication result that include failure of the embryo to implant, early embryonic mortality, retarded development and growth, and accelerated development and growth. These complications all appear to be induced within the first week of embryo development and not withstanding the immediate endpoint of large or small size at birth, may alter the course of development throughout the life of the animal. Progesterone appears to play a causative role in establishing the abnormal growth of the fetus by decelerating or accelerating embryonic development. This may act through increasing the transport of blood born growth factors into the uterine lumen or by stimulating the release of growth factors from the endometrium directly. It can not be ruled out that progesterone mediated abundance of, or absence of, appropriate nutrition may bring about the same lifelong outcome. In vitro culture situations that include serum and/or co-culture can also bring about these abnormalities of growth. It is hypothesized that exposure to growth factors "out of phase" may result in an irreversible induction of abnormal development. The described abnormalities that occur in sheep and cattle have not yet been described for children resulting from IVF.

  5. Stress Induces AMP-Dependent Loss of Potency Factors Id2 and Cdx2 in Early Embryos and Stem Cells

    PubMed Central

    Xie, Yufen; Awonuga, Awoniyi; Liu, Jian; Rings, Edmond; Puscheck, Elizabeth Ella

    2013-01-01

    The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5 h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo. PMID:23316940

  6. The flipflop orphan genes are required for limb bud eversion in the Tribolium embryo.

    PubMed

    Thümecke, Susanne; Beermann, Anke; Klingler, Martin; Schröder, Reinhard

    2017-01-01

    Unlike Drosophila but similar to other arthropod and vertebrate embryos, the flour beetle Tribolium castaneum develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia are only poorly understood. Here we show that the newly discovered genes Tc-flipflop1 and Tc-flipflop2 are involved in regulating the directional budding of appendages. RNAi-knockdown of Tc-flipflop results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The Tc-flipflop genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that Tc-RhoGEF2 , a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a Tc-flipflop -like RNAi-phenotype. The similarity of the inverted appendage phenotype in both the flipflop - and the RhoGEF2 RNAi gene knockdown led us to conclude that the Tc-flipflop orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.

  7. P-TEFb, the Super Elongation Complex and Mediator Regulate a Subset of Non-paused Genes during Early Drosophila Embryo Development

    PubMed Central

    Dahlberg, Olle; Shilkova, Olga; Tang, Min; Holmqvist, Per-Henrik; Mannervik, Mattias

    2015-01-01

    Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos. PMID:25679530

  8. Vertebrate sex-determining genes play musical chairs

    PubMed Central

    Pan, Qiaowei; Anderson, Jennifer; Bertho, Sylvain; Herpin, Amaury; Wilson, Catherine; Postlethwait, John H.; Schartl, Manfred; Guiguen, Yann

    2017-01-01

    Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome. PMID:27291506

  9. Expression of phosphatidylcholine biosynthetic enzymes during early embryogenesis in the amphibian Bufo arenarum.

    PubMed

    Fernández-Bussy, Rodrigo; Mouguelar, Valeria; Banchio, Claudia; Coux, Gabriela

    2015-04-01

    In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.

  10. Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

    PubMed Central

    McConnell, Sean C.; Hernandez, Kyle M.; Wcisel, Dustin J.; Kettleborough, Ross N.; Stemple, Derek L.; Andrade, Jorge; de Jong, Jill L. O.

    2016-01-01

    Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e. We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates. PMID:27493218

  11. Optical tracking of embryonic vertebrates behavioural responses using automated time-resolved video-microscopy system

    NASA Astrophysics Data System (ADS)

    Walpitagama, Milanga; Kaslin, Jan; Nugegoda, Dayanthi; Wlodkowic, Donald

    2016-12-01

    The fish embryo toxicity (FET) biotest performed on embryos of zebrafish (Danio rerio) has gained significant popularity as a rapid and inexpensive alternative approach in chemical hazard and risk assessment. The FET was designed to evaluate acute toxicity on embryonic stages of fish exposed to the test chemical. The current standard, similar to most traditional methods for evaluating aquatic toxicity provides, however, little understanding of effects of environmentally relevant concentrations of chemical stressors. We postulate that significant environmental effects such as altered motor functions, physiological alterations reflected in heart rate, effects on development and reproduction can occur at sub-lethal concentrations well below than LC10. Behavioral studies can, therefore, provide a valuable integrative link between physiological and ecological effects. Despite the advantages of behavioral analysis development of behavioral toxicity, biotests is greatly hampered by the lack of dedicated laboratory automation, in particular, user-friendly and automated video microscopy systems. In this work we present a proof-of-concept development of an optical system capable of tracking embryonic vertebrates behavioral responses using automated and vastly miniaturized time-resolved video-microscopy. We have employed miniaturized CMOS cameras to perform high definition video recording and analysis of earliest vertebrate behavioral responses. The main objective was to develop a biocompatible embryo positioning structures that were suitable for high-throughput imaging as well as video capture and video analysis algorithms. This system should support the development of sub-lethal and behavioral markers for accelerated environmental monitoring.

  12. Toxicity of glyphosate as Glypro and LI700 to red-eared slider (trachemys scripta elegans) embryos and early hatchlings.

    PubMed

    Sparling, Donald W; Matson, Cole; Bickham, John; Doelling-Brown, Paige

    2006-10-01

    More than 8.2 billion ha of cropland, gardens, and forests are treated with the herbicide glyphosate each year. Whereas the toxicity of glyphosate and associated adjuvants has been measured in other vertebrates, few, if any, studies have looked at their effects in reptiles. In some instances, management of turtle habitat requires control of successional stages through application of herbicides. Adults and juvenile turtles may be exposed directly, whereas embryos may contact the chemicals through the soil. In the present study, we exposed eggs of red-eared sliders (Trachemys scripta elegans) to single applications of herbicide ranging from 0 to 11,206 ppm wet weight of glyphosate in Glypro and 0 to 678 ppm of the surfactant, LI700. Hatching success at the highest concentration was significantly lower (73%) than in other treatments (80-100%). At hatch, turtles at the highest concentration weighed less than those at other concentrations. During a 14-d holding period, we observed dose-response relationships in the ability of hatchlings to right themselves when turned on their backs. At the end of the holding period, hatchlings at the highest dose level were still lighter, and somatic indices were lower, than those in other treatments. Genetic damage, as measured by flow cytometry, increased with treatment concentration except for the highest dose. We conclude that because of the high concentrations needed to produce effects and the protection offered by several centimeters of soil or sediment, glyphosate with LI700 poses low levels of risk to red-eared slider embryos under normal field operations with regards to the endpoints measured in the present study. Carelessness in handling glyphosate or failure to follow label directions may produce adverse effects. There also is a risk that the health of turtle embryos may be affected in ways not measured in the present study.

  13. [Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

    PubMed

    Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

    2008-10-14

    To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

  14. The immunologic and antioxidant effects of L-phenylalanine on the uterine implantation of mice embryos during early pregnancy.

    PubMed

    Dong, Yulan; Bai, Yongping; Liu, Guanhui; Wang, Zixu; Cao, Jing; Chen, Yaoxing; Yang, Hongliang

    2014-10-01

    L-phenylalanine (L-PHE) is a synthetic precursor of catecholamines. Because it cannot be synthesised by an organism, it must be absorbed from the environment. Despite the wide use of L-PHE, whether L-PHE has a negative impact on embryo implantation and development is poorly understood. This study attempted to determine the roles of L-PHE in embryo implantation and development and in the immune response and antioxidant status of the uterus in early pregnancy mice injected intraperitoneally with 320 mg/kg L-PHE. The embryo number of treated mice decreased by 57.6%, and the size of their embryos was reduced by 2.8% (P⟩0.05) along the long diameter and 11.9% (P⟨0.05) along the short diameter at E9 compared with control mice. In addition, L-PHE significantly suppressed B lymphocyte proliferation. L-PHE increased IL-2 secretion but decreased the IL-4 concentration, thereby up-regulating the ratio of IL-2/IL-4 to 1.37-8.45. An analysis of the oxidant and antioxidant status showed that, compared with the control mice, the level of superoxide dismutase activity decreased by 21.54-39.94% and the glutathione peroxidase activity decreased by 15.27-18.96% among the L-PHE-treated mice at E1-E9. However, the malonaldehyde content increased by 14.29%-90.11% among the L-PHE-treated mice. Therefore, L-PHE impaired embryo implantation by disrupting cytokine-based immunity and oxidative stress in the uterus.

  15. Transcriptome analysis of PCOS arrested 2-cell embryos.

    PubMed

    Lu, Cuiling; Chi, Hongbin; Wang, Yapeng; Feng, Xue; Wang, Lina; Huang, Shuo; Yan, Liying; Lin, Shengli; Liu, Ping; Qiao, Jie

    2018-06-18

    In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos. A total of 49 differently expressed genes (DEGs) were annotated with GO terms in the up-regulated genes between PCOS arrested and non-PCOS arrested embryos after GO enrichment. A total of 29 DEGs were annotated with GO terms in the down-regulated genes between PCOS arrested and non-arrested 2-cell embryos after GO enrichment. These data can provide a reference for screening specific genes involved in the arrest of PCOS embryos.

  16. A homeobox gene involved in node, notochord and neural plate formation of chick embryos.

    PubMed

    Stein, S; Kessel, M

    1995-01-01

    We have isolated a chicken cDNA clone, Cnot, resembling in sequence and expression pattern the Xenopus homeobox gene Xnot. The major, early transcription domains of Cnot are the node, the notochord and prenodal and postnodal neural plate caudal from the prospective hindbrain level. All these cell populations appear to be descendants of the Cnot-expressing cells of the node, suggesting a cell lineage relationship. After the onset of somitogenesis, a second, independent expression domain appears in the neural folds at the prospective mid- and forebrain levels, and further transcripts are found in the epiphysis, the ventral diencephalon, the preoral gut and the limb buds. Transplantation of nodes from extended streak embryos leads to the formation of ectopic notochords, which express Cnot in the typical, cranially decreasing gradient. Transplantation of young nodes to young hosts has previously been described to induce secondary embryos. We observed that secondary chick embryos express Cnot in node derived, notochord-like structures and in the anterior neural plate, similar to the domains seen in primary embryos. However, expression was absent from the posterior neural plate, which in the induction experiments is excluded from the node lineage. This finding corroborates our initial conclusion about a cell lineage relationship between node, notochord, and neural plate defined by Cnot expression. The midline mesoderm of vertebrate embryos consists of two tissues, the prechordal mesoderm and the notochord. The anterior notochord, the head process, may represent an intermediate form. The transition from prechordal to chordal mesoderm can be followed by the expression of the two marker homeobox genes goosecoid and Cnot, first in the primitive streak, and then in the head process. We suggest that expression of goosecoid or Cnot is involved in the specification of a prechordal or notochordal identity, respectively. A transition from goosecoid to Cnot expression may proceed

  17. Spatial distribution of the capacity to initiate a secondary embryo in the 32-cell embryo of Xenopus laevis.

    PubMed

    Kageura, H

    1990-12-01

    To examine the spatial distribution of dorsal determinants in the early embryos of Xenopus laevis, individual cells from the 32-cell embryo were transplanted into the same tier of the ventral side of a synchronous recipient. Their abilities to initiate a secondary embryo were measured by the incidence of secondary embryos and by the length of the secondary axis relative to the primary embryo. The ability was found to be localized in all cells (A1, B1, C1, and D1) of the dorsal most column and in the vegetal cells (C2 and D2) of the dorsolateral column. Transplanted C1 (subequatorial) cells caused the highest incidence of a secondary embryo and the average relative length of the secondary embryo was also greatest. Effectiveness decreased in the order: D1, B1, D2, C2, and A1. When these results were compared with Dale and Slack's fate map of the 32-cell embryo, it was concluded that the distribution of dorsal determinants is unique and does not coincide with the prospective regions for any tissues, though it is somewhat similar to the prospective region of dorsal endoderm or notochord. From these results it seems that dorsal determinants do not determine a particular tissue in an embryo but rather the "dorsal" region of an embryo.

  18. Dynamic expression of the LAP family of genes during early development of Xenopus tropicalis.

    PubMed

    Yang, Qiutan; Lv, Xiaoyan; Kong, Qinghua; Li, Chaocui; Zhou, Qin; Mao, Bingyu

    2011-10-01

    The leucine-rich repeats and PDZ (LAP) family of genes are crucial for the maintenance of cell polarity as well as for epithelial homeostasis and tumor suppression in both vertebrates and invertebrates. Four members of this gene family are known: densin, erbin, scribble and lano. Here, we identified the four members of the LAP gene family in Xenopus tropicalis and studied their expression patterns during embryonic development. The Xenopus LAP proteins show a conserved domain structure that is similar to their homologs in other vertebrates. In Xenopus embryos, these genes were detected in animal cap cells at the early gastrula stage. At later stages of development, they were widely expressed in epithelial tissues that are highly polar in nature, including the neural epithelia, optic and otic vesicles, and in the pronephros. These data suggest that the roles of the Xenopus LAP genes in the control of cell polarity and morphogenesis are conserved during early development. Erbin and lano show similar expression patterns in the developing head, suggesting potential functional interactions between the two molecules in vivo.

  19. Early life sensory ability-ventilatory responses of thornback ray embryos (Raja clavata) to predator-type electric fields.

    PubMed

    Ball, Rachel Emma; Oliver, Matthew Kenneth; Gill, Andrew Bruce

    2016-07-01

    Predator avoidance is fundamental for survival and it can be particularly challenging for prey animals if physical movement away from a predatory threat is restricted. Many sharks and rays begin life within an egg capsule that is attached to the sea bed. The vulnerability of this sedentary life stage is exacerbated in skates (Rajidae) as the compulsory ventilatory activity of embryos makes them conspicuous to potential predators. Embryos can reduce this risk by mediating ventilatory activity if they detect the presence of a predator using an acute electrosense. To determine how early in embryonic life predator elicited behavioral responses can occur, the reactions of three different age groups (1/3 developed, 2/3 developed, and near hatching) of embryonic thornback rays Raja clavata were tested using predator-type electric field stimuli. Egg capsules were exposed to continuous or intermittent stimuli in order to assess varying predator-type encounter scenarios on the ventilatory behavior of different developmental stages. All embryos reacted with a "freeze response" following initial electric field (E-field) exposure, ceasing ventilatory behavior in response to predator presence, demonstrating electroreceptive functionality for the first time at the earliest possible stage in ontogeny. This ability coincided with the onset of egg ventilatory behavior and may represent an effective means to enhance survival. A continuous application of stimuli over time revealed that embryos can adapt their behavior and resume normal activity, whereas when presented intermittently, the E-field resulted in a significant reduction in overall ventilatory activity across all ages. Recovery from stimuli was significantly quicker in older embryos, potentially indicative of the trade-off between avoiding predation and adequate respiration. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 721-729, 2016. © 2015 Wiley Periodicals, Inc.

  20. Deep cytoplasmic rearrangements in ventralized Xenopus embryos

    NASA Technical Reports Server (NTRS)

    Brown, E. E.; Denegre, J. M.; Danilchik, M. V.

    1993-01-01

    Following fertilization in Xenopus, dramatic rearrangements of the egg cytoplasm relocalize maternally synthesized egg components. During the first cell cycle the vegetal yolk mass rotates relative to the egg surface, toward the sperm entry point (SEP) (J. P. Vincent, G. F. Oster, and J. C. Gerhart, 1986, Dev. Biol. 113, 484-500), while concomitant deep cytoplasmic rearrangements occur in the animal hemisphere (M. V. Danilchik and J. M. Denegre, 1991, Development 111, 845-856). In this paper we examine the role of vegetal yolk mass rotation in producing the animal cytoplasmic rearrangements. We inhibited rotation by uv-irradiating embryos during the first cell cycle, a treatment that yields an extremely ventralized phenotype. Both uv-irradiated embryos and unirradiated control embryos show cytoplasmic rearrangements in the animal hemisphere during the first cell cycle. Cytoplasmic rearrangements on the SEP side of the embryo associated with the path of the sperm pronucleus, plus a swirl on the anti-SEP (dorsal) side, are seen, whether or not yolk mass rotation has occurred. This result suggests a role for the expanding sperm aster in directing animal hemisphere cytoplasmic movements. In unirradiated control embryos the anti-SEP (dorsal) swirl is larger than that in uv-irradiated embryos and often extends into the vegetal hemisphere, consistent with the animal cytoplasm having been pulled dorsally and vegetally by the sliding vegetal yolk mass. Thus the yolk mass rotation may normally enhance the dorsalward cytoplasmic movement, begun by the sperm aster, enough to induce normal axis formation. We extended our observations of unirradiated control and uv-irradiated embryos through early cleavages. The vegetal extent of the anti-SEP (dorsal) swirl pattern seen in control embryos persists through the early cleavage period, such that labeled animal cytoplasm extends deep into dorsal third-tier blastomeres at the 32-cell stage. Significantly, in uv-irradiated embryos

  1. Regulation of early Xenopus development by ErbB signaling

    PubMed Central

    Nie, Shuyi; Chang, Chenbei

    2008-01-01

    ErbB signaling has long been implicated in cancer formation and progression and is shown to regulate cell division, migration and death during tumorigenesis. The functions of the ErbB pathway during early vertebrate embryogenesis, however, are not well understood. Here we report characterization of ErbB activities during early frog development. Gain-of-function analyses show that EGFR, ErbB2 and ErbB4 induce ectopic tumor-like cell mass that contains increased numbers of mitotic cells. Both the muscle and the neural markers are expressed in these ectopic protrusions. ErbBs also induce mesodermal markers in ectodermal explants. Loss-of-function studies using carboxyl terminal-truncated dominant-negative ErbB receptors demonstrate that blocking ErbB signals leads to defective gastrulation movements and malformation of the embryonic axis with a reduction in the head structures in early frog embryos. These data, together with the observation that ErbBs are expressed early during frog embryogenesis, suggest that ErbBs regulate cell proliferation, movements and embryonic patterning during early Xenopus development. PMID:16258939

  2. Climatic factors affecting quantity and quality grade of in vivo derived embryos of cattle.

    PubMed

    Chinchilla-Vargas, Josué; Jahnke, Marianna M; Dohlman, Tyler M; Rothschild, Max F; Gunn, Patrick J

    2018-05-01

    The present study investigated the effects of climatic variables on the quality grade and quantity of in vivo derived cattle embryos in the Midwestern United States. Climatic information included greatest and least daily temperature, average daily wind speed and average temperature-humidity index for each of the 765 records. The response variables included the number of ovarian structures, viable embryos, quality grade 1 embryos, quality grade 2 embryos, quality grade 3 embryos, freezable embryos (sum of quality grade 1 and quality grade 2 embryos), transferable embryos (sum of quality grade 1-3 embryos), degenerate embryos and unfertilized ova. Measures for variables among the breeds of donors and sires grouped by geographical origin were compared. A negative effect of greater temperatures during the early embryonic development stage tended (P < 0.10) to be associated with a decrease in the quality of embryos recovered. Interestingly, the greater the Temperature-Humidity Index (THI) during the early ovarian antral follicular development stage 40-45 days prior to ovulation was associated with a tendency for greater numbers of total number of freezable and transferable embryos recovered per uterine flushing (P < 0.10). Increased wind speed at the early antral follicular phase 40-45 days prior to ovulation was associated with an increase in the percentage of quality grade 1 embryos recovered (P < 0.05). Wind speed during the estrous synchronization period was also associated with a lesser number of embryos recovered (P < 0.05). This retrospective study confirms that climatic variables have significant effects on the in vivo production of cattle embryos and that wind speed should be considered in future analyses of factors affecting embryo quality. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Enhance beef cattle improvement by embryo biotechnologies.

    PubMed

    Wu, B; Zan, L

    2012-10-01

    Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer (in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. © 2011 Blackwell Verlag GmbH.

  4. Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo.

    PubMed Central

    Blangy, A; Léopold, P; Vidal, F; Rassoulzadegan, M; Cuzin, F

    1991-01-01

    We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development. Images PMID:1766880

  5. Toxicity and cardiac effects of carbaryl in early developing zebrafish (Danio rerio) embryos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lin, C.C.; Hui, Michelle N.Y.; Cheng, S.H. E-mail: bhcheng@cityu.edu.hk

    2007-07-15

    Carbaryl, an acetylcholinesterase inhibitor, is known to be moderately toxic to adult zebrafish and has been reported to cause heart malformations and irregular heartbeat in medaka. We performed experiments to study the toxicity of carbaryl, specifically its effects on the heart, in early developing zebrafish embryos. LC50 and EC50 values for carbaryl at 28 h post-fertilization were 44.66 {mu}g/ml and 7.52 {mu}g/ml, respectively, and 10 {mu}g/ml carbaryl was used in subsequent experiments. After confirming acetylcholinesterase inhibition by carbaryl using an enzymatic method, we observed red blood cell accumulation, delayed hatching and pericardial edema, but not heart malformation as described inmore » some previous reports. Our chronic exposure data also demonstrated carbaryl-induced bradycardia, which is a common effect of acetylcholinesterase inhibitors due to the accumulation of acetylcholine, in embryos from 1 day post-fertilization (dpf) to 5 dpf. The distance between the sinus venosus, the point where blood enters the atrium, and the bulbus arteriosus, the point where blood leaves the ventricle, indicated normal looping of the heart tube. Immunostaining of myosin heavy chains with the ventricle-specific antibody MF20 and the atrium-specific antibody S46 showed normal development of heart chambers. At the same time, acute exposure resulted in carbaryl-induced bradycardia. Heart rate dropped significantly after a 10-min exposure to 100 {mu}g/ml carbaryl but recovered when carbaryl was removed. The novel observation of carbaryl-induced bradycardia in 1- and 2-dpf embryos suggested that carbaryl affected cardiac function possibly through an alternative mechanism other than acetylcholinesterase inhibition such as inhibition of calcium ion channels, since acetylcholine receptors in zebrafish are not functional until 3 dpf. However, the exact nature of this mechanism is currently unknown, and thus further studies are required.« less

  6. Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos

    PubMed Central

    Gupta, Prabuddha; Martin, René; Knölker, Hans-Joachim; Nihalani, Deepak; Kumar Sinha, Deepak

    2017-01-01

    Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis. PMID:28678859

  7. Complementary Gli activity mediates early patterning of the mouse visual system.

    PubMed

    Furimsky, Marosh; Wallace, Valerie A

    2006-03-01

    The Sonic hedgehog (Shh) signaling pathway plays a key role in the development of the vertebrate central nervous system, including the eye. This pathway is mediated by the Gli transcription factors (Gli1, Gli2, and Gli3) that differentially activate and repress the expression of specific downstream target genes. In this study, we investigated the roles of the three vertebrate Glis in mediating midline Shh signaling in early ocular development. We examined the ocular phenotypes of Shh and Gli combination mutant mouse embryos and monitored proximodistal and dorsoventral patterning by the expression of specific eye development regulatory genes using in situ hybridization. We show that midline Shh signaling relieves the repressor activity of Gli3 adjacent to the midline and then promotes eye pattern formation through the nonredundant activities of all three Gli proteins. Gli3, in particular, is required to specify the dorsal optic stalk and to define the boundary between the optic stalk and the optic cup.

  8. A right-handed signalling pathway drives heart looping in vertebrates

    PubMed Central

    Ocaña, Oscar H.; Coskun, Hakan; Minguillón, Carolina; Murawala, Prayag; Tanaka, Elly M.; Galcerán, Joan; Muñoz-Chapuli, Ramón; Nieto, M. Angela

    2017-01-01

    The majority of animals show external bilateral symmetry, precluding the observation of multiple internal left-right (L/R) asymmetries that are fundamental for organ packaging and function1,2. In vertebrates, left identity is mediated by the left-specific Nodal-Pitx2 axis that is repressed on the right-hand side by the epithelial-mesenchymal transition (EMT) inducer Snail13,4. Despite some existing evidence3,5, it remains unclear whether an equivalent instructive pathway provides right-hand specific information to the embryo. Here we show that in zebrafish, BMP mediates the L/R asymmetric activation of another EMT inducer, Prrx1a, in the lateral plate mesoderm (LPM) with higher levels on the right. Prrx1a drives L/R differential cell movements towards the midline leading to a leftward displacement of the cardiac posterior pole through an actomyosin-dependent mechanism. Downregulation of Prrx1a prevents heart looping and leads to mesocardia. Two parallel and mutually repressed pathways, respectively driven by Nodal and BMP on the left and right LPM, converge on the asymmetric activation of Pitx2 and Prrx1, two transcription factors that integrate left and right information to govern heart morphogenesis. This mechanism is conserved in the chicken embryo and, in the mouse, Snail1 fulfills the role played by Prrx1 in fish and chick. Thus, a differential L/R EMT produces asymmetric cell movements and forces, more prominent from the right, that drive heart laterality in vertebrates. PMID:28880281

  9. Ectoderm gene activation in sea urchin embryos mediated by the CCAAT-binding factor.

    PubMed

    Li, Xiaotao; Bhattacharya, Chitralekha; Dayal, Sandeep; Maity, Sankar; Klein, William H

    2002-05-01

    Transcriptional enhancers are short stretches of DNA that function to achieve highly specific patterns of gene expression. To identify the mechanisms by which enhancers achieve their specificity, we made use of an enhancer from the aboral ectoderm-specific spec2a gene of the sea urchin Strongylocentrotus purpuratus. The spec2a enhancer contains five cis-regulatory elements within 78 base pairs that interact with five distinct DNA-binding proteins to confer aboral ectoderm expression. Here, we present an analysis of the sea urchin CCAAT binding factor (CBF), which binds to a CCAAT motif within the spec2a enhancer. S. purpuratus CBF and SpOtx, a ubiquitously expressed factor, act together at closely placed cis-regulatory elements to mediate spec2a transcription in the ectoderm. SpCBF was the sole factor that bound to the spec2a CCAAT element, and two of the three subunits that make up the CBF holoprotein were cloned and shown to have high sequence conservation with their vertebrate orthologs. Based on its involvement in the regulation of several other sea urchin genes, SpCBF appears to be a major transcription factor in the sea urchin embryo for positive regulation of ectoderm gene expression. In addition to its role in vertebrate cell growth and proliferation, our results indicate that CBF also functions at the early stages of germ layer formation, namely ectoderm differentiation.

  10. Constrained vertebrate evolution by pleiotropic genes.

    PubMed

    Hu, Haiyang; Uesaka, Masahiro; Guo, Song; Shimai, Kotaro; Lu, Tsai-Ming; Li, Fang; Fujimoto, Satoko; Ishikawa, Masato; Liu, Shiping; Sasagawa, Yohei; Zhang, Guojie; Kuratani, Shigeru; Yu, Jr-Kai; Kusakabe, Takehiro G; Khaitovich, Philipp; Irie, Naoki

    2017-11-01

    Despite morphological diversification of chordates over 550 million years of evolution, their shared basic anatomical pattern (or 'bodyplan') remains conserved by unknown mechanisms. The developmental hourglass model attributes this to phylum-wide conserved, constrained organogenesis stages that pattern the bodyplan (the phylotype hypothesis); however, there has been no quantitative testing of this idea with a phylum-wide comparison of species. Here, based on data from early-to-late embryonic transcriptomes collected from eight chordates, we suggest that the phylotype hypothesis would be better applied to vertebrates than chordates. Furthermore, we found that vertebrates' conserved mid-embryonic developmental programmes are intensively recruited to other developmental processes, and the degree of the recruitment positively correlates with their evolutionary conservation and essentiality for normal development. Thus, we propose that the intensively recruited genetic system during vertebrates' organogenesis period imposed constraints on its diversification through pleiotropic constraints, which ultimately led to the common anatomical pattern observed in vertebrates.

  11. Studies of Weak, ELF Electromagnetic Fields Effects on the Early Embryonic Development

    DTIC Science & Technology

    1988-12-29

    characters: - General aspect: stage; size of the embryo; size of the head relatively to the trunk. - Head : morphology of the skull; development of...the eyes; size and morphology of the beak; size of the neck . - Trunk: morphology of the vertebral column; development of the tail; closure of thorax and...mishandled, the orientation of the embryo was not taken into account. As indicated in Fig. 7, the head -tail axis orientation of an embryo was North (N

  12. Vertebral architecture in the earliest stem tetrapods.

    PubMed

    Pierce, Stephanie E; Ahlberg, Per E; Hutchinson, John R; Molnar, Julia L; Sanchez, Sophie; Tafforeau, Paul; Clack, Jennifer A

    2013-02-14

    The construction of the vertebral column has been used as a key anatomical character in defining and diagnosing early tetrapod groups. Rhachitomous vertebrae--in which there is a dorsally placed neural arch and spine, an anteroventrally placed intercentrum and paired, posterodorsally placed pleurocentra--have long been considered the ancestral morphology for tetrapods. Nonetheless, very little is known about vertebral anatomy in the earliest stem tetrapods, because most specimens remain trapped in surrounding matrix, obscuring important anatomical features. Here we describe the three-dimensional vertebral architecture of the Late Devonian stem tetrapod Ichthyostega using propagation phase-contrast X-ray synchrotron microtomography. Our scans reveal a diverse array of new morphological, and associated developmental and functional, characteristics, including a possible posterior-to-anterior vertebral ossification sequence and the first evolutionary appearance of ossified sternal elements. One of the most intriguing features relates to the positional relationships between the vertebral elements, with the pleurocentra being unexpectedly sutured or fused to the intercentra that directly succeed them, indicating a 'reverse' rhachitomous design. Comparison of Ichthyostega with two other stem tetrapods, Acanthostega and Pederpes, shows that reverse rhachitomous vertebrae may be the ancestral condition for limbed vertebrates. This study fundamentally revises our current understanding of vertebral column evolution in the earliest tetrapods and raises questions about the presumed vertebral architecture of tetrapodomorph fish and later, more crownward, tetrapods.

  13. Gene Expression in Pre-MBT Embryos and Activation of Maternally-Inherited Program of Apoptosis to be Executed at around MBT as a Fail-Safe Mechanism in Xenopus Early Embryogenesis

    PubMed Central

    Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Uchiyama, Hiroaki; Kuroyanagi, Shinsaku; Takai, Jun-Ichi; Takahashi, Senji; Kajitani, Masayuki; Kaito, Chikara; Sekimizu, Kazuhisa; Takayama, Eiji; Igarashi, Kazuei; Hara, Hiroshi

    2008-01-01

    S-adenosylmethionine decarboxylase (SAMDC) is an enzyme which converts S-adenosylmethione (SAM), a methyl donor, to decarboxylated SAM (dcSAM), an aminopropyl donor for polyamine biosynthesis. In our studies on gene expression control in Xenopus early embryogenesis, we cloned the mRNA for Xenopus SAMDC, and overexpressed the enzyme by microinjecting its mRNA into Xenopus fertilized eggs. In the mRNA-injected embryos, the level of SAMDC was enormously increased, the SAM was exhausted, and protein synthesis was greatly inhibited, but cellular polyamine content did not change appreciably. SAMDC-overexpressed embryos cleaved and developed normally up to the early blastula stage, but at the midblastula stage, or the stage of midblastula transition (MBT), all the embryos were dissociated into cells, and destroyed due to execution of apoptosis. During cleavage SAMDC-overexpressed embryos transcribed caspase-8 gene, and this was followed by activation of caspase-9. When we overexpressed p53 mRNA in fertilized eggs, similar apoptosis took place at MBT, but in this case, transcription of caspase-8 did not occur, however activation of caspase-9 took place. Apoptosis induced by SAMDC-overexpression was completely suppressed by Bcl-2, whereas apoptosis induced by p53 overexpression or treatments with other toxic agents was only partially rescued. When we injected SAMDC mRNA into only one blastomere of 8- to 32-celled embryos, descendant cells of the mRNA-injected blastomere were segregated into the blastocoel and underwent apoptosis within the blastocoel, although such embryos continued to develop and became tadpoles with various extents of anomaly, reflecting the developmental fate of the eliminated cells. Thus, embryonic cells appear to check themselves at MBT and if physiologically severely-damaged cells occur, they are eliminated from the embryo by activation and execution of the maternally-inherited program of apoptosis. We assume that the apoptosis executed at MBT is a

  14. The chick embryo: a leading model in somitogenesis studies.

    PubMed

    Pourquié, Olivier

    2004-09-01

    The vertebrate body is built on a metameric organization which consists of a repetition of functionally equivalent units, each comprising a vertebra, its associated muscles, peripheral nerves and blood vessels. This periodic pattern is established during embryogenesis by the somitogenesis process. Somites are generated in a rhythmic fashion from the presomitic mesoderm and they subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somitogenesis has been very actively studied in the chick embryo since the 19th century and many of the landmark experiments that led to our current understanding of the vertebrate segmentation process have been performed in this organism. Somite formation involves an oscillator, the segmentation clock whose periodic signal is converted into the periodic array of somite boundaries by a spacing mechanism relying on a traveling threshold of FGF signaling regressing in concert with body axis extension.

  15. Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.

    PubMed

    Varela, A S; Cardoso, T F; Fernandes E Silva, E; Goularte, K L; Okamoto, M H; Sampaio, L A; Jardim, R D; Corcini, C D

    Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.

  16. RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates

    PubMed Central

    Forest, David; Nishikawa, Ryuhei; Kobayashi, Hiroshi; Parton, Angela; Bayne, Christopher J.; Barnes, David W.

    2007-01-01

    We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3′ UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3′ mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology. PMID:17227856

  17. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

    PubMed

    Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

    2013-10-01

    approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence. This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest. ClinicalTrials.gov Identifier: NCT01397136.

  18. Types of neural cells in the spinal ganglia of human embryos and early fetuses.

    PubMed

    Olszewska, B; Woźniak, W; Gardner, E; O'Rahilly, R

    1979-01-01

    Spinal ganglial of human embryos and fetuses ranging in C.-R. length from 15 to 74 mm and in age from 6 1/2 to 11 postovulatory weeks were studied by light and electron microscopy. A sequence of events in differentiation and maturation enabled five types of cells to be distinguished: 1. apolar, undifferentiated neuroblasts are the main cells at 6 1/2 to 7 1/2 weeks; 2. early bipolar neuroblasts (strictly speaking, types 2 to 5 are immature neurons) predominate at the end of the embryonic period proper (8 postovulatory weeks); 3. intermediate bipolar neuroblasts are characteristic of the early fetal period; 4. late bipolar neuroblasts, in which two proceses arise separately from one pole of the cell, appear at about 10 postovulatory weeks; 5. unipolar neuroblasts are found within another week and, by that time, cells of types 1 and 2 are no longer present.

  19. Biphasic influence of dexamethasone exposure on embryonic vertebrate skeleton development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cheng, Xin; Chen, Jian-long; Ma, Zheng-lai

    slightly increased in mesenchymal cell mass treated by low concentration of Dex. Mmp-13 expression was obviously up-regulated by Dex in both mesenchymal cells and primary chondrocyte cultures. And Col10a1 expression was also increased by Dex exposure in chondrocyte. In summary, we have revealed that different concentrations of Dex exposure during early gestation could exert a biphasic effect on vertebrate skeletal development. - Highlights: • Chick embryos occurred shortening of the long bone following Dex exposure. • Dex suppressed chondrocytes proliferation and promoted apoptosis. • Dex exposure decreased ALP production and up-regulated Runx-2 and Mmp-13. • Dex exhibited biphasic effects on chondrogenic proliferation and nodule formation. • The hypertrophy and ossification were accelerated by Dex both in vivo and in vitro.« less

  20. Vertebrate sex-determining genes play musical chairs.

    PubMed

    Pan, Qiaowei; Anderson, Jennifer; Bertho, Sylvain; Herpin, Amaury; Wilson, Catherine; Postlethwait, John H; Schartl, Manfred; Guiguen, Yann

    2016-01-01

    Sexual reproduction is one of the most highly conserved processes in evolution. However, the genetic and cellular mechanisms making the decision of whether the undifferentiated gonad of animal embryos develops either towards male or female are manifold and quite diverse. In vertebrates, sex-determining mechanisms range from environmental to simple or complex genetic mechanisms and different mechanisms have evolved repeatedly and independently. In species with simple genetic sex-determination, master sex-determining genes lying on sex chromosomes drive the gonadal differentiation process by switching on a developmental program, which ultimately leads to testicular or ovarian differentiation. So far, very few sex-determining genes have been identified in vertebrates and apart from mammals and birds, these genes are apparently not conserved over a larger number of related orders, families, genera, or even species. To fill this knowledge gap and to better explore genetic sex-determination, we propose a strategy (RAD-Sex) that makes use of next-generation sequencing technology to identify genetic markers that define sex-specific segments of the male or female genome. Copyright © 2016 Académie des sciences. All rights reserved.

  1. Spatial Anisotropies and Temporal Fluctuations in Extracellular Matrix Network Texture during Early Embryogenesis

    PubMed Central

    Loganathan, Rajprasad; Potetz, Brian R.; Rongish, Brenda J.; Little, Charles D.

    2012-01-01

    Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level, extracellular matrix (ECM) networks facilitate cell motility. At the tissue level, ECM networks provide material properties required to accommodate the large-scale deformations and forces that shape amniote embryos. In other words, the primordial biomaterial from which reptilian, avian, and mammalian embryos are molded is a dynamic composite comprised of cells and ECM. Despite its central importance during early morphogenesis we know little about the intrinsic micrometer-scale surface properties of primordial ECM networks. Here we computed, using avian embryos, five textural properties of fluorescently tagged ECM networks — (a) inertia, (b) correlation, (c) uniformity, (d) homogeneity, and (e) entropy. We analyzed fibronectin and fibrillin-2 as examples of fibrous ECM constituents. Our quantitative data demonstrated differences in the surface texture between the fibronectin and fibrillin-2 network in Day 1 (gastrulating) embryos, with the fibronectin network being relatively coarse compared to the fibrillin-2 network. Stage-specific regional anisotropy in fibronectin texture was also discovered. Relatively smooth fibronectin texture was exhibited in medial regions adjoining the primitive streak (PS) compared with the fibronectin network investing the lateral plate mesoderm (LPM), at embryonic stage 5. However, the texture differences had changed by embryonic stage 6, with the LPM fibronectin network exhibiting a relatively smooth texture compared with the medial PS-oriented network. Our data identify, and partially characterize, stage-specific regional anisotropy of fibronectin texture within tissues of a warm-blooded embryo. The data suggest that changes in ECM textural properties reflect orderly time

  2. The evolution of porcine embryo in vitro production.

    PubMed

    Grupen, Christopher G

    2014-01-01

    The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. The Armadillo Repeat Gene ZAK IXIK Promotes Arabidopsis Early Embryo and Endosperm Development through a Distinctive Gametophytic Maternal Effect[C][W][OA

    PubMed Central

    Ngo, Quy A.; Baroux, Celia; Guthörl, Daniela; Mozerov, Peter; Collinge, Margaret A.; Sundaresan, Venkatesan; Grossniklaus, Ueli

    2012-01-01

    The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin–dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of MINISEED3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways. PMID:23064319

  4. Progesterone is critical for the development of mouse embryos.

    PubMed

    Zhang, Cong; Murphy, Bruce D

    2014-08-01

    Infertility affects approximately 10-15 % of reproductive-aged couples, and embryo loss due to preimplantation death is common to many mammals. Previous studies showed that a complex series of interactive molecular events are associated with this process, especially hormones (progesterone and estrogens) and growth factors, and are important for the cleavage and differentiation of the blastocysts. Yet, the mechanism of preimplantation embryo development is unclear. Using conditional knockout mice (CKO), we showed the development of blastocyst is tightly controlled by the level of progesterone (P4); furthermore, we found that the time when P4 should increase is also crucial for the formation of blastocysts. In CKO mice whose Lrh1 (liver receptor homolog 1) is deleted under the expression of Cre recombinase driven by progesterone receptor promoter, which reduced P4 synthesis, few of their embryos can reach blastocyst stage. When these CKO mice were supplied with P4 in the afternoon of dpc 1 (day post copulation), most of the embryos can form blastocysts; when CKO mice were supplied with P4 from the morning of dpc1, one-third of the embryos can reach blastocyst stage; however, the supplement of P4 in the morning of dpc 2 made very few of the embryos become blastocysts. We conclude that early exposure to P4 is essential for timely progression of early embryogenesis in the mouse.

  5. Starch Turnover and Metabolism during Flower and Early Embryo Development1[CC-BY

    PubMed Central

    Pazmino, Diana; Gagliardini, Valeria

    2016-01-01

    The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed. PMID:27794100

  6. Sex determination of duck embryos: observations on syrinx development

    USGS Publications Warehouse

    Wilson, Robert E.; Sonsthagen, Sarah A.; Franson, J. Christian

    2013-01-01

    Ducks exhibit sexual dimorphism in vocal anatomy. Asymmetrical ossification of the syrinx (bulla syringealis) is discernable at about 10 days of age in male Pekin duck (Anas platyrhynchos domestica) embryos, but information is lacking on the early development of the bulla in wild ducks. To evaluate the reliability of this characteristic for sexing developing embryos, we examined the syrinx of dead embryos and compared results with molecular sexing techniques in high arctic nesting Common Eiders (Somateria mollissima). Embryos 8 days or older were accurately (100%) sexed based on the presence/absence of a bulla, 2 days earlier than Pekin duck. The use of the tracheal bulla can be a valuable technique when sex identification of embryos or young ducklings is required.

  7. The C. elegans homolog of Drosophila Lethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo.

    PubMed

    Beatty, Alexander; Morton, Diane; Kemphues, Kenneth

    2010-12-01

    Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.

  8. 5Alpha-Reduced Steroids Are Major Metabolites in the Early Equine Embryo Proper and Its Membranes.

    PubMed

    Raeside, James I; Christie, Heather L; Betteridge, Keith J

    2015-09-01

    Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro- and 3beta,5alpha-tetrahydro- steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta,5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta,5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids--for example, 5alpha-dihydrotestosterone in male sexual differentiation--provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in

  9. Fatal fat embolism in isolated vertebral compression fracture

    PubMed Central

    Saldanha, Vilas; Balasubramanian, Manjula; Handal, John

    2010-01-01

    Fat embolism after long bone and pelvic fractures as well as orthopedic interventions is a well-documented phenomenon, but it is highly unusual after isolated vertebral fractures. We report a case of fatal fat embolism in a 78-year-old man after an isolated vertebral compression fracture with no related orthopedic intervention. A high index of suspicion is necessary for early diagnosis and successfully treating this unusual complication. PMID:20229119

  10. Fatal fat embolism in isolated vertebral compression fracture.

    PubMed

    Lastra, Ricardo R; Saldanha, Vilas; Balasubramanian, Manjula; Handal, John

    2010-07-01

    Fat embolism after long bone and pelvic fractures as well as orthopedic interventions is a well-documented phenomenon, but it is highly unusual after isolated vertebral fractures. We report a case of fatal fat embolism in a 78-year-old man after an isolated vertebral compression fracture with no related orthopedic intervention. A high index of suspicion is necessary for early diagnosis and successfully treating this unusual complication.

  11. Embryology of Early Jurassic dinosaur from China with evidence of preserved organic remains.

    PubMed

    Reisz, Robert R; Huang, Timothy D; Roberts, Eric M; Peng, ShinRung; Sullivan, Corwin; Stein, Koen; LeBlanc, Aaron R H; Shieh, DarBin; Chang, RongSeng; Chiang, ChengCheng; Yang, Chuanwei; Zhong, Shiming

    2013-04-11

    Fossil dinosaur embryos are surprisingly rare, being almost entirely restricted to Upper Cretaceous strata that record the late stages of non-avian dinosaur evolution. Notable exceptions are the oldest known embryos from the Early Jurassic South African sauropodomorph Massospondylus and Late Jurassic embryos of a theropod from Portugal. The fact that dinosaur embryos are rare and typically enclosed in eggshells limits their availability for tissue and cellular level investigations of development. Consequently, little is known about growth patterns in dinosaur embryos, even though post-hatching ontogeny has been studied in several taxa. Here we report the discovery of an embryonic dinosaur bone bed from the Lower Jurassic of China, the oldest such occurrence in the fossil record. The embryos are similar in geological age to those of Massospondylus and are also assignable to a sauropodomorph dinosaur, probably Lufengosaurus. The preservation of numerous disarticulated skeletal elements and eggshells in this monotaxic bone bed, representing different stages of incubation and therefore derived from different nests, provides opportunities for new investigations of dinosaur embryology in a clade noted for gigantism. For example, comparisons among embryonic femora of different sizes and developmental stages reveal a consistently rapid rate of growth throughout development, possibly indicating that short incubation times were characteristic of sauropodomorphs. In addition, asymmetric radial growth of the femoral shaft and rapid expansion of the fourth trochanter suggest that embryonic muscle activation played an important role in the pre-hatching ontogeny of these dinosaurs. This discovery also provides the oldest evidence of in situ preservation of complex organic remains in a terrestrial vertebrate.

  12. A transcriptional blueprint for a spiral-cleaving embryo.

    PubMed

    Chou, Hsien-Chao; Pruitt, Margaret M; Bastin, Benjamin R; Schneider, Stephan Q

    2016-08-05

    The spiral cleavage mode of early development is utilized in over one-third of all animal phyla and generates embryonic cells of different size, position, and fate through a conserved set of stereotypic and invariant asymmetric cell divisions. Despite the widespread use of spiral cleavage, regulatory and molecular features for any spiral-cleaving embryo are largely uncharted. To address this gap we use RNA-sequencing on the spiralian model Platynereis dumerilii to capture and quantify the first complete genome-wide transcriptional landscape of early spiral cleavage. RNA-sequencing datasets from seven stages in early Platynereis development, from the zygote to the protrochophore, are described here including the de novo assembly and annotation of ~17,200 Platynereis genes. Depth and quality of the RNA-sequencing datasets allow the identification of the temporal onset and level of transcription for each annotated gene, even if the expression is restricted to a single cell. Over 4000 transcripts are maternally contributed and cleared by the end of the early spiral cleavage phase. Small early waves of zygotic expression are followed by major waves of thousands of genes, demarcating the maternal to zygotic transition shortly after the completion of spiral cleavages in this annelid species. Our comprehensive stage-specific transcriptional analysis of early embryonic stages in Platynereis elucidates the regulatory genome during early spiral embryogenesis and defines the maternal to zygotic transition in Platynereis embryos. This transcriptome assembly provides the first systems-level view of the transcriptional and regulatory landscape for a spiral-cleaving embryo.

  13. Transient Overexpression of adh8a Increases Allyl Alcohol Toxicity in Zebrafish Embryos

    PubMed Central

    Klüver, Nils; Ortmann, Julia; Paschke, Heidrun; Renner, Patrick; Ritter, Axel P.; Scholz, Stefan

    2014-01-01

    Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in

  14. Twist functions in vertebral column formation in medaka, Oryzias latipes.

    PubMed

    Yasutake, Junichi; Inohaya, Keiji; Kudo, Akira

    2004-07-01

    Medaka twist, a basic helix-loop-helix (bHLH) transcription factor, is expressed in the sclerotome during embryogenesis. We previously established a line of twist-EGFP transgenic medaka, whose EGFP expression is regulated by the twist promoter; therefore, we could observe the behavior of sclerotomal cells in vivo. In the transgenic medaka embryos, EGFP-positive sclerotomal cells migrated dorsally around the notochord and the neural tube, where at a later stage the vertebral column would be formed. This finding strongly suggests that twist-expressing sclerotomal cells participate in vertebral column formation in medaka. To clarify the function of twist gene in the sclerotome, we performed knockdown analysis of twist by using two kinds of morpholino antisense oligonucleotides targeted against twist (MO1 and MO2). Both the MO1 and MO2 morphants exhibited absence of neural arches, which are bilaterally paired, dorsomedially oriented bones on the dorsal aspect of the centrum. In addition, MO2, which blocks translation of only endogenous twist mRNA in the twist-EGFP transgenic medaka, did not affect the migration pattern of EGFP-positive cells, revealing that the migration of sclerotome-derived cells were normal in the absence of twist gene function. These results demonstrate that medaka twist functions in vertebral column formation by regulating the sclerotomal cell differentiation.

  15. Hatching, survival and deformities of piracanjuba (Brycon orbignyanus) embryos subjected to different cooling protocols.

    PubMed

    Paes, Maria do Carmo Faria; da Silva, Regiane Cristina; do Nascimento, Nivaldo Ferreira; Valentin, Fernanda Nogueira; Senhorini, José Augusto; Nakaghi, Laura Satiko Okada

    2014-12-01

    Groups of one hundred Brycon orbignyanus embryos at the stage of blastopore closure were subjected to different cooling protocols. Different combinations and concentrations of cryoprotectants were tested: sucrose, methanol, ethylene glycol and dimethyl sulfoxide (Me2SO); at different temperatures (0.0±2.0°C and 8.0±2.0°C) and refrigeration times (6, 10, 24, 72 and 168 h), with the exception of the positive control (incubation without previous cooling). At the end of each refrigeration time, the embryos were acclimatized, rehydrated and incubated to determine hatching, survival and deformity rates. Morphological analysis of embryos was also carried out. The results showed that temperature and refrigeration time are critical factors for embryo survival. No embryos survived after 24, 72 and 168 h of refrigeration. Furthermore, when the refrigeration time increased from 6 to 10h and the temperature decreased from 8.0±2.0°C to 0.0±2.0°C, mortality rates increased significantly. It was also found that in all protocols dead eggs and/or larvae with some degree of deformity were present. The main larval deformities observed were the malformation of the head, tail, yolk sac, vertebral column and eyes. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. The conserved role of Krox-20 in directing Hox gene expression during vertebrate hindbrain segmentation.

    PubMed

    Nonchev, S; Maconochie, M; Vesque, C; Aparicio, S; Ariza-McNaughton, L; Manzanares, M; Maruthainar, K; Kuroiwa, A; Brenner, S; Charnay, P; Krumlauf, R

    1996-09-03

    Transient segmentation in the hindbrain is a fundamental morphogenetic phenomenon in the vertebrate embryo, and the restricted expression of subsets of Hox genes in the developing rhombomeric units and their derivatives is linked with regional specification. Here we show that patterning of the vertebrate hindbrain involves the direct upregulation of the chicken and pufferfish group 2 paralogous genes, Hoxb-2 and Hoxa-2, in rhombomeres 3 and 5 (r3 and r5) by the zinc finger gene Krox-20. We identified evolutionarily conserved r3/r5 enhancers that contain high affinity Krox-20. binding sites capable of mediating transactivation by Krox-20. In addition to conservation of binding sites critical for Krox-20 activity in the chicken Hoxa-2 and pufferfish Hoxb-2 genes, the r3/r5 enhancers are also characterized by the presence of a number of identical motifs likely to be involved in cooperative interactions with Krox-20 during the process of hindbrain patterning in vertebrates.

  17. Terrestrial origin of viviparity in mesozoic marine reptiles indicated by early triassic embryonic fossils.

    PubMed

    Motani, Ryosuke; Jiang, Da-yong; Tintori, Andrea; Rieppel, Olivier; Chen, Guan-bao

    2014-01-01

    Viviparity in Mesozoic marine reptiles has traditionally been considered an aquatic adaptation. We report a new fossil specimen that strongly contradicts this traditional interpretation. The new specimen contains the oldest fossil embryos of Mesozoic marine reptile that are about 10 million years older than previous such records. The fossil belongs to Chaohusaurus (Reptilia, Ichthyopterygia), which is the oldest of Mesozoic marine reptiles (ca. 248 million years ago, Early Triassic). This exceptional specimen captures an articulated embryo in birth position, with its skull just emerged from the maternal pelvis. Its headfirst birth posture, which is unlikely to be a breech condition, strongly indicates a terrestrial origin of viviparity, in contrast to the traditional view. The tail-first birth posture in derived ichthyopterygians, convergent with the conditions in whales and sea cows, therefore is a secondary feature. The unequivocally marine origin of viviparity is so far not known among amniotes, a subset of vertebrate animals comprising mammals and reptiles, including birds. Therefore, obligate marine amniotes appear to have evolved almost exclusively from viviparous land ancestors. Viviparous land reptiles most likely appeared much earlier than currently thought, at least as early as the recovery phase from the end-Permian mass extinction.

  18. Breathing air in air: in what ways might extant amphibious fish biology relate to prevailing concepts about early tetrapods, the evolution of vertebrate air breathing, and the vertebrate land transition?

    PubMed

    Graham, Jeffrey B; Lee, Heather J

    2004-01-01

    The air-breathing fishes have heuristic importance as possible models for the Paleozoic evolution of vertebrate air breathing and the transition to land. A recent hypothesis about this transition suggests that the diverse assemblage of marine amphibious fishes occurring primarily in tropical, high intertidal zone habitats are analogs of early tetrapods and that the intertidal zone, not tropical freshwater lowlands, was the springboard habitat for the Devonian land transition by vertebrates. Here we argue that selection pressures imposed by life in the intertidal zone are insufficient to have resulted in the requisite aerial respiratory capacity or the degree of separation from water required for the vertebrate land transition. The extant marine amphibious fishes, which occur mainly on rocky shores or mudflats, have reached the limit of their niche expansion onto land and remain tied to water by respiratory structures that are less efficient in air and more vulnerable to desiccation than lungs. We further argue that evolutionary contingencies actuated by the Devonian origin of the tetrapods marked a critical point of divergence for a way of life in which selection pressures would operate on the physiology, morphology, and natural history of the different vertebrate groups. While chronically hypoxic and shallow water conditions in the habitats of some primitive bony fishes and some amphibians appear similar to the conditions that prevailed in the Devonian, markedly different selection pressures have operated on other amphibians and bony fishes over the 300 million years since the vertebrate land transition. For example, both egg development and larval metamorphosis in extant amphibians are geared mainly toward compensating for the uncertainty of habitat water quality or even the absence of water by minimizing the time required to develop there. In contrast, reproduction by most intertidal (and amphibious) fishes, all of which are teleosts, remains dependent on a

  19. Optical coherence microscopy as a novel, non-invasive method for the 4D live imaging of early mammalian embryos.

    PubMed

    Karnowski, Karol; Ajduk, Anna; Wieloch, Bartosz; Tamborski, Szymon; Krawiec, Krzysztof; Wojtkowski, Maciej; Szkulmowski, Maciej

    2017-06-23

    Imaging of living cells based on traditional fluorescence and confocal laser scanning microscopy has delivered an enormous amount of information critical for understanding biological processes in single cells. However, the requirement for a high numerical aperture and fluorescent markers still limits researchers' ability to visualize the cellular architecture without causing short- and long-term photodamage. Optical coherence microscopy (OCM) is a promising alternative that circumvents the technical limitations of fluorescence imaging techniques and provides unique access to fundamental aspects of early embryonic development, without the requirement for sample pre-processing or labeling. In the present paper, we utilized the internal motion of cytoplasm, as well as custom scanning and signal processing protocols, to effectively reduce the speckle noise typical for standard OCM and enable high-resolution intracellular time-lapse imaging. To test our imaging system we used mouse and pig oocytes and embryos and visualized them through fertilization and the first embryonic division, as well as at selected stages of oogenesis and preimplantation development. Because all morphological and morphokinetic properties recorded by OCM are believed to be biomarkers of oocyte/embryo quality, OCM may represent a new chapter in imaging-based preimplantation embryo diagnostics.

  20. IGF-1 REGULATES VERTEBRAL BONE AGING THROUGH SEX-SPECIFIC AND TIME-DEPENDENT MECHANISMS

    PubMed Central

    Ashpole, Nicole M; Herron, Jacquelyn C; Mitschelen, Matthew C; Farley, Julie A; Logan, Sreemathi; Yan, Han; Ungvari, Zoltan; Hodges, Erik L.; Csiszar, Anna; Ikeno, Yuji; Humphrey, Mary Beth; Sonntag, William E

    2016-01-01

    Advanced aging is associated with increased risk of bone fracture, especially within the vertebrae, which exhibit significant reductions in trabecular bone structure. Aging is also associated with a reduction in circulating levels of insulin-like growth factor (IGF-1). Studies have suggested that the reduction in IGF-1 compromises healthspan, while others report that loss of IGF-1 is beneficial as it increases healthspan and lifespan. To date, the effect of decreases in circulating IGF-1 on vertebral bone aging has not been thoroughly investigated. Here, we delineate the consequences of a loss of circulating IGF-1 on vertebral bone aging in male and female Igff/f mice. IGF-1 was reduced at multiple specific time points during the mouse lifespan- early in postnatal development (crossing albumin-Cre mice with Igff/f mice), or early adulthood, and late adulthood using hepatic-specific viral vectors (AAV8-TBG-Cre). Vertebrae bone structure was analyzed at 27 months of age using microCT and quantitative bone histomorphometry. Consistent with previous studies, both male and female mice exhibited age-related reductions in vertebral bone structure. In male mice, reduction of circulating IGF-1 induced at any age did not diminish vertebral bone loss. Interestingly, early-life loss of IGF-1 in females resulted in a 67% increase in vertebral bone volume fraction, as well as increased connectivity density and increased trabecular number. The maintenance of bone structure in the early-life IGF-1-deficient females was associated with increased osteoblast surface and an increased ratio of osteoprotegerin/receptor-activator of NFkB-ligand levels in circulation. Within 3 months of a loss of IGF-1, there was a 2.2 fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF-1. Together, these data suggest the age-related loss of vertebral bone density in females can be

  1. Climate change, extinction risks, and reproduction of terrestrial vertebrates.

    PubMed

    Carey, Cynthia

    2014-01-01

    This review includes a broad, but superficial, summary of our understanding about current and future climate changes, the predictions about how these changes will likely affect the risks of extinction of organisms, and how current climate changes are already affecting reproduction in terrestrial vertebrates. Many organisms have become extinct in the last century, but habitat destruction, disease and man-made factors other than climate change have been implicated as the causal factor in almost all of these. Reproduction is certain to be negatively impacted in all vertebrate groups for a variety of reasons, such as direct thermal and hydric effects on mortality of embryos, mismatches between optimal availability of food supplies, frequently determined by temperature, and reproductive capacities, sometimes determined by rigid factors such as photoperiod, and disappearance of appropriate foraging opportunities, such as melting sea ice. The numbers of studies documenting correlations between climate changes and biological phenomena are rapidly increasing, but more direct information about the consequences of these changes for species survival and ecosystem health is needed than is currently available.

  2. Chest Low-Dose Computed Tomography for Early Lung Cancer Diagnosis as an Opportunity to Diagnose Vertebral Fractures in HIV-Infected Smokers, an ANRS EP48 HIV CHEST Substudy.

    PubMed

    Thouvenin, Yann; Makinson, Alain; Cournil, Amandine; Eymard-Duvernay, Sabrina; Lentz, Pierre; Delemazure, Anne-Sophie; Corneloup, Olivier; Fabre, Sylvie; Quesnoy, Mylène; Poire, Sylvain; Brillet, Pierre-Yves; Cyteval, Catherine; Reynes, Jacques; Le Moing, Vincent

    2015-07-01

    To estimate the prevalence of vertebral fractures on chest low-dose computed tomography (LDCT) in HIV-infected smokers. Cross-sectional study of vertebral fractures visualized on chest LDCT from a multicenter prospective cohort evaluating feasibility of chest LDCT for early lung cancer diagnosis in HIV-infected subjects. Subjects were included if 40 years or older, had been active smokers within the last 3 years of at least 20 pack-years, and had a CD4 T-lymphocyte nadir cell count <350 per microliter and an actual CD4 T-cell count >100 cells per microliter. Spinal reconstructed sagittal planes obtained from chest axial native acquisitions were blindly read by a musculoskeletal imaging specialist. Assessment of the fractured vertebra used Genant semiquantitative method. The study end point was the prevalence of at least 1 vertebral fracture. Three hundred ninety-seven subjects were included. Median age was 49.5 years, median smoking history was 30 pack-years, median last CD4 count was 584 cells per microliter, and median CD4 nadir count was 168 cells per microliter; 90% of subjects had a viral load below 50 copies per milliliter. At least 1 fracture was visible in 46 (11.6%) subjects. In multivariate analysis, smoking ≥40 packs-years [OR = 2.5; 95% CI: (1.2 to 5.0)] was associated with an increased risk of vertebral fracture, while HIV viral load <200 copies per milliliter [OR = 0.3; 95% CI: (0.1 to 0.9)] was protective. Prevalence of vertebral fractures on chest LDCT was 11.6% in this high-risk population. Smoking cessation and early introduction of antiretroviral therapy for prevention of vertebral fractures could be beneficial. Chest LDCT is an opportunity to diagnose vertebral fractures.

  3. Glucocorticoid teratogenesis in mouse whole embryo culture.

    PubMed

    Pratt, R M; Perry, E L; Chapman, L M; Goulding, E H

    1984-08-01

    Glucocorticoids, such as triamcinolone acetonide (TAC-A) and triamcinolone hexacetonide (TAC-HA), are potent inducers of cleft palate in vivo in various mouse strains when administered on day 11 of gestation, whereas they are poor or ineffective inducers of cleft lip when given on day 7. The purpose of the present study was to determine whether glucocorticoids are capable of interfering with early embryonic development in culture. CD-1 mouse embryos were cultured for 48 hours starting either on day 8 (plug day 0) with the embryo inside the yolk sac, or on day 10 with the embryo exteriorized from its functional yolk sac. At the end of the culture period, embryos were examined grossly for malformations and biochemically for altered DNA and protein levels. With the day 8 cultures, TAC-A produced a dose-dependent inhibition of growth along with malformations consisting of cardiac irregularities, abnormal rotation, and irregular neural tube closure. With the day 10 cultures, these malformations were not observed, presumably due to the advanced stage of development when the embryos were exposed to TAC-A; however, TAC-A did produce growth inhibition along with cleft lip. When TAC-HA was administered in vivo to pregnant donor females on day 7, in combination with TAC-A added on day 10 to the culture medium, there was a dramatic increase in the frequency of cleft lip along with other alterations in craniofacial appearance. Our results demonstrate that glucocorticoids are capable of directly affecting embryonic growth and development during the early stages of organogenesis.

  4. Setting up equine embryo gender determination by preimplantation genetic diagnosis in a commercial embryo transfer program.

    PubMed

    Herrera, C; Morikawa, M I; Bello, M B; von Meyeren, M; Centeno, J Eusebio; Dufourq, P; Martinez, M M; Llorente, J

    2014-03-15

    Preimplantation genetic diagnosis (PGD) allows identifying genetic traits in early embryos. Because in some equine breeds, like Polo Argentino, females are preferred to males for competition, PGD can be used to determine the gender of the embryo before transfer and thus allow the production of only female pregnancies. This procedure could have a great impact on commercial embryo production programs. The present study was conducted to adapt gender selection by PGD to a large-scale equine embryo transfer program. To achieve this, we studied (i) the effect on pregnancy rates of holding biopsied embryos for 7 to 10 hours in holding medium at 32 °C before transfer, (ii) the effect on pregnancy rates of using embryos of different sizes for biopsy, and (iii) the efficiency of amplification by heating biopsies before polymerase chain reaction. Equine embryos were classified by size (≤300, 300-1000, and >1000 μm), biopsied, and transferred 1 to 2 or 7 to 10 hours after flushing. Some of the biopsy samples obtained were incubated for 10 minutes at 95 °C and the rest remained untreated. Pregnancy rates were recorded at 25 days of gestation; fetal gender was determined using ultrasonography and compared with PGD results. Holding biopsied embryos for 7 to 10 hours before transfer produced pregnancy rates similar to those for biopsied embryos transferred within 2 hours (63% and 57%, respectively). These results did not differ from pregnancy rates of nonbiopsied embryos undergoing the same holding times (50% for 7-10 hours and 63% for 1-2 hours). Pregnancy rates for biopsied and nonbiopsied embryos did not differ between size groups or between biopsied and nonbiopsied embryos within the same size group (P > 0.05). Incubating biopsy samples for 10 minutes at 95 °C before polymerase chain reaction significantly increased the diagnosis rate (78.5% vs. 45.5% for treated and nontreated biopsy samples respectively). Gender determination using incubated biopsy samples matched the

  5. Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

    NASA Astrophysics Data System (ADS)

    Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

    2015-11-01

    Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

  6. Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster.

    PubMed

    de Vega-Bartol, José J; Simões, Marta; Lorenz, W Walter; Rodrigues, Andreia S; Alba, Rob; Dean, Jeffrey F D; Miguel, Célia M

    2013-08-30

    It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in

  7. Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

    PubMed

    Morel, Alexandre; Teyssier, Caroline; Trontin, Jean-François; Eliášová, Kateřina; Pešek, Bedřich; Beaufour, Martine; Morabito, Domenico; Boizot, Nathalie; Le Metté, Claire; Belal-Bessai, Leila; Reymond, Isabelle; Harvengt, Luc; Cadene, Martine; Corbineau, Françoise; Vágner, Martin; Label, Philippe; Lelu-Walter, Marie-Anne

    2014-09-01

    Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets. © 2014 Scandinavian Plant Physiology Society.

  8. Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

    PubMed

    Aït-Azzouzene, D; Langkopf, A; Cohen, J; Bleux, C; Gendron, M C; Kanellopoulos-Langevin, C

    1998-05-01

    Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

  9. Undernutrition affects embryo quality of superovulated ewes.

    PubMed

    Abecia, J A; Forcada, F; Palacín, I; Sánchez-Prieto, L; Sosa, C; Fernández-Foren, A; Meikle, A

    2015-02-01

    To determine the effect of undernutrition on embryo production and quality in superovulated sheep, 45 ewes were allocated into two groups to be fed diets that provided 1.5 (control, C; n = 20) or 0.5 (low nutrition, L; n = 25) times daily requirements for maintenance, from oestrous synchronization with intravaginal sponges to embryo collection. Embryos were collected 7 days after the onset of oestrus (day 0). Low nutrition resulted in lower live weight and body condition at embryo collection (P < 0.05). Diet (P < 0.01) and day of sampling (P < 0.001) significantly affected plasma non-esterified fatty acid (NEFA) and insulin concentrations. Plasma leptin concentrations decreased on day 7 only in L ewes. A significant effect of dietary treatment (P < 0.05) and day (P < 0.0001) was observed on plasma insulin-like growth factor (IGF)-I concentrations. The number of recovered oocytes and embryos did not differ between the groups (L: 15.4 ± 0.4; C: 12.4 ± 0.4). Recovery rate was lower (P < 0.05) in the L (60%) than in the C group (73%). The total number of embryos and number of viable-transferable embryos (5.0 ± 0.3 and 3.4 ± 0.3 embryos, respectively) of the L group were lower (P < 0.1) when compared with controls (8.4 ± 0.4 and 6.2 ± 0.4 embryos, respectively). Undernutrition during the period of superovulation and early embryonic development reduced total and viable number of embryos. These effects might be mediated by disruption of endocrine homeostasis, oviduct environment and/or oocyte quality.

  10. Experimenting with embryos: can philosophy help?

    PubMed

    Heyd, David

    1996-10-01

    Beyond the well-known ethical issues involved in medical experimentation on human subjects, experimenting with embryos raises unique and particularly hard problems. Besides the psychological obstacles connected with the fear of "playing God" and the awe with which we hold the process of the creation of human beings, there are three philosophical problems which are the main subject of the article: 1. The logical problem of circularity: the morality of experimenting on embryos is dependent on the status of the embryo, which in turn is partly decided by experimentation; 2. The metaphysical problem: experiments are justified by the benefits they bring to human subjects; but it is doubtful whether an early embryo is a "subject" and whether coming into being is a "benefit"; 3. The moral problem: the standard constraint on medical experiments is that they benefit either the individual subject or at least members of a relevantly defined group of patients suffering from the same syndrome. But embryo experimentation is often associated with potential cure to people of a completely different category (like geriatric patients). Finally, the article discusses the limits of the force of philosophical arguments in the formation of actual policies for regulating such practices as experimenting with embryos. The widely-shared fourteen-day limit is shown to be a sound practical compromise despite the difficulties in justifying it philosophically.

  11. Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells.

    PubMed

    Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

    2017-08-01

    Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA

  12. Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells

    PubMed Central

    Guo, Fan; Li, Lin; Li, Jingyun; Wu, Xinglong; Hu, Boqiang; Zhu, Ping; Wen, Lu; Tang, Fuchou

    2017-01-01

    Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA

  13. Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

    PubMed

    Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

    2012-11-01

    Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

  14. Histone deacetylase is required for the activation of Wnt/β-catenin signaling crucial for heart valve formation in zebrafish embryos.

    PubMed

    Kim, Young-Seop; Kim, Myoung-Jin; Koo, Tae-Hee; Kim, Jun-Dae; Koun, Soonil; Ham, Hyung Jin; Lee, You Mie; Rhee, Myungchull; Yeo, Sang-Yeob; Huh, Tae-Lin

    2012-06-22

    During vertebrate heart valve formation, Wnt/β-catenin signaling induces BMP signals in atrioventricular canal (AVC) myocardial cells and underlying AVC endocardial cells then undergo endothelial-mesenchymal transdifferentiation (EMT) by receiving this BMP signals. Histone deacetylases (HDACs) have been implicated in numerous developmental processes by regulating gene expression. However, their specific roles in controlling heart valve development are largely unexplored. To investigate the role of HDACs in vertebrate heart valve formation, we treated zebrafish embryos with trichostatin A (TSA), an inhibitor of class I and II HDACs, from 36 to 48 h post-fertilization (hpf) during which heart looping and valve formation occur. Following TSA treatment, abnormal linear heart tube development was observed. In these embryos, expression of AVC myocardial bmp4 and AVC endocardial notch1b genes was markedly reduced with subsequent failure of EMT in the AVC endocardial cells. However, LiCl-mediated activation of Wnt/β-catenin signaling was able to rescue defective heart tube formation, bmp4 and notch1b expression, and EMT in the AVC region. Taken together, our results demonstrated that HDAC activity plays a pivotal role in vertebrate heart tube formation by activating Wnt/β-catenin signaling which induces bmp4 expression in AVC myocardial cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Glycoconjugate distribution in early human notochord and axial mesenchyme.

    PubMed

    Götz, W; Quondamatteo, F

    2001-02-01

    Glycosylation patterns of cells and tissues give insights into spatially and temporally regulated developmental processes and can be detected histochemically using plant lectins with specific affinities for sugar moieties. The early development of the vertebral column in man is a process which has never been investigated by lectin histochemistry. Therefore, we studied binding of several lectins (AIA, Con A, GSA II, LFA, LTA, PNA, RCA I, SBA, SNA, WGA) in formaldehyde-fixed sections of the axial mesenchyme of 5 human embryos in Carnegie stages 12-15. During these developmental stages, an unsegmented mesenchyme covers the notochord. Staining patterns did not show striking temporal variations except for SBA which stained the cranial axial mesenchyme only in the early stage 12 embryo and for PNA, of which the staining intensity in the mesenchyme decreased with age. The notochord appeared as a highly glycosylated tissue. Carbohydrates detected may correspond to adhesion molecules or to secreted substances like proteoglycans or proteins which could play an inductive role, for example, for the neural tube. The axial perinotochordal unsegmented mesenchyme showed strong PNA binding. Therefore, its function as a PNA-positive "barrier" tissue is discussed. The endoderm of the primitive gut showed a lectin-binding pattern that was similar to that of the notochord, which may correlate with interactions between these tissues during earlier developmental stages.

  16. Studying the effects of microgravity on lower vertebrate development and behavior

    NASA Technical Reports Server (NTRS)

    Wassersug, Richard J.; Pronych, Scott; Souza, Kenneth A.

    1991-01-01

    Lower vertebrates have been used in space research for at least 3 decades, and have a number of advantages such as the ability to be maintained safely in space conditions, high development rates, easy observability, and small size. Several major investigations with lower vertebrates are in the flight queue in various countries, and some of this research with amphibians that has Canadian Space Agency sponsorship is reviewed. In connection with NASA's frog embryology experiment, Canadian scientists will conduct postflight experiments on live tadpoles brought back from space to determine whether larvae that develop from these embryos are behaviorally normal. Swimming kinematics in particular will be examined, since a distinctive looping behavior of Xenopus tadpoles under microgravity has been noted. A collaborative study with the Institute of Biomedical Problems in Moscow is designed to elucidate the relationship between buoyancy regulation and lung development in tadpoles, and is scheduled to fly on a Biocosmos satellite in 1992.

  17. A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

    PubMed

    Cho, J W

    1999-08-31

    In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.

  18. Small Fish Species as Powerful Model Systems to Study Vertebrate Physiology in Space

    NASA Astrophysics Data System (ADS)

    Muller, M.; Aceto, J.; Dalcq, J.; Alestrom, P.; Nourizadeh-Lillabadi, R.; Goerlich, R.; Schiller, V.; Winkler, C.; Renn, J.; Eberius, M.; Slenzka, K.

    2008-06-01

    Small fish models, mainly zebrafish (Danio rerio) and medaka (Oryzias latipes), have been used for many years as powerful model systems for vertebrate developmental biology. Moreover, these species are increasingly recognized as valuable systems to study vertebrate physiology, pathology, pharmacology and toxicology, including in particular bone physiology. The biology of small fishes presents many advantages, such as transparency of the embryos, external and rapid development, small size and easy reproduction. Further characteristics are particularly useful for space research or for large scale screening approaches. Finally, many technologies for easily characterizing bones are available. Our objective is to investigate the changes induced by microgravity in small fish. By combining whole genome analysis (microarray, DNA methylation, chromatin modification) with live imaging of selected genes in transgenic animals, a comprehensive and integrated characterization of physiological changes in space could be gained, especially concerning bone physiology.

  19. Wnt signaling balances specification of the cardiac and pharyngeal muscle fields

    PubMed Central

    Mandal, Amrita; Holowiecki, Andrew; Song, Yuntao Charlie; Waxman, Joshua S.

    2017-01-01

    Canonical Wnt/β-catenin (Wnt) signaling plays multiple conserved roles during fate specification of cardiac progenitors in developing vertebrate embryos. Although lineage analysis in ascidians and mice has indicated there is a close relationship between the cardiac second heart field (SHF) and pharyngeal muscle (PM) progenitors, the signals underlying directional fate decisions of the cells within the cardio-pharyngeal muscle field in vertebrates are not yet understood. Here, we examined the temporal requirements of Wnt signaling in cardiac and PM development. In contrast to a previous report in chicken embryos that suggested Wnt inhibits PM development during somitogenesis, we find that in zebrafish embryos Wnt signaling is sufficient to repress PM development during anterior-posterior patterning. Importantly, the temporal sensitivity of dorso-anterior PMs to increased Wnt signaling largely overlaps with when Wnt signaling promotes specification of the adjacent cardiac progenitors. Furthermore, we find that excess early Wnt signaling can cell autonomously promote expansion of the first heart field (FHF) progenitors at the expense of PM and SHF within the anterior lateral plate mesoderm (ALPM). Our study provides insight into an antagonistic developmental mechanism that balances the sizes of the adjacent cardiac and PM progenitor fields in early vertebrate embryos. PMID:28087459

  20. Gene expression analysis in zebrafish embryos: a potential approach to predict effect concentrations in the fish early life stage test.

    PubMed

    Weil, Mirco; Scholz, Stefan; Zimmer, Michaela; Sacher, Frank; Duis, Karen

    2009-09-01

    Based on the hypothesis that analysis of gene expression could be used to predict chronic fish toxicity, the zebrafish (Danio rerio) embryo test (DarT), developed as a replacement method for the acute fish test, was expanded to a gene expression D. rerio embryo test (Gene-DarT). The effects of 14 substances on lethal and sublethal endpoints of the DarT and on expression of potential marker genes were investigated: the aryl hydrocarbon receptor 2, cytochrome P450 1A (cypla), heat shock protein 70, fizzy-related protein 1, the transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene family protein g (avian) 1 and NF-E2-p45-related factor, and heme oxygenase 1 (hmox1). After exposure of zebrafish embryos for 48 h, differential gene expression was evaluated using reverse transcriptase-polymerase chain reaction, gel electrophoresis, and densitometric analysis of the gels. All tested compounds significantly affected the expression of at least one potential marker gene, with cyp1a and hmox1 being most sensitive. Lowest-observed-effect concentrations (LOECs) for gene expression were below concentrations resulting in 10% lethal effects in the DarT. For 10 (3,4- and 3,5-dichloroaniline, 1,4-dichlorobenzene, 2,4-dinitrophenol, atrazine, parathion-ethyl, chlorotoluron, genistein, 4-nitroquinoline-1-oxide, and cadmium) out of the 14 tested substances, LOEC values derived with the Gene-DarT differ by a factor of less than 10 from LOEC values of fish early life stage tests with zebrafish. For pentachloroaniline and pentachlorobenzene, the Gene-DarT showed a 23- and 153-fold higher sensitivity, respectively, while for lindane, it showed a 13-fold lower sensitivity. For ivermectin, the Gene-DarT was by a factor of more than 1,000 less sensitive than the acute fish test. The results of the present study indicate that gene expression analysis in zebrafish embryos could principally be used to predict effect concentrations in the fish early life stage test.

  1. Size-dependent regulation of dorsal-ventral patterning in the early Drosophila embryo

    PubMed Central

    Garcia, Mayra; Nahmad, Marcos; Reeves, Gregory T.; Stathopoulos, Angelike

    2013-01-01

    How natural variation in embryo size affects patterning of the Drosophila embryo dorsal-ventral (DV) axis is not known. Here we examined quantitatively the relationship between nuclear distribution of the Dorsal transcription factor, boundary positions for several target genes, and DV axis length. Data were obtained from embryos of a wild-type background as well as from mutant lines inbred to size select embryos of smaller or larger sizes. Our data show that the width of the nuclear Dorsal gradient correlates with DV axis length. In turn, for some genes expressed along the DV axis, the boundary positions correlate closely with nuclear Dorsal levels and with DV axis length; while the expression pattern of others is relatively constant and independent of the width of the Dorsal gradient. In particular, the patterns of snail (sna) and ventral nervous-system defective (vnd) correlate with nuclear Dorsal levels and exhibit scaling to DV length; while the pattern of intermediate neuroblasts defective (ind) remains relatively constant with respect to changes in Dorsal and DV length. However, in mutants that exhibit an abnormal expansion of the Dorsal gradient which fails to scale to DV length, only sna follows the Dorsal distribution and exhibits overexpansion; in contrast, vnd and ind do not overexpand suggesting some additional mechanism acts to refine the dorsal boundaries of these two genes. Thus, our results argue against the idea that the Dorsal gradient works as a global system of relative coordinates along the DV axis and suggest that individual targets respond to changes in embryo size in a gene-specific manner. PMID:23800450

  2. In vitro and in vivo effects of ulipristal acetate on fertilization and early embryo development in mice.

    PubMed

    Gómez-Elías, Matías D; Munuce, María J; Bahamondes, Luis; Cuasnicú, Patricia S; Cohen, Débora J

    2016-01-01

    Does ulipristal acetate (UPA), a selective progesterone receptor modulator used for emergency contraception (EC), interfere with fertilization or early embryo development in vitro and in vivo? At doses similar to those used for EC, UPA does not affect mouse gamete transport, fertilization or embryo development. UPA acts as an emergency contraceptive mainly by inhibiting or delaying ovulation. However, there is little information regarding its effects on post-ovulatory events preceding implantation. This was an in vitro and in vivo experimental study involving the use of mouse gametes and embryos from at least three animals in each set of experiments. For in vitro fertilization experiments, mouse epididymal spermatozoa capacitated in the presence of different concentrations of UPA (0-1000 ng/ml) were used to inseminate cumulus-intact or cumulus-free eggs in the presence or absence of UPA during gamete co-incubation, and the percentage of fertilized eggs was determined. For in vivo fertilization experiments, superovulated females caged with proven fertile males were injected with UPA (40 mg/kg) or vehicle just before or just after mating and the percentage of fertilized eggs recovered from the ampulla was determined. To investigate the effect of UPA on embryo development, zygotes were recovered from mated females, cultured in the presence of UPA (1000 ng/ml) for 4 days and the progression of embryo development was monitored daily. In vitro studies revealed that the presence of UPA during capacitation and/or gamete co-incubation does not affect fertilization. Whereas the in vivo administration of UPA at the same time as hCG injection produced a decrease in the number of eggs ovulated compared with controls (vehicle injected animals, P < 0.05), no effects on fertilization were observed when UPA was administered shortly before or after mating. No differences were observed in either the percentage of cleaved embryos or the cleavage speed when UPA was present during in

  3. Cilia in vertebrate left–right patterning

    PubMed Central

    Dasgupta, Agnik

    2016-01-01

    Understanding how left–right (LR) asymmetry is generated in vertebrate embryos is an important problem in developmental biology. In humans, a failure to align the left and right sides of cardiovascular and/or gastrointestinal systems often results in birth defects. Evidence from patients and animal models has implicated cilia in the process of left–right patterning. Here, we review the proposed functions for cilia in establishing LR asymmetry, which include creating transient leftward fluid flows in an embryonic ‘left–right organizer’. These flows direct asymmetric activation of a conserved Nodal (TGFβ) signalling pathway that guides asymmetric morphogenesis of developing organs. We discuss the leading hypotheses for how cilia-generated asymmetric fluid flows are translated into asymmetric molecular signals. We also discuss emerging mechanisms that control the subcellular positioning of cilia and the cellular architecture of the left–right organizer, both of which are critical for effective cilia function during left–right patterning. Finally, using mosaic cell-labelling and time-lapse imaging in the zebrafish embryo, we provide new evidence that precursor cells maintain their relative positions as they give rise to the ciliated left–right organizer. This suggests the possibility that these cells acquire left–right positional information prior to the appearance of cilia. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821522

  4. Cilia in vertebrate left-right patterning.

    PubMed

    Dasgupta, Agnik; Amack, Jeffrey D

    2016-12-19

    Understanding how left-right (LR) asymmetry is generated in vertebrate embryos is an important problem in developmental biology. In humans, a failure to align the left and right sides of cardiovascular and/or gastrointestinal systems often results in birth defects. Evidence from patients and animal models has implicated cilia in the process of left-right patterning. Here, we review the proposed functions for cilia in establishing LR asymmetry, which include creating transient leftward fluid flows in an embryonic 'left-right organizer'. These flows direct asymmetric activation of a conserved Nodal (TGFβ) signalling pathway that guides asymmetric morphogenesis of developing organs. We discuss the leading hypotheses for how cilia-generated asymmetric fluid flows are translated into asymmetric molecular signals. We also discuss emerging mechanisms that control the subcellular positioning of cilia and the cellular architecture of the left-right organizer, both of which are critical for effective cilia function during left-right patterning. Finally, using mosaic cell-labelling and time-lapse imaging in the zebrafish embryo, we provide new evidence that precursor cells maintain their relative positions as they give rise to the ciliated left-right organizer. This suggests the possibility that these cells acquire left-right positional information prior to the appearance of cilia.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

  5. A novel gene's role in an ancient mechanism: secreted Frizzled-related protein 1 is a critical component in the anterior-posterior Wnt signaling network that governs the establishment of the anterior neuroectoderm in sea urchin embryos.

    PubMed

    Khadka, Anita; Martínez-Bartolomé, Marina; Burr, Stephanie D; Range, Ryan C

    2018-01-01

    The anterior neuroectoderm (ANE) in many deuterostome embryos (echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates) is progressively restricted along the anterior-posterior axis to a domain around the anterior pole. In the sea urchin embryo, three integrated Wnt signaling branches (Wnt/β-catenin, Wnt/JNK, and Wnt/PKC) govern this progressive restriction process, which begins around the 32- to 60-cell stage and terminates by the early gastrula stage. We previously have established that several secreted Wnt modulators of the Dickkopf and secreted Frizzled-related protein families (Dkk1, Dkk3, and sFRP-1/5) are expressed within the ANE and play important roles in modulating the Wnt signaling network during this process. In this study, we use morpholino and dominant-negative interference approaches to characterize the function of a novel Frizzled-related protein, secreted Frizzled-related protein 1 (sFRP-1), during ANE restriction. sFRP-1 appears to be related to a secreted Wnt modulator, sFRP3/4, that is essential to block Wnt signaling and establish the ANE in vertebrates. Here, we show that the sea urchin sFRP3/4 orthologue is not expressed during ANE restriction in the sea urchin embryo. Instead, our results indicate that ubiquitously expressed maternal sFRP-1 and Fzl1/2/7 signaling act together as early as the 32- to 60-cell stage to antagonize the ANE restriction mechanism mediated by Wnt/β-catenin and Wnt/JNK signaling. Then, starting from the blastula stage, Fzl5/8 signaling activates zygotic sFRP-1 within the ANE territory, where it works with the secreted Wnt antagonist Dkk1 (also activated by Fzl5/8 signaling) to antagonize Wnt1/Wnt8-Fzl5/8-JNK signaling in a negative feedback mechanism that defines the outer ANE territory boundary. Together, these data indicate that maternal and zygotic sFRP-1 protects the ANE territory by antagonizing the Wnt1/Wnt8-Fzl5/8-JNK signaling pathway throughout ANE restriction, providing precise

  6. Effect of progesterone supplementation in the first week post conception on embryo survival in beef heifers.

    PubMed

    Beltman, M E; Lonergan, P; Diskin, M G; Roche, J F; Crowe, M A

    2009-04-15

    Progesterone is essential for establishment and maintenance of pregnancy in mammals. The objective of this study was to examine the effect of elevating progesterone during the different physiological stages of early embryo development on embryo survival. Estrus was synchronized in cross-bred beef heifers (n=197, approximately 2-years old) and they were inseminated 12-18h after estrus onset (=Day 0). Inseminated heifers were randomly assigned to 1 of 3 treatments: (1) Control, n=69; (2) progesterone supplementation using a Controlled Internal Drug Release Device (CIDR) from Day 3 to 6.5, n=64; or (3) progesterone supplementation using a CIDR from Day 4.5 to 8, n=64. Body condition (BCS) and locomotion scores (scale of 1-5) were recorded for all animals. Animals with a locomotion score >/=4 (very lame) were excluded. Embryo survival rate was determined at slaughter on Day 25. Conceptus length and weight were recorded and the corpus luteum (CL) of all pregnant animals was dissected and weighed. Supplementation with exogenous progesterone increased (P<0.05) peripheral progesterone concentrations, but did not affect embryo survival rate compared with controls. Mean CL weight, conceptus length and conceptus weight were not different between treatments. There was a positive relationship (P<0.04) between the increase in progesterone concentrations from Days 3 to 6.5 and embryo survival rate in treated heifers and a similar trend existed between the increase from Days 4.5 to 8 (P<0.06). There was also a positive relationship (P<0.05) between the progesterone concentration on Day 6.5 and the embryo survival rate in treated heifers. A direct correlation was seen between locomotion score and embryo survival rate, with higher (P<0.05) early embryo survival rates in heifers with a lower locomotion score. In conclusion, supplementation with progesterone at different stages of early embryo development increased peripheral progesterone concentration and resulted in a positive

  7. Analysis of global gene expression profiles to identify differentially expressed genes critical for embryo development in Brassica rapa.

    PubMed

    Zhang, Yu; Peng, Lifang; Wu, Ya; Shen, Yanyue; Wu, Xiaoming; Wang, Jianbo

    2014-11-01

    Embryo development represents a crucial developmental period in the life cycle of flowering plants. To gain insights into the genetic programs that control embryo development in Brassica rapa L., RNA sequencing technology was used to perform transcriptome profiling analysis of B. rapa developing embryos. The results generated 42,906,229 sequence reads aligned with 32,941 genes. In total, 27,760, 28,871, 28,384, and 25,653 genes were identified from embryos at globular, heart, early cotyledon, and mature developmental stages, respectively, and analysis between stages revealed a subset of stage-specific genes. We next investigated 9,884 differentially expressed genes with more than fivefold changes in expression and false discovery rate ≤ 0.001 from three adjacent-stage comparisons; 1,514, 3,831, and 6,633 genes were detected between globular and heart stage embryo libraries, heart stage and early cotyledon stage, and early cotyledon and mature stage, respectively. Large numbers of genes related to cellular process, metabolism process, response to stimulus, and biological process were expressed during the early and middle stages of embryo development. Fatty acid biosynthesis, biosynthesis of secondary metabolites, and photosynthesis-related genes were expressed predominantly in embryos at the middle stage. Genes for lipid metabolism and storage proteins were highly expressed in the middle and late stages of embryo development. We also identified 911 transcription factor genes that show differential expression across embryo developmental stages. These results increase our understanding of the complex molecular and cellular events during embryo development in B. rapa and provide a foundation for future studies on other oilseed crops.

  8. The current status and future of commercial embryo transfer in cattle.

    PubMed

    Hasler, John F

    2003-12-15

    A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen

  9. Fossilized embryos are widespread but the record is temporally and taxonomically biased

    USGS Publications Warehouse

    Donoghue, P.C.J.; Kouchinsky, A.; Waloszek, Dieter; Bengtson, S.; Dong, X.-P.; Val'Kov, A.K.; Cunningham, J.A.; Repetski, J.E.

    2006-01-01

    We report new discoveries of embryos and egg capsules from the Lower Cambrian of Siberia, Middle Cambrian of Australia and Lower Ordovician of North America. Together with existing records, embryos have now been recorded from four of the seven continents. However, the new discoveries highlight secular and systematic biases in the fossil record of embryonic stages. The temporal window within which the embryos and egg capsules are found is of relatively short duration; it ends in the Early Ordovician and is roughly coincident with that of typical "Orsten"-type faunas. The reduced occurrence of such fossils has been attributed to reducing levels of phosphate in marine waters during the early Paleozoic, but may also be owing to the increasing depth of sediment mixing by infaunal metazoans. Furthermore, most records younger than the earliest Cambrian are of a single kind - large eggs and embryos of the priapulid-like scalidophoran Markuelia. We explore alternative explanations for the low taxonomic diversity of embryos recovered thus far, including sampling, size, anatomy, ecology, and environment, concluding that the preponderance of Markuelia embryos is due to its precocious development of cuticle at an embryonic stage, predisposing it to preservation through action as a substrate on which microbially mediated precipitation of authigenic calcium phosphate may occur. The fossil record of embryos may be limited to a late Neoproterozoic to early Ordovician snapshot that is subject to dramatic systematic bias. Together, these biases must be considered seriously in attempts to use the fossil record to arbitrate between hypotheses of developmental and life history evolution implicated in the origin of metazoan clades. ?? 2006 Blackwell Publishing Ltd.

  10. A potential determinant role of adiponectin and receptors for the early embryo development in PCOS patients with obesity hinted by quantitative profiling.

    PubMed

    Zhang, Ning; Hao, Cuifang; Liu, Xiaoyan; Zhang, Shouxin; Zhang, Fengrong; Zhuang, Lili; Zhao, Dongmei

    2017-02-01

    To identify the quantitative profiling of adiponectin and its receptors (AdipoR1, AdipoR2, and T-cadherin) in cumulus cells (CCs) and to evaluate their roles in the early embryo development of polycystic ovary syndrome (PCOS) patients, in part, with obesity. Fifty-five subjects were divided into two groups according to the body mass index. Oocytes were further inseminated and only mature and normal fertilized oocytes (2PN) were included in this research. Real-time PCR and western blot were performed to identify adiponectin and its receptors in CCs. Adiponectin and receptors were ubiquitously expressed in CCs of PCOS and non-PCOS patients. The level of AdipoR2 in CCs from the oocytes yielding blastocyst after 5/6 days in vitro culture was markedly higher than in those from oocytes could not develop to blastocyst stage after Day 6, for non-obese or obese PCOS patients (0.1647 ± 0.0161 versus 0.0783 ± 0.0385, 0.1948 ± 0.0307 versus 0.1057 ± 0.0236, respectively, p < 0.05). In addition, only in patients with PCOS and concurrent obesity the AdipoR1 in CCs was considerably increased in CC-B + compared with CC-B - subgroup (0.5162 ± 0.0371 versus 0.2448 ± 0.0333, p < 0.01). The development of early embryo was associated with the up-regulation of AdipoR1 and AdipoR2 in PCOS patients. Our results suggested that adiponectin could positively modulate embryo development in humans. Further investigations should be carried out to unlock the crucial role that adiponectin plays in embryo development.

  11. Cervical vertebral erosion caused by bilateral vertebral artery tortuosity, predisposing to spinal, sprain: A medieval case study.

    PubMed

    Darton, Yves

    2014-03-01

    Bone resorption within the cervical spine due to vertebral arterial tortuosities is rarely observed in medical practice because the condition often lacks clinical symptoms. Traumatic complications involving the vertebral arteries are relatively common and occasionally very serious, but very few affect bone, appearing only when survival has been sufficiently long for a pseudoaneurysm to form. CT scans and MRI screening, practised increasingly today following traffic and sports accidents, incidentally show that arterial tortuosities that had stimulated bone resorption are relatively frequent. Only rarely do such tortuosities cause nerve compression or trigger orthopaedic problems, while large pseudoaneurysms and congenital absence of a vertebral pedicle may require surgery to stabilize the spine. There are few publications by palaeopathologists reporting such conditions of the cervical vertebrae. This contribution reports a case of a tiered bilateral tortuosity of the vertebral artery dating from the Early Middle Ages; it provides a basis by which to recognize this type of lesion in osteoarchaeology, and it attests to the fact that multiple tortuosities may lead to spinal instability in the form of spine sprain. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Early evolution of vertebrate photoreception: lessons from lampreys and lungfishes.

    PubMed

    Collin, Shaun P

    2009-03-01

    Lampreys (Agnatha) and lungfish (Dipnoi) are representatives of the earliest and the intermediate stages in vertebrate evolution, respectively, and survived in the Cambrian (approximately 540 mA, lampreys) and Devonian (approximately 400 mA, lungfishes) Periods. The unique phylogenetic position of these two groups presents us with an exciting opportunity to understand life in ancient times and to begin to trace the evolution of vision and photoreception in vertebrates. Using a multidisciplinary approach employing anatomical and molecular techniques, the evolution of photoreception is explored in these extant, living fossils to predict the environmental lighting conditions to which our vertebrate ancestors were exposed. Contrary to expectations, the retinae of the southern hemisphere lamprey (Geotria australis Gray, 1851) and the Australian lungfish (Neoceratodus forsteri Krefft, 1870) are far from "primitive," each possessing five types of photoreceptors, many with spectral filters for tuning the light. Detailed ultrastructural analysis reveals that all five receptor types in G. australis are cone-like, whereas N. forsteri possesses four cone types and a single type of rod. Each receptor type also contains a different visual pigment (opsin gene); that is, LWS, SWS1, SWS2, RhA and RhB in G. australis and LWS, SWS1, SWS2, Rh1 and Rh2 in N. forsteri, all of which are expressed within the retina and are sensitive to different parts of the electromagnetic spectrum, providing the potential for pentachromatic and tetrachromatic color vision, respectively. © 2009 ISZS, Blackwell Publishing and IOZ/CAS.

  13. Early somatic embryo induction events in alfalfa callus cultures. [Medicago sativa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    El-Bakry, A.A.; Hildebrand, D.F.

    1987-04-01

    High and low regenerating alfalfa Medicago sativa L. cv Regen S full sibs were isolated from a callus culture screen on modified Blaydes medium. The average number of embryos per ovary were thirty and zero for the high and low genotypes respectively after six weeks in culture. Proembryonic cell masses (4-8 celled) were observed after 4-5 days in culture and maximum meristematic activity was at 6-7 days in culture, for the high regenerating genotypes. Well formed globular embryos, both epidermal and subepidermal in origin, were observed after 2 weeks is culture. Samples in culture for 3, 6 and 14 daysmore » from the high and low regenerating genotypes were radiolabeled in vivo with /sup 35/S-methionine and run both on one and two dimension gels. The results will be discussed in relation to differences in proteins between the high and low regenerating genotypes at the stage of maximum meristematic activity (day 6) and differences occurring relative to the appearance of globular stage embryos (day 14) will be presented.« less

  14. Properties of Barley Seed Chitinases and Release of Embryo-Associated Isoforms during Early Stages of Imbibition 1

    PubMed Central

    Swegle, Mark; Kramer, Karl J.; Muthukrishnan, Subbaratnam

    1992-01-01

    Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity. Two of these (CH1 and CH2) are found primarily in the aleurone and endosperm tissues, and the other three (CH3, CH4, and CH5) are enriched in the embryo. From the bran fraction, three of these CHs (CH1, CH2, and CH3) were purified to apparent homogeneity. These three CHs have apparent molecular masses of 27, 34, and 35 kilodaltons and isoelectric points of 9.3, 9.2, and 8.7, respectively. CH2 and CH3 have amino terminal sequences resembling a portion of the chitin-binding domain of lectins and other plant defense proteins. CH1 lacks this domain. All three CHs exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro. During the early period of imbibition by seeds, two of the embryo-associated CHs are selectively released into the surrounding aqueous medium. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668964

  15. Transcriptomic analysis highlights epigenetic and transcriptional regulation during zygotic embryo development of Pinus pinaster

    PubMed Central

    2013-01-01

    Background It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Results Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning

  16. Differential expression of Toll-like receptor pathway genes in chicken embryo fibroblasts from chickens resistant and susceptible to Marek’s disease

    USDA-ARS?s Scientific Manuscript database

    The Toll-like receptor (TLR) signaling pathway is one of the innate immune defense mechanisms against pathogens in vertebrates and invertebrates. However, the role of TLR in non-MHC genetic resistance or susceptibility to Marek’s disease (MD) in the chicken is yet to be elucidated. Chicken embryo fi...

  17. IGF-1 Regulates Vertebral Bone Aging Through Sex-Specific and Time-Dependent Mechanisms.

    PubMed

    Ashpole, Nicole M; Herron, Jacquelyn C; Mitschelen, Matthew C; Farley, Julie A; Logan, Sreemathi; Yan, Han; Ungvari, Zoltan; Hodges, Erik L; Csiszar, Anna; Ikeno, Yuji; Humphrey, Mary Beth; Sonntag, William E

    2016-02-01

    Advanced aging is associated with increased risk of bone fracture, especially within the vertebrae, which exhibit significant reductions in trabecular bone structure. Aging is also associated with a reduction in circulating levels of insulin-like growth factor (IGF-1). Studies have suggested that the reduction in IGF-1 compromises healthspan, whereas others report that loss of IGF-1 is beneficial because it increases healthspan and lifespan. To date, the effect of decreases in circulating IGF-1 on vertebral bone aging has not been thoroughly investigated. Here, we delineate the consequences of a loss of circulating IGF-1 on vertebral bone aging in male and female Igf(f/f) mice. IGF-1 was reduced at multiple specific time points during the mouse lifespan: early in postnatal development (crossing albumin-cyclic recombinase [Cre] mice with Igf(f/f) mice); and in early adulthood and in late adulthood using hepatic-specific viral vectors (AAV8-TBG-Cre). Vertebrae bone structure was analyzed at 27 months of age using micro-computed tomography (μCT) and quantitative bone histomorphometry. Consistent with previous studies, both male and female mice exhibited age-related reductions in vertebral bone structure. In male mice, reduction of circulating IGF-1 induced at any age did not diminish vertebral bone loss. Interestingly, early-life loss of IGF-1 in females resulted in a 67% increase in vertebral bone volume fraction, as well as increased connectivity density and increased trabecular number. The maintenance of bone structure in the early-life IGF-1-deficient females was associated with increased osteoblast surface and an increased ratio of osteoprotegerin/receptor-activator of NF-κB-ligand (RANKL) levels in circulation. Within 3 months of a loss of IGF-1, there was a 2.2-fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF-1. Together, these data

  18. Lethal effect of dehydroleucodine (DhL) on amphibian Bufo arenarum embryos.

    PubMed

    Moreno, Liliana Elizabeth; Juárez, Américo Osvaldo; Pelzer, Lilian Eugenia

    2012-03-01

    The dehydroleucodine is a sesquiterpene lactone isolated from Artemisia douglasiana Besser which is used in popular medicine. Toxicity tests using embryos of amphibian have been widely used in order to predict toxic effects of different compounds. However, to our knowledge, there are not studies focussed on the toxic effects of dehydroleucodine on Bufo arenarum, which is an anuran widely distributed in South America. The effect of dehydroleucodine on the survival of embryos was evaluated in an acute test during the early life stage of B. arenarum embryos. Lethality and the degree of adverse effects were dehydroleucodine dose-dependent. Overall, amphibian early life stages appeared to be more susceptible to the embryotoxicity associated with exposure to dehydroleucodine, especially at concentration greater that 3mM. This increased susceptibility may result from the relatively high rate of cellular differentiation and morphogenesis that occurs at this early stage of development. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates.

    PubMed

    Plouhinec, Jean-Louis; Medina-Ruiz, Sofía; Borday, Caroline; Bernard, Elsa; Vert, Jean-Philippe; Eisen, Michael B; Harland, Richard M; Monsoro-Burq, Anne H

    2017-10-01

    During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

  20. A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates

    PubMed Central

    Borday, Caroline; Bernard, Elsa; Vert, Jean-Philippe; Eisen, Michael B.; Harland, Richard M.

    2017-01-01

    During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research. PMID:29049289

  1. Developmental consequences of cryopreservation of mammalian oocytes and embryos.

    PubMed

    Smith, Gary D; Silva E Silva, Cristine Ane

    2004-08-01

    During the last three decades, significant advances have been made in successful cryopreservation of mammalian preimplantation embryos, and more recently oocytes. The ability to cryopreserve, thaw, and establish pregnancies with supernumerary preimplantation embryos has become an important tool in fertility treatment. Human oocyte cryopreservation has practical application in preserving fertility for individuals at risk of compromised egg quality due to cancer treatments or advanced maternal age. While oocyte/embryo cryopreservation success has increased over time, there is still room for improvement. Oocytes and embryos are susceptible to cryo-damage, which collectively entails cellular damage caused by mechanical, chemical, or thermal forces during the freeze-thaw process. Basic studies focused on understanding cellular structures, their composition, and more importantly their functions, in normal cell developments will continue to be critical in assessing, understanding, and correcting oocyte/embryo cryo-damage. This review will delineate many of the oocyte/embryo intracellular and extracellular structures that are or may be compromised during cryopreservation. A global theme presented throughout this review is that many structural components of the oocyte/embryo also have essential functional roles in development. Compromising these cellular structures, and thus their cellular homeostatic functions, can deleteriously influence initial cryo-survival or compromise subsequent normal development through effects on the oocyte and/or early embryo.

  2. Viability of bovine demi- and quarter-embryos after transfer.

    PubMed

    Bredbacka, P; Huhtinen, M; Aalto, J; Rainio, V

    1992-07-01

    The viability of bovine demi- and quarter-embryos was investigated. Early compacting morulae were nonsurgically flushed from superovulated donor cows and were bisected by two microneedles. One of the halves was then split further into two quarters. Each demi- and quarter-embryo was placed in an evacuated zona pellucida. One demi- or two quarter-embryos were transferred non-surgically into cow or heifer recipients. Viability was measured by ultrasound scanning of the fetuses on Days 35, 48 and 60 of pregnancy. The pregnancy rates at Day 60 were 46.2% (6/13) for heifers and 33.3% (4/12) for cows after the transfer of a single demi-embryo. The transfer of two quarter-embryos resulted in a pregnancy rate of 61.5% (8/13) for heifers and 8.3% (1/12) for cows. Seven (53.8%) and four (33.3%) live fetuses were found on Day 60 following the transfer of demi-embryos into heifers and cows, respectively. The transfer of quarter-embryos resulted in 10 fetuses (38.5%) in the heifer recipients and only one fetus (4.2%) in the cow recipients. The results of this study suggest that heifers are more suitable than cows as recipients for quarter-embryos.

  3. Effects of gamma radiation on the early developmental stages of Zebrafish (Danio rerio).

    PubMed

    Praveen Kumar, M K; Shyama, S K; Kashif, Shamim; Dubey, S K; Avelyno, D'costa; Sonaye, B H; Kadam Samit, B; Chaubey, R C

    2017-08-01

    The zebrafish is gaining importance as a popular vertebrate model organism and is widely employed in ecotoxicological studies, especially for the biomonitoring of pollution in water bodies. There is limited data on the genetic mechanisms governing the adverse health effects in regards to an early developmental exposure to gamma radiation. In the present study zebrafish (Danio rerio) embryos were exposed to 1, 2.5, 5, 7.5 and 10Gy of gamma radiation at 3h post fertilization (hpf). Different developmental toxicity endpoints were investigated. Further, expression of genes associated with the development and DNA damage i.e. (sox2 sox19a and p53) were evaluated using Quantitative PCR (qPCR). The significant changes in the expression of sox2 sox19a and p53 genes were observed. This data was supported the developmental defects observed in the zebrafish embryo exposed to gamma radiation such as i.e. increased DNA damage, decreased hatching rate, increase in median hatching time, decreased body length, increased mortality rate, increased morphological deformities. Further, study shows that the potential ecotoxicological threat of gamma radiation on the early developmental stages of zebrafish. Further, it revealed that the above parameters can be used as predictive biomarkers of gamma radiation exposure. Copyright © 2017. Published by Elsevier Inc.

  4. Estrogen receptor-mediated effects of a xenoestrogen, bisphenol A, on preimplantation mouse embryos.

    PubMed

    Takai, Y; Tsutsumi, O; Ikezuki, Y; Hiroi, H; Osuga, Y; Momoeda, M; Yano, T; Taketani, Y

    2000-04-21

    The effects of bisphenol A, a xenoestrogen widely used in industry and dentistry, were studied in early preimplantation mouse embryos. Two-cell mouse embryos were cultured with 100 pM to 100 microM bisphenol A with or without 100 nM tamoxifen and evaluated at 24-h intervals for their development to eight-cell and blastocyst stages. At 72 h, blastocysts were cultured for another 48 h without bisphenol A, and surface areas of trophoblast spread were measured. At 24 h, more embryos exposed to 3 nM bisphenol A than to controls had reached the eight-cell stage. At 48 h, more embryos exposed to 1 nM and 3 nM bisphenol A than to controls had become blastocysts. At 100 microM, bisphenol A decreased frequency of development to blastocysts. Tamoxifen counteracted both stimulatory and inhibitory effects of bisphenol A on blastocyst formation. Although bisphenol A did not alter blastocyst morphology or cell number, early exposure to 100 microM bisphenol A increased subsequent trophoblast areas. These findings suggest that bisphenol A may not only effect early embryonic development via estrogen receptors even at low, environmentally relevant doses, but also exert some late effects on subsequent development of these embryos. Copyright 2000 Academic Press.

  5. Early miscarriage rate in lean polycystic ovary syndrome women after euploid embryo transfer - a matched-pair study.

    PubMed

    Luo, Lu; Gu, Fang; Jie, Huying; Ding, Chenhui; Zhao, Qiang; Wang, Qiong; Zhou, Canquan

    2017-11-01

    The early miscarriage rate is reported to be higher in patients with polycystic ovary syndrome (PCOS) compared with non-PCOS patients. However, whether PCOS is an independent risk factor for early miscarriage is still controversial; to what extent embryonic aneuploidy accounts for miscarriages of PCOS is still unknown. In this 1:3 matched-pair study, 67 lean PCOS patients and 201 controls matched for age, body mass index (BMI) and embryo scores undergoing a single euploid blastocyst transfer in vitrified-warmed cycles were analysed. Clinical pregnancy, early miscarriage and live birth rates were compared. Logistic regression analysis was performed to further evaluate the factors associated with early miscarriage and live birth. Clinical pregnancy rates were 50.7% in PCOS and 55.2% in control groups. Early miscarriage rate was significantly (P = 0.029) increased in the PCOS group compared with controls; non-PCOS patients had a significantly higher live birth rate than PCOS patients, P < 0.001. Further regression analyses showed that PCOS was significantly associated with a higher risk of early miscarriage and decreased chance of live birth. In conclusion, PCOS in women undergoing pre-implantation genetic diagnosis may, independently from BMI and karyotype, increase the risk of miscarriage. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  6. Effect of Culture Conditions on Viability of Mouse and Rat Embryos Developed in Vitro

    PubMed Central

    Popova, Elena; Bader, Michael; Krivokharchenko, Alexander

    2011-01-01

    Currently in vitro culture of mouse preimplantation embryos has become a very important technique to investigate different mechanisms of early embryogenesis. However, there is a big difference in the preimplantation development between mammalian species. Despite close relatedness to mice, in vitro cultivation of rat preimplantation embryos is still delicate and needs further investigation and optimizations. In this study we have compared the in vitro developmental potential of mouse and rat embryos cultured at different culture conditions in parallel experiments. Interestingly, mouse zygotes developed in vitro until blastocyst stage even in inadequate medium without any phosphates and with low osmolarity which was formulated especially for cultivation of rat embryos. Rat parthenotes and zygotes developed in M16 medium formulated for mouse embryos only till 2-cell stage and further development is blocked completely at this stage. Moreover, developmental ability of rat embryos in vitro was significantly lower in comparison with mouse even in special rat mR1ECM medium. Mouse and rat embryos at 2-cell stage obtained in vivo developed until blastocyst stages significantly more efficiently compared to zygotes. Culture of mouse zygotes in glass capillaries resulted in a significantly higher rate of morula and blastocyst development compared with dishes. The Well-of-the-Well system resulted in a significant improvement when compared with dishes for the culture of rat zygotes only until morula stage. Reduced oxygen tension increased the developmental rate of rat but not mouse zygotes until blastocyst stage. This study demonstrates that development of early preimplantation embryos is altered by different culture conditions and show strong differences even between two related species such as mice and rats. Therefore, for understanding the fundamental mechanisms of early mammalian development it is very important to use embryos of various species. PMID:24710194

  7. Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo

    PubMed Central

    Woda, Juliana M; Calzonetti, Teresa; Hilditch-Maguire, Paige; Duyao, Mabel P; Conlon, Ronald A; MacDonald, Marcy E

    2005-01-01

    Background Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5 huntingtin deficient embryos. Results In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Conclusion Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease. PMID:16109169

  8. Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo.

    PubMed

    Woda, Juliana M; Calzonetti, Teresa; Hilditch-Maguire, Paige; Duyao, Mabel P; Conlon, Ronald A; MacDonald, Marcy E

    2005-08-18

    Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdh(ex4/5) huntingtin deficient embryos. In the absence of huntingtin, expression of nutritive genes appears normal but E7.0-7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease.

  9. Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

    PubMed

    Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo

    2016-10-01

    . Furthermore, the developed blastocysts were scored using the blastocyst quality score (BQS), and the correlation with oxygen consumption rate was also assessed. The device enabled the oxygen consumption rate of an embryo to be measured automatically within a minute. The oxygen concentration gradient profile showed excellent linearity in a distance-dependent change. A close correlation in the oxygen consumption rates of bovine embryos was observed between the SECM measuring system and CERMs, with a determination coefficient of 0.8203 (P = 0.0008). Oxygen consumption rates of human embryos that have reached the blastocyst stage were significantly higher than those of arrested embryos at 48, 72 and 96 h after thawing (P = 0.039, 0.004 and 0.049, respectively). Thus, in vitro development of frozen-thawed human embryos to the blastocyst stage would be predicted at 48 h after thawing (day 4) by measuring the oxygen consumption using CERMs. Although a positive linear relationship between BQS and the oxygen consumption rate was observed [the determination coefficient was R(2) = 0.6537 (P = 0.008)], two blastocysts exhibited low oxygen consumption rates considering their relatively high BQS. This suggests that morphology and metabolism in human embryos might not correlate consistently. Transfer of the embryo and pregnancy evaluation was not performed. Thus, a correlation between oxygen consumption and the in vivo viability of embryos remains unknown. Clinical trials, including embryo transfer, would be desirable to determine a threshold value to elect clinically relevant, quality embryos for transfer. We utilized frozen-thawed human embryos in this study. The effect of these manipulations on the respiratory activity of the embryo is also unknown. Selection of quality embryos, especially in a single embryo transfer cycle, by CERMs may have an impact on obtaining better clinical outcomes, albeit with clinical trials being required. Furthermore, the early determination of quality

  10. The red tide toxin, brevetoxin, induces embryo toxicity and developmental abnormalities.

    PubMed Central

    Kimm-Brinson, K L; Ramsdell, J S

    2001-01-01

    Brevetoxins are lipophilic polyether toxins produced by the red tide dinoflagellate Gymnodinium breve, and their neurotoxic effects on adult animals have been documented. In this study, we characterized adverse developmental effects of brevetoxin-1 (PbTx-1) using an exposure paradigm that parallels the maternal oocyte transfer of toxin. Medaka fish (Oryzias latipes) embryos were exposed to PbTx-1 via microinjection of toxin reconstituted in a triolein oil droplet. Embryos microinjected with doses of 0.1-8.0 ng/egg (ppm) of brevetoxin-1 exhibited pronounced muscular activity (hyperkinesis) after embryonic day 4. Upon hatching, morphologic abnormalities were commonly found in embryos at the following lowest adverse effect levels: 1.0-3.0 ppm, lateral curvature of the spinal column; 3.1-3.4 ppm, herniation of brain meninges through defects in the skull; and 3.4-4.0 ppm, malpositioned eye. Hatching abnormalities were also commonly observed at brevetoxin doses of 2.0 ppm and higher with head-first, as opposed to the normal tail-first, hatching, and doses > 4.1 ng/egg produced embryos that developed but failed to hatch. Given the similarity of developmental processes found between higher and lower vertebrates, teratogenic effects of brevetoxins have the potential to occur among different phylogenetic classes. The observation of developmental abnormalities after PbTx-1 exposure identifies a new spectrum of adverse effects that may be expected to occur following exposure to G. breve red tide events. PMID:11335186

  11. Equine cloning: in vitro and in vivo development of aggregated embryos.

    PubMed

    Gambini, Andrés; Jarazo, Javier; Olivera, Ramiro; Salamone, Daniel F

    2012-07-01

    The production of cloned equine embryos remains highly inefficient. Embryo aggregation has not yet been tested in the equine, and it might represent an interesting strategy to improve embryo development. This study evaluated the effect of cloned embryo aggregation on in vitro and in vivo equine embryo development. Zona-free reconstructed embryos were individually cultured in microwells (nonaggregated group) or as 2- or 3-embryo aggregates (aggregated groups). For in vitro development, they were cultured until blastocyst stage and then either fixed for Oct-4 immunocytochemical staining or maintained in in vitro culture where blastocyst expansion was measured daily until Day 17 or the day on which they collapsed. For in vivo assays, Day 7-8 blastocysts were transferred to synchronized mares and resultant vesicles, and cloned embryos were measured by ultrasonography. Embryo aggregation improved blastocyst rates on a per well basis, and aggregation did not imply additional oocytes to obtain blastocysts. Embryo aggregation improved embryo quality, nevertheless it did not affect Day 8 and Day 16 blastocyst Oct-4 expression patterns. Equine cloned blastocysts expanded and increased their cell numbers when they were maintained in in vitro culture, describing a particular pattern of embryo growth that was unexpectedly independent of embryo aggregation, as all embryos reached similar size after Day 7. Early pregnancy rates were higher using blastocysts derived from aggregated embryos, and advanced pregnancies as live healthy foals also resulted from aggregated embryos. These results indicate that the strategy of aggregating embryos can improve their development, supporting the establishment of equine cloned pregnancies.

  12. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    PubMed

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

  13. The surgical treatment of vertebral deformities in achondroplastic dwarfism.

    PubMed

    Parisini, P; Greggi, T; Casadei, R; Martini, A; De Zerbi, M; Campanacci, L; Perozzi, M

    1996-01-01

    The authors analyzed 15 patients affected with achondroplastic dwarfism with vertebral deformity treated surgically between 1976 and 1994. The forms represented were: achondroplasia; diastrophic dwarfism; spondyloepiphyseal achondroplasia. The types of vertebral deformity were: kyphosis: 12 (angular: 6; regular: 6); scoliosis: 1; kyphoscoliosis: 2. Neurological symptoms were present in 10 patients. Treatment was as follows: laminectomy: 8; posterior fusion with instrumentation: 2; anterior fusion: 2; anterior fusion with laminectomy and posterior fusion: 3. There were postoperative neurological complications in 4 cases (27%). Fusion must be performed early in angular kyphosis in the adult in order to prevent neurological symptoms. Wide laminectomies do not require associated fusion because they do not cause late vertebral instability.

  14. Transcription of fgf8 is regulated by activating and repressive cis-elements at the midbrain-hindbrain boundary in zebrafish embryos.

    PubMed

    Inoue, Fumitaka; Parvin, Mst Shahnaj; Yamasu, Kyo

    2008-04-15

    Fgf8 is expressed in the isthmic region of the developing brain, serving an organizing function in vertebrate embryos. We previously identified S4.2 downstream to the zebrafish fgf8 gene as a regulatory region that drives transcription in the anterior hindbrain. Here, we investigated the mechanism of fgf8 regulation by the S4.2 region during development. Reporter analyses in embryos revealed that S4.2 closely recapitulates fgf8 expression in the anteriormost hindbrain during somitogenesis. This region contains a sequence highly conserved in fgf8 of diverse vertebrates. Further analyses of S4.2 revealed a 342-bp core region composed of three subregions (#2, #3, and #4). Regions #3 and #4 drove expression broadly in the brain from the midbrain to r5 of the hindbrain, whereas a 28-bp sequence in #2 repressed ectopic expression in the midbrain and in r2 to r5. The enhancer function of S4.2 was absent in pax2a mutant embryos, while it was activated ectopically by pax2a misexpression in the hindbrain. We identified two sites in the core region that are bound by Pax2a in vitro and in vivo, the disruption of which abrogated the S4.2 activity. Thus, fgf8 expression in the anteriormost hindbrain involves activation and repression, with Pax2a as a pivotal regulator.

  15. Maternal organism and embryo biosensoring: insights from ruminants.

    PubMed

    Sandra, Olivier; Constant, Fabienne; Vitorino Carvalho, Anais; Eozénou, Caroline; Valour, Damien; Mauffré, Vincent; Hue, Isabelle; Charpigny, Gilles

    2015-04-01

    In terms of contribution to pregnancy, the mother not only produces gametes, but also hosts gestation, whose progression in the uterus is conditioned by early events during implantation. In ruminants, this period is associated with elongation of the extra-embryonic tissues, gastrulation of the embryonic disk and cross-talk with the endometrium. Recent data have prompted the need for accurate staging of the bovine conceptus and shown that asynchrony between elongation and gastrulation processes may account for pregnancy failure. Data mining of endometrial gene signatures has allowed the identification of molecular pathways and new factors regulated by the conceptus (e.g. FOXL2, SOCS6). Interferon-tau has been recognised to be the major signal of pregnancy recognition, but prostaglandins and lysophospholipids have also been demonstrated to be critical players at the conceptus-endometrium interface. Interestingly, up-regulation of interferon-regulated gene expression has been identified in circulating immune cells during implantation, making these factors a potential source of non-invasive biomarkers for early pregnancy. Distinct endometrial responses have been shown to be elicited by embryos produced by artificial insemination, in vitro fertilisation or somatic cell nuclear transfer. These findings have led to the concept that endometrium is an early biosensor of embryo quality. This biological property first demonstrated in cattle has been recently extended and associated with embryo selection in humans. Hence, compromised or suboptimal endometrial quality can subtly or deeply affect embryo development, with visible and sometimes severe consequences for placentation, foetal development, pregnancy outcome and the long-term health of the offspring. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Protein Phosphorylation during Coconut Zygotic Embryo Development1

    PubMed Central

    Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

    1998-01-01

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

  17. Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function.

    PubMed

    Faivre-Sarrailh, Catherine; Banerjee, Swati; Li, Jingjun; Hortsch, Michael; Laval, Monique; Bhat, Manzoor A

    2004-10-01

    Septate junctions (SJs) in epithelial and neuronal cells play an important role in the formation and maintenance of charge and size selective barriers. They form the basis for the ensheathment of nerve fibers in Drosophila and for the attachment of myelin loops to axonal surface in vertebrates. The cell-adhesion molecules NRX IV/Caspr/Paranodin (NCP1), contactin and Neurofascin-155 (NF-155) are all present at the vertebrate axo-glial SJs. Mutational analyses have shown that vertebrate NCP1 and its Drosophila homolog, Neurexin IV (NRX IV) are required for the formation of SJs. In this study, we report the genetic, molecular and biochemical characterization of the Drosophila homolog of vertebrate contactin, CONT. Ultrastructural and dye-exclusion analyses of Cont mutant embryos show that CONT is required for organization of SJs and paracellular barrier function. We show that CONT, Neuroglian (NRG) (Drosophila homolog of NF-155) and NRX IV are interdependent for their SJ localization and these proteins form a tripartite complex. Hence, our data provide evidence that the organization of SJs is dependent on the interactions between these highly conserved cell-adhesion molecules.

  18. The Zebrafish G12 Gene is required for Nuclear Positioning and Cell Migrations during Early Development

    NASA Technical Reports Server (NTRS)

    Reinsch, S. S.; Conway, G. C.

    2003-01-01

    After fertilization Zebrafish embryos undergo synchronous cleavage to form a blastula of cells sitting upon a single multinucleate yolk cell. At the beginning of gastrulation these cells undergo extensive cell migrations to form the major body axes. We have discovered a gene, G12, which is required for cell migrations and positioning of nuclei in the large syncytial yolk cell. Overexpression of a G12-GFP fusion protein is not toxic and shows that the protein localizes inside the yolk cell to the yolk nuclei, microtubules, and to the margin between the blastomeres and the large yolk cell. Morpholino (MO) injection into the 1-cell embryo or into just the yolk syncytium conipletely inhibits cell migrations, doming of the yolk cell, and positioning of nuclei around the margin. This effect can be partially rescued by injection of G12-GFP encoding RNA. Given the known role of microtubules in nuclear positioning of yolk nuclei in Zebrafish, we investigated the microtubules in morpholiiio injected and rescued embryos. We find that microtubules are sparse and disorganized in MO-injected embryos and are restored to normal organization upon G12-GFP rescue. G12 plays a pivotal role in organization of inicrotubules during early development. G12 is highly conserved in vertebrates and two homologues exist in the human genome. One of the human hoinologues is amplified in aggressive breast tumors.

  19. Terrestrial Origin of Viviparity in Mesozoic Marine Reptiles Indicated by Early Triassic Embryonic Fossils

    PubMed Central

    Motani, Ryosuke; Jiang, Da-yong; Tintori, Andrea; Rieppel, Olivier; Chen, Guan-bao

    2014-01-01

    Viviparity in Mesozoic marine reptiles has traditionally been considered an aquatic adaptation. We report a new fossil specimen that strongly contradicts this traditional interpretation. The new specimen contains the oldest fossil embryos of Mesozoic marine reptile that are about 10 million years older than previous such records. The fossil belongs to Chaohusaurus (Reptilia, Ichthyopterygia), which is the oldest of Mesozoic marine reptiles (ca. 248 million years ago, Early Triassic). This exceptional specimen captures an articulated embryo in birth position, with its skull just emerged from the maternal pelvis. Its headfirst birth posture, which is unlikely to be a breech condition, strongly indicates a terrestrial origin of viviparity, in contrast to the traditional view. The tail-first birth posture in derived ichthyopterygians, convergent with the conditions in whales and sea cows, therefore is a secondary feature. The unequivocally marine origin of viviparity is so far not known among amniotes, a subset of vertebrate animals comprising mammals and reptiles, including birds. Therefore, obligate marine amniotes appear to have evolved almost exclusively from viviparous land ancestors. Viviparous land reptiles most likely appeared much earlier than currently thought, at least as early as the recovery phase from the end-Permian mass extinction. PMID:24533127

  20. Culture of Cells from Amphibian Embryos.

    ERIC Educational Resources Information Center

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  1. Telomere lengthening early in development.

    PubMed

    Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

    2007-12-01

    Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

  2. The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice.

    PubMed

    Zernicka-Goetz, Magdalena

    2006-08-01

    Development of the early mouse embryo has always been classified as regulative, meaning that when parts or blastomeres of the embryo are isolated they change their developmental fate and can even reconstruct the whole. However, regulative development does not mean that, in situ, these parts or blastomeres are equivalent; it does not mean that the early mammalian embryo is a ball of identical cells without any bias. Regulative development simply means that whatever bias the regions of the embryo might have they still remain flexible and can respond to experimental interference by changes of fate. This realization -- that regulative development and patterning can co-exist -- has led to a renaissance of interest in the first days of development of the mouse embryo, and several laboratories have provided evidence for some early bias. Now the challenge is to gain some understanding of the molecular basis of this bias.

  3. Live imaging of rat embryos with Doppler swept-source optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Larina, Irina V.; Furushima, Kenryo; Dickinson, Mary E.; Behringer, Richard R.; Larin, Kirill V.

    2009-09-01

    The rat has long been considered an excellent system to study mammalian embryonic cardiovascular physiology, but has lacked the extensive genetic tools available in the mouse to be able to create single gene mutations. However, the recent establishment of rat embryonic stem cell lines facilitates the generation of new models in the rat embryo to link changes in physiology with altered gene function to define the underlying mechanisms behind congenital cardiovascular birth defects. Along with the ability to create new rat genotypes there is a strong need for tools to analyze phenotypes with high spatial and temporal resolution. Doppler OCT has been previously used for 3-D structural analysis and blood flow imaging in other model species. We use Doppler swept-source OCT for live imaging of early postimplantation rat embryos. Structural imaging is used for 3-D reconstruction of embryo morphology and dynamic imaging of the beating heart and vessels, while Doppler-mode imaging is used to visualize blood flow. We demonstrate that Doppler swept-source OCT can provide essential information about the dynamics of early rat embryos and serve as a basis for a wide range of studies on functional evaluation of rat embryo physiology.

  4. Live imaging of rat embryos with Doppler swept-source optical coherence tomography

    PubMed Central

    Larina, Irina V.; Furushima, Kenryo; Dickinson, Mary E.; Behringer, Richard R.; Larin, Kirill V.

    2009-01-01

    The rat has long been considered an excellent system to study mammalian embryonic cardiovascular physiology, but has lacked the extensive genetic tools available in the mouse to be able to create single gene mutations. However, the recent establishment of rat embryonic stem cell lines facilitates the generation of new models in the rat embryo to link changes in physiology with altered gene function to define the underlying mechanisms behind congenital cardiovascular birth defects. Along with the ability to create new rat genotypes there is a strong need for tools to analyze phenotypes with high spatial and temporal resolution. Doppler OCT has been previously used for 3-D structural analysis and blood flow imaging in other model species. We use Doppler swept-source OCT for live imaging of early postimplantation rat embryos. Structural imaging is used for 3-D reconstruction of embryo morphology and dynamic imaging of the beating heart and vessels, while Doppler-mode imaging is used to visualize blood flow. We demonstrate that Doppler swept-source OCT can provide essential information about the dynamics of early rat embryos and serve as a basis for a wide range of studies on functional evaluation of rat embryo physiology. PMID:19895102

  5. Use of the Coelomic Grafting Technique for Prolonged ex utero Cultivation of Late Preprimitive Streak-Stage Rabbit Embryos.

    PubMed

    Püschel, Bernd; Männer, Jörg

    2016-01-01

    Due to its morphological similarity with the early human embryo, the pregastrulation-stage rabbit may represent an appropriate mammalian model for studying processes involved in early human development. The usability of mammalian embryos for experimental studies depends on the availability of whole embryo culture methods facilitating prolonged ex utero development. While currently used culture methods yield high success rates for embryos from primitive streak stages onward, the success rate of extended cultivation of preprimitive streak-stage mammalian embryos is low for all previously established methods and for all studied species. This limits the usability of preprimitive streak-stage rabbit embryos in experimental embryology. We have tested whether the extraembryonic coelom of 4-day-old chick embryos may be used for prolonged ex utero culture of preprimitive streak-stage rabbit embryos (stage 2, 6.2 days post coitum). We found that, within this environment, stage 2 rabbit blastocysts can be cultured at decreasing success rates (55% after 1 day, 35% after 2 days, 15% after 3 days) up to a maximum of 72 h. Grafted blastocysts can continue development from the onset of gastrulation to early organogenesis and thereby form all structures characterizing age-matched controls (e.g. neural tube, somites, beating heart). Compared to normal controls, successfully cultured embryos developed at a slower rate and finally showed some structural and gross morphological anomalies. The method presented here was originally developed for whole embryo culture of mouse embryos by Gluecksohn-Schoenheimer in 1941. It is a simple and inexpensive method that may represent a useful extension to presently available ex utero culture systems for rabbit embryos. © 2016 S. Karger AG, Basel.

  6. Aberrant ligand-induced activation of G protein-coupled estrogen receptor 1 (GPER) results in developmental malformations during vertebrate embryogenesis.

    PubMed

    Jayasinghe, B Sumith; Volz, David C

    2012-01-01

    G protein-coupled estrogen receptor 1 (GPER) is a G protein-coupled receptor (GPCR) unrelated to nuclear estrogen receptors but strongly activated by 17β-estradiol in both mammals and fish. To date, the distribution and functional characterization of GPER within reproductive and nonreproductive vertebrate organs have been restricted to juvenile and adult animals. In contrast, virtually nothing is known about the spatiotemporal distribution and function of GPER during vertebrate embryogenesis. Using zebrafish as an animal model, we investigated the potential functional role and expression of GPER during embryogenesis. Based on real-time PCR and whole-mount in situ hybridization, gper was expressed as early as 1 h postfertilization (hpf) and exhibited strong stage-dependent expression patterns during embryogenesis. At 26 and 38 hpf, gper mRNA was broadly distributed throughout the body, whereas from 50 to 98 hpf, gper expression was increasingly localized to the heart, brain, neuromasts, craniofacial region, and somite boundaries of developing zebrafish. Continuous exposure to a selective GPER agonist (G-1)-but not continuous exposure to a selective GPER antagonist (G-15)-from 5 to 96 hpf, or within three developmental windows ranging from 10 to 72 hpf, resulted in adverse concentration-dependent effects on survival, gross morphology, and somite formation within the trunk of developing zebrafish embryos. Importantly, based on co-exposure studies, G-15 blocked severe G-1-induced developmental toxicity, suggesting that G-1 toxicity is mediated via aberrant activation of GPER. Overall, our findings suggest that xenobiotic-induced GPER activation represents a potentially novel and understudied mechanism of toxicity for environmentally relevant chemicals that affect vertebrate embryogenesis.

  7. Low cost labeling with highlighter ink efficiently visualizes developing blood vessels in avian and mouse embryos.

    PubMed

    Takase, Yuta; Tadokoro, Ryosuke; Takahashi, Yoshiko

    2013-12-01

    To understand how blood vessels form to establish the intricate network during vertebrate development, it is helpful if one can visualize the vasculature in embryos. We here describe a novel labeling method using highlighter ink, easily obtained in stationery stores with a low cost, to visualize embryo-wide vasculatures in avian and mice. We tested 50 different highlighters for fluorescent microscopy with filter sets equipped in a standard fluorescent microscope. The yellow and violet inks yielded fluorescent signals specifically detected by the filters used for green fluorescent protein (GFP) and red fluorescent protein (RFP) detections, respectively. When the ink solution was infused into chicken/quail and mouse embryos, vasculatures including large vessels and capillaries were labeled both in living and fixed embryos. Ink-infused embryos were further subjected to histological sections, and double stained with antibodies including QH-1 (quail), α smooth muscle actin (αSMA), and PECAM-1 (mouse), revealing that the endothelial cells were specifically labeled by the infused highlighter ink. Highlighter-labeled signals were detected with a resolution comparable to or higher than signals of fluorescein isothiocyanate (FITC)-lectin and Rhodamine-dextran, conventionally used for angiography. Furthermore, macroconfocal microscopic analyses with ink-infused embryos visualized fine vascular structures of both embryo proper and extra-embryonic plexus in a Z-stack image of 2400 μm thick with a markedly high resolution. Together, the low cost highlighter ink serves as an alternative reagent useful for visualization of blood vessels in developing avian and mouse embryos and possibly in other animals. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  8. Effects of growth hormone on the ultrastructure of bovine preimplantation embryos.

    PubMed

    Kölle, Sabine; Stojkovic, Miodrag; Reese, Sven; Reichenbach, Horst-Dieter; Wolf, Eckhard; Sinowatz, Fred

    2004-07-01

    Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.

  9. Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos

    PubMed Central

    D’Souza, Fiona; Pudakalakatti, Shivanand M.; Uppangala, Shubhashree; Honguntikar, Sachin; Salian, Sujith Raj; Kalthur, Guruprasad; Pasricha, Renu; Appajigowda, Divya; Atreya, Hanudatta S.; Adiga, Satish Kumar

    2016-01-01

    Early development of certain mammalian embryos is protected by complex checkpoint systems to maintain the genomic integrity. Several metabolic pathways are modulated in response to genetic insults in mammalian cells. The present study investigated the relationship between the genetic integrity, embryo metabolites and developmental competence in preimplantation stage mouse embryos with the aim to identify early biomarkers which can predict embryonic genetic integrity using spent medium profiling by NMR spectroscopy. Embryos carrying induced DNA lesions (IDL) developed normally for the first 2.5 days, but began to exhibit a developmental delay at embryonic day 3.5(E3.5) though they were morphologically indistinguishable from control embryos. Analysis of metabolites in the spent medium on E3.5 revealed a significant association between pyruvate, lactate, glucose, proline, lysine, alanine, valine, isoleucine and thymine and the extent of genetic instability observed in the embryos on E4.5. Further analysis revealed an association of apoptosis and micronuclei frequency with P53 and Bax transcripts in IDL embryos on the E4.5 owing to delayed induction of chromosome instability. We conclude that estimation of metabolites on E3.5 in spent medium may serve as a biomarker to predict the genetic integrity in pre-implantation stage embryos which opens up new avenues to improve outcomes in clinical IVF programs. PMID:27853269

  10. Morphogenesis and evolution of vertebrate appendicular muscle

    PubMed Central

    HAINES, LYNN; CURRIE, PETER D.

    2001-01-01

    Two different modes are utilised by vertebrate species to generate the appendicular muscle present within fins and limbs. Primitive Chondricthyan or cartilaginous fishes use a primitive mode of muscle formation to generate the muscle of the fins. Direct epithelial myotomal extensions invade the fin and generate the fin muscles while remaining in contact with the myotome. Embryos of amniotes such as chick and mouse use a similar mechanism to that deployed in the bony teleost species, zebrafish. Migratory mesenchymal myoblasts delaminate from fin/limb level somites, migrate to the fin/limb field and differentiate entirely within the context of the fin/limb bud. Migratory fin and limb myoblasts express identical genes suggesting that they possess both morphogenetic and molecular identity. We conclude that the mechanisms controlling tetrapod limb muscle formation arose prior to the Sarcopterygian or tetrapod radiation. PMID:11523824

  11. Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

    PubMed Central

    Belacortu, Yaiza; Weiss, Ron; Kadener, Sebastian; Paricio, Nuria

    2012-01-01

    Background Cabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. Methodology/Principal Findings To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. Conclusions/Significance Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of

  12. An amphioxus winged helix/forkhead gene, AmphiFoxD: insights into vertebrate neural crest evolution

    NASA Technical Reports Server (NTRS)

    Yu, Jr-Kai; Holland, Nicholas D.; Holland, Linda Z.

    2002-01-01

    During amphioxus development, the neural plate is bordered by cells expressing many genes with homologs involved in vertebrate neural crest induction. However, these amphioxus cells evidently lack additional genetic programs for the cell delaminations, migrations, and differentiations characterizing definitive vertebrate neural crest. We characterize an amphioxus winged helix/forkhead gene (AmphiFoxD) closely related to vertebrate FoxD genes. Phylogenetic analysis indicates that the AmphiFoxD is basal to vertebrate FoxD1, FoxD2, FoxD3, FoxD4, and FoxD5. One of these vertebrate genes (FoxD3) consistently marks neural crest during development. Early in amphioxus development, AmphiFoxD is expressed medially in the anterior neural plate as well as in axial (notochordal) and paraxial mesoderm; later, the gene is expressed in the somites, notochord, cerebral vesicle (diencephalon), and hindgut endoderm. However, there is never any expression in cells bordering the neural plate. We speculate that an AmphiFoxD homolog in the common ancestor of amphioxus and vertebrates was involved in histogenic processes in the mesoderm (evagination and delamination of the somites and notochord); then, in the early vertebrates, descendant paralogs of this gene began functioning in the presumptive neural crest bordering the neural plate to help make possible the delaminations and cell migrations that characterize definitive vertebrate neural crest. Copyright 2002 Wiley-Liss, Inc.

  13. Quantifying Three-Dimensional Morphology and RNA from Individual Embryos

    PubMed Central

    Green, Rebecca M.; Leach, Courtney L.; Hoehn, Natasha; Marcucio, Ralph S.; Hallgrímsson, Benedikt

    2017-01-01

    Quantitative analysis of morphogenesis aids our understanding of developmental processes by providing a method to link changes in shape with cellular and molecular processes. Over the last decade many methods have been developed for 3D imaging of embryos using microCT scanning to quantify the shape of embryos during development. These methods generally involve a powerful, cross-linking fixative such as paraformaldehyde to limit shrinkage during the CT scan. However, the extended time frames that these embryos are incubated in such fixatives prevent use of the tissues for molecular analysis after microCT scanning. This is a significant problem because it limits the ability to correlate variation in molecular data with morphology at the level of individual embryos. Here, we outline a novel method that allows RNA, DNA or protein isolation following CT scan while also allowing imaging of different tissue layers within the developing embryo. We show shape differences early in craniofacial development (E11.5) between common mouse genetic backgrounds, and demonstrate that we are able to generate RNA from these embryos after CT scanning that is suitable for downstream RT-PCR and RNAseq analyses. PMID:28152580

  14. A modified culture method significantly improves the development of mouse somatic cell nuclear transfer embryos.

    PubMed

    Dai, Xiangpeng; Hao, Jie; Zhou, Qi

    2009-08-01

    Many strategies have been established to improve the efficiency of somatic cell nuclear transfer (SCNT), but relatively few focused on improving culture conditions. The effect of different culture media on preimplantation development of mouse nuclear transfer embryos was investigated. A modified sequential media method, named D media (M16/KSOM and CZB-EG/KSOM), was successfully established that significantly improves SCNT embryo development. Our result demonstrated that while lacking any adverse effect on in vivo fertilized embryos, the D media dramatically improves the blastocyst development of SCNT embryos compared with other commonly used media, including KSOM, M16, CZB, and alphaMEM. Specifically, the rate of blastocyst formation was 62.3% for D1 (M16/KSOM) versus 10-30% for the other media. An analysis of media components indicated that removing EDTA and glutamine from the media can be beneficial for early SCNT embryo development. Our results suggest that in vitro culture environment plays an important role in somatic cell reprogramming, and D media represent the most efficient culture method reported to date to support mouse SCNT early embryo development in vitro.

  15. Trimethyltin chloride inhibits neuronal cell differentiation in zebrafish embryo neurodevelopment.

    PubMed

    Kim, Jin; Kim, C-Yoon; Song, Juha; Oh, Hanseul; Kim, Cheol-Hee; Park, Jae-Hak

    2016-01-01

    Trimethyltin chloride (TMT) is a neurotoxicant widely present in the aquatic environment, primarily from effluents of the plastic industry. It is known to cause acute neuronal death in the limbic-cerebellar system, particularly in the hippocampus. However, relatively few studies have estimated the effects of TMT toxicity on neurodevelopment. In this study, we confirmed the dose-dependent effects of TMT on neurodevelopmental stages through analysis of morphological changes and fluorescence assays using HuC-GFP and olig2-dsRed transgenic zebrafish embryos. In addition, we analyzed the expression of genes and proteins related to neurodevelopment. Exposure of embryos to TMT for 4 days post fertilization (dpf) elicited a concentration-related decrease in body length and increase in axial malformation. TMT affected the fluorescent CNS structure by decreasing pattern of HuC-GFP and olig2-dsRed transgenic zebrafish. In addition, it significantly modulated the expression patterns of Sonic hedgehog a (Shha), Neurogenin1 (Ngn1), Embryonic lethal abnormal vision like protein 3 (Elavl3), and Glial fibrillary acidic protein (Gfap). The overexpression of Shha and Ngn1, and downregulation of Elavl3 and Gfap, indicate repression of proneural cell differentiation. Our study demonstrates that TMT inhibits specific neurodevelopmental stages in zebrafish embryos and suggests a possible mechanism for the toxicity of TMT in vertebrate neurodevelopment. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

    PubMed

    Binder, N K; Evans, J; Gardner, D K; Salamonsen, L A; Hannan, N J

    2014-10-10

    Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and

  17. Sulphonated phthalocyanine induced caudal malformative syndrome in the chick embryo.

    PubMed

    Sandor, S; Prelipceanu, O; Checiu, I

    1985-01-01

    Sulphonated phthalocyanine (Pht.) has been tested for its possible noxious effect on the developing chick embryo. When injected into the subembryonic cavity of 40-45 hours incubated chick embryos (mainly 10-20 somite pairs), Pht. induces a highly reproducible caudal malformative syndrome (trunk and taillessness, various anomalies of the limbs). The main effect is--in about 15% of the malformed specimens--associated with unilateral microphthalmy and, less frequently, with coelosomy. Microscopically developmental disturbances of the caudal axial organs, of the mesonephros and of the limbs are observed. The initial pathological changes, at microscopic level, are necrosis and hemorrhages in the caudal axial and paraxial area. The allantois is poorly developed or even absent. Skeletal changes involve anomalies of the ribs and of the vertebral column and total or partial absence of the pelvic girdle bones. The high mortality, mainly during the first week, is due--first of all--to the developmental disturbances including the poor development or absence of the allantois. Control experiments with CuCl2 suggest the ethiological role of Cu. Pathogenetic aspects are discussed.

  18. New evidence on the anatomy and phylogeny of the earliest vertebrates.

    PubMed Central

    Xian-guang, Hou; Aldridge, Richard J; Siveter, David J; Siveter, Derek J; Xiang-hong, Feng

    2002-01-01

    We report the discovery of a new agnathan specimen from the Lower Cambrian Chengjiang Lagerstätte of China and thereby provide new evidence on the myomeres (V-shaped), the branchial apparatus (gill filaments and arches), the dorsal fin and the gonads (24-26) of the earliest vertebrates. The new specimen and the co-occurring Myllokunmingia fengjiaoa and Haikouichthys ercaicunensis represent a single species, which is a primitive member of the crown group craniates (vertebrates) and post-dates the origin of the myxinoids (hagfish). The origin of the vertebrate clade is at least as old as Early Cambrian. PMID:12350247

  19. Loss of Bmal1 decreases oocyte fertilization, early embryo development and implantation potential in female mice.

    PubMed

    Xu, Jian; Li, Yan; Wang, Yizi; Xu, Yanwen; Zhou, Canquan

    2016-10-01

    Biological clock genes expressed in reproductive tissues play important roles in maintaining the normal functions of reproductive system. However, disruption of female circadian rhythm on oocyte fertilization, preimplantation embryo development and blastocyst implantation potential is still unclear. In this study, ovulation, in vivo and in vitro oocyte fertilization, embryo development, implantation and intracellular reactive oxygen species (ROS) levels in ovary and oviduct were studied in female Bmal1+/+ and Bmal1-/- mice. The number of naturally ovulated oocyte in Bmal1-/- mice decreased (5.2 ± 0.8 vs 7.8 ± 0.8, P < 0.001), with an increasing abnormal oocyte ratio (20.4 ± 3.5 vs 11.7 ± 2.0%, P = 0.001) after superovulation. Significantly lower fertilization rate and obtained blastocyst number were observed in Bmal1-/- female mice either mated with wild-type in vivo or fertilized by sperm from wild-type male mice in vitro (all P < 0.05). Interestingly, in vitro fertilization rate of oocytes derived from Bmal1-/- increased significantly compared with in vivo study (P < 0.01). After transferring blastocysts derived from Bmal1+/+ and Bmal1-/- female mice to pseudopregnant mice, the implantation sites of the latter decreased 5 days later (8.0 ± 0.8 vs 5.3 ± 1.0, P = 0.005). The intracellular ROS levels in the ovary on proestrus day and in the oviduct on metestrus day increased significantly in Bmal1-/- mice compared with that of Bmal1+/+ mice. Deletion of the core biological clock gene Bmal1 significantly decreases oocyte fertilization rate, early embryo development and implantation potential in female mice, and these may be possibly caused by excess ROS levels generated in ovary and oviduct.

  20. Synaptic scaffold evolution generated components of vertebrate cognitive complexity

    PubMed Central

    Nithianantharajah, J.; Komiyama, N.H.; McKechanie, A.; Johnstone, M.; Blackwood, D. H.; St Clair, D.; Emes, R.D.; van de Lagemaat, L. N.; Saksida, L.M.; Bussey, T.J.; Grant, S.G.N.

    2014-01-01

    The origins and evolution of higher cognitive functions including complex forms of learning, attention and executive functions are unknown. A potential mechanism driving the evolution of vertebrate cognition early in the vertebrate lineage (550 My ago) was genome duplication and subsequent diversification of postsynaptic genes. Here we report the first genetic analysis of a vertebrate gene family in cognitive functions measured using computerized touchscreens. Comparison of mice carrying mutations in all four Dlg paralogs show simple associative learning required Dlg4, while Dlg2 and Dlg3 diversified to play opposing roles in complex cognitive processes. Exploiting the translational utility of touchscreens in humans and mice, testing Dlg2 mutations in both species showed Dlg2’s role in complex learning, cognitive flexibility and attention has been highly conserved over 100 My. Dlg family mutations underlie psychiatric disorders suggesting genome evolution expanded the complexity of vertebrate cognition at the cost of susceptibility to mental illness. PMID:23201973

  1. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

    PubMed Central

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

    2016-01-01

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  2. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

    PubMed

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Swann, Karl; Borri, Paola

    2016-06-15

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. © 2016. Published by The Company of Biologists Ltd.

  3. PGF₂α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus.

    PubMed

    Gomez, E; Martin, D; Carrocera, S; Muñoz, M

    2015-08-01

    In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo-maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (β-actin, NFkB, clusterin and immunoproteosome 20S β5i subunit-i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F2 α and PGE2 concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF2 α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE2. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo-maternal interactions in vivo in monotocous species.

  4. Epigenetic regulation of left-right asymmetry by DNA methylation.

    PubMed

    Wang, Lu; Liu, Zhibin; Lin, Hao; Ma, Dongyuan; Tao, Qinghua; Liu, Feng

    2017-10-16

    DNA methylation is a major epigenetic modification; however, the precise role of DNA methylation in vertebrate development is still not fully understood. Here, we show that DNA methylation is essential for the establishment of the left-right (LR) asymmetric body plan during vertebrate embryogenesis. Perturbation of DNA methylation by depletion of DNA methyltransferase 1 ( dnmt1 ) or dnmt3bb.1 in zebrafish embryos leads to defects in dorsal forerunner cell (DFC) specification or collective migration, laterality organ malformation, and disruption of LR patterning. Knockdown of dnmt1 in Xenopus embryos also causes similar defects. Mechanistically, loss of dnmt1 function induces hypomethylation of the lefty2 gene enhancer and promotes lefty2 expression, which consequently represses Nodal signaling in zebrafish embryos. We also show that Dnmt3bb.1 regulates collective DFC migration through cadherin 1 (Cdh1). Taken together, our data uncover dynamic DNA methylation as an epigenetic mechanism to control LR determination during early embryogenesis in vertebrates. © 2017 The Authors.

  5. Cloning, expression pattern, and potential role of apoptosis inhibitor 5 in the termination of embryonic diapause and early embryo development of Artemia sinica.

    PubMed

    Zhang, Shuang; Yao, Feng; Jing, Ting; Zhang, Mengchen; Zhao, Wei; Zou, Xiangyang; Sui, Linlin; Hou, Lin

    2017-09-10

    During the embryonic development of Artemia sinica, the diapause phenomenon can be induced by high salinity or low temperature conditions. The diapause embryo at the gastrula stage is maintained under the threat of apoptosis to guarantee the embryo's normal development. In this process, apoptosis inhibitor proteins play vital roles in protecting embryos against apoptosis. Apoptosis inhibitor5 (API5) plays a pivotal role in regulating the cell cycle and preventing programmed cell death after growth factor starvation. In the present study, we cloned the full-length cDNA representing the api5 gene from A. sinica (As-api5), which encodes a 372-amino acid protein. In situ hybridization experiments revealed that As-api5 expression is not tissue or organ specific. Quantitative real-time PCR analyses of the developmental expression of As-api5 showed that it reached its highest level at 10h, after which its expression decreased. High salinity and low temperature treatments increased the expression of As-api5. Western blotting was used to assess the abundance of As-API5 and related proteins (As-CyclinA, As-CyclinE, As-E2F1, As-CDK2, As-APAF1, and As-Caspase9). Downregulation of As-api5 expression using a short interfering RNA resulted in increased mortality and embryo malformation of A. sinica. Taken together, the results indicated that API5 plays a crucial role in embryonic diapause termination and early embryo development of A. sinica. Copyright © 2017. Published by Elsevier B.V.

  6. Short communication: expression and alternative splicing of POU1F1 pathway genes in preimplantation bovine embryos.

    PubMed

    Laporta, J; Driver, A; Khatib, H

    2011-08-01

    Early embryo loss is a major contributing factor to cow infertility and that 70 to 80% of this loss occurs between d 8 and 16 postfertilization. However, little is known about the molecular mechanisms and the nature of genes involved in normal and abnormal embryonic development. Moreover, information is limited on the contributions of the genomes of dams and of embryos to the development and survival of preimplantation embryos. We hypothesized that proper gene expression level in the developing embryo is essential for embryo survival and pregnancy success. As such, the characterization of expression profiles in early embryos could lead to a better understanding of the mechanisms involved in normal and abnormal embryo development. To test this hypothesis, 2 d-8 embryo populations (degenerate embryos and blastocysts) that differed in morphology and developmental status were investigated. Expression levels of POU1F1 pathway genes were estimated in 4 sets of biological replicate pools of degenerate embryos and blastocysts. The OPN and STAT5A genes were found to be upregulated in degenerate embryos compared with blastocysts, whereas STAT5B showed similar expression levels in both embryo groups. Analysis of splice variants of OPN and STAT5A revealed expression patterns different from the total expression values of these genes. As such, measuring expression of individual transcripts should be considered in gene expression studies. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  7. Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo apoptosis.

    PubMed

    Ji, Qianqian; Zhu, Kongju; Liu, Zhiguo; Song, Zhenwei; Huang, Yuankai; Zhao, Haijing; Chen, Yaosheng; He, Zuyong; Mo, Delin; Cong, Peiqing

    2013-03-15

    Trichostain A (TSA), an inhibitor of histone deacetylases, improved developmental competence of SCNT embryos in many species, apparently by improved epigenetic reprogramming. The objective of the present study was to determine the effects of TSA-induced apoptosis in cloned porcine embryos. At various developmental stages, a comet assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were used to detect apoptosis, and real-time polymerase chain reaction was used to assess expression of genes related to apoptosis and pluripotency. In this study, TSA significantly induced apoptosis (in a dose-dependent manner) at the one-, two-, and four-cell stages. However, in blastocyst stage embryos, TSA decreased the apoptotic index (P < 0.05). Expression levels of Caspase 3 were higher in TSA-treated versus control embryos at the two-cell stage (not statistically significant). The expression ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of antiapoptotic potential, was higher in TSA-treated groups at the one-, two-, and four-cell and blastocyst stages. Furthermore, expression levels of pluripotency-related genes, namely, Oct4 and Nanog, were elevated at the morula stage (P < 0.05) in TSA treatment groups. We concluded that inducing apoptosis might be a mechanism by which TSA promotes development of reconstructed embryos. At the initial stage of apoptosis induction, abnormal cells were removed, thereby enhancing proliferation of healthy cells and improving embryo quality. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Loss of col8a1a Function during Zebrafish Embryogenesis Results in Congenital Vertebral Malformations

    PubMed Central

    Gray, Ryan S.; Wilm, Thomas; Smith, Jeff; Bagnat, Michel; Dale, Rodney M.; Topczewski, Jacek; Johnson, Stephen L.; Solnica-Krezel, Lilianna

    2014-01-01

    Congenital vertebral malformations (CVM) occur in 1 in 1,000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles (m531, vu41, vu105) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue. PMID:24333517

  9. Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations.

    PubMed

    Gray, Ryan S; Wilm, Thomas P; Smith, Jeff; Bagnat, Michel; Dale, Rodney M; Topczewski, Jacek; Johnson, Stephen L; Solnica-Krezel, Lilianna

    2014-02-01

    Congenital vertebral malformations (CVM) occur in 1 in 1000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles ((m531, vu41, vu105)) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. The painted turtle, Chrysemys picta: a model system for vertebrate evolution, ecology, and human health.

    PubMed

    Valenzuela, Nicole

    2009-07-01

    Painted turtles (Chrysemys picta) are representatives of a vertebrate clade whose biology and phylogenetic position hold a key to our understanding of fundamental aspects of vertebrate evolution. These features make them an ideal emerging model system. Extensive ecological and physiological research provide the context in which to place new research advances in evolutionary genetics, genomics, evolutionary developmental biology, and ecological developmental biology which are enabled by current resources, such as a bacterial artificial chromosome (BAC) library of C. picta, and the imminent development of additional ones such as genome sequences and cDNA and expressed sequence tag (EST) libraries. This integrative approach will allow the research community to continue making advances to provide functional and evolutionary explanations for the lability of biological traits found not only among reptiles but vertebrates in general. Moreover, because humans and reptiles share a common ancestor, and given the ease of using nonplacental vertebrates in experimental biology compared with mammalian embryos, painted turtles are also an emerging model system for biomedical research. For example, painted turtles have been studied to understand many biological responses to overwintering and anoxia, as potential sentinels for environmental xenobiotics, and as a model to decipher the ecology and evolution of sexual development and reproduction. Thus, painted turtles are an excellent reptilian model system for studies with human health, environmental, ecological, and evolutionary significance.

  11. Ancient duplications and functional divergence in the interferon regulatory factors of vertebrates provide insights into the evolution of vertebrate immune systems.

    PubMed

    Du, Kang; Zhong, Zaixuan; Fang, Chengchi; Dai, Wei; Shen, Yanjun; Gan, Xiaoni; He, Shunping

    2018-04-01

    Interferon regulatory factors (IRFs) were first discovered as transcription factors that regulate the transcription of human interferon (IFN)-β. Increasing evidence shows that they might be important players involved in Adaptive immune system (AIS) evolution. Although numbers of IRFs have been identified in chordates, the evolutionary history and functional diversity of this gene family during the early evolution of vertebrates have remained obscure. Using IRF HMM profile and HMMER searches, we identified 148 IRFs in 11 vertebrates and 4 protochordates. For them, we reconstructed the phylogenetic relationships, determined the synteny conservation, investigated the profile of natural selection, and analyzed the expression patterns in four "living fossil" vertebrates: lamprey, elephant shark, coelacanth and bichir. The results from phylogeny and synteny analysis imply that vertebrate IRFs evolved from three predecessors, instead of four as suggested in a previous study, as results from an ancient duplication followed by special expansions and lost during the vertebrate evolution. The profile of natural selection and expression reveals functional dynamics during the process. Together, they suggest that the 2nd whole-genome duplication (2WGD) provided raw materials for innovation in the IRF family, and that the birth of type-I IFN might be an important factor inducing the establishment of IRF-mediated immune networks. As a member involved in the AIS evolution, IRF provide insights into the process and mechanism involved in the complexity and novelties of vertebrate immune systems. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Elucidating the origin of chromosomal aberrations in IVF embryos by preimplantation genetic analysis.

    PubMed

    Frumkin, Tsvia; Malcov, Mira; Yaron, Yuval; Ben-Yosef, Dalit

    2008-01-30

    Preimplantation genetic screening (PGS) has been proposed as a method for improving success rates in patients with repeated IVF failures. This approach is based on the hypothesis that such failures are the result of aneuploid embryos. It has been suggested that FISH analysis of blastomeres removed from preimplantation embryos represent the chromosomal constitution of the entire embryo. However, it is not yet clear whether it also represents the chromosomal constitution of the implanted embryo. PGS reanalysis on day 5 of embryos designated as "aneuploid" on day 3 may demonstrate a high rate of mosaicism for chromosomal aberration. Some of these mosaic embryos are capable of developing into normal embryos by "self-correction". Others, however, may accumulate additional chromosomal anomalies. It is therefore concluded that the chromosomal constitution of a preimplantation embryo may evolve during early cleavages. Meiotic and post zygotic mitotic errors may account for these chromosomal aberrations. This review will focus on elucidating the origin of chromosomal changes during preimplantation embryo development by studying their chromosomal constitution at different stages.

  13. [Calcium in the developing skeletal muscles of the chick embryo].

    PubMed

    Samosudova, N V; Enenko, S O; Larin, Iu S; Shungskaia, V E

    1982-07-01

    The osmium-pyroantimonate technique was used for the ultrastructural study of Ca2+-localization in two types of chick embryo skeletal muscles: m. pectoralis and m. soleus. In 8- and 12-day old embryos the pyroantimonate precipitate was found on plasmalemma, condensed chromatine and ribosomes and in N-lines of I-band. During myogenesis (15-, 21-day old embryos) the calcium precipitate is redistributed from the above mentioned sites to terminal cisternae and N-line of I-band. It is proposed that calcium of N-lines may be involved in the glycogenolysis, its association with the muscle contraction occurring particularly at early developmental stages.

  14. Evolution of the β-adrenoreceptors in vertebrates.

    PubMed

    Zavala, Kattina; Vandewege, Michael W; Hoffmann, Federico G; Opazo, Juan C

    2017-01-01

    The study of the evolutionary history of genes related to human disease lies at the interface of evolution and medicine. These studies provide the evolutionary context on which medical researchers should work, and are also useful in providing information to suggest further genetic experiments, especially in model species where genetic manipulations can be made. Here we studied the evolution of the β-adrenoreceptor gene family in vertebrates with the aim of adding an evolutionary framework to the already abundant physiological information. Our results show that in addition to the three already described vertebrate β-adrenoreceptor genes there is an additional group containing cyclostome sequences. We suggest that β-adrenoreceptors diversified as a product of the two whole genome duplications that occurred in the ancestor of vertebrates. Gene expression patterns are in general consistent across species, suggesting that expression dynamics were established early in the evolutionary history of vertebrates, and have been maintained since then. Finally, amino acid polymorphisms that are associated to pathological conditions in humans appear to be common in non-human mammals, suggesting that the phenotypic effects of these mutations depend on epistatic interaction with other positions. The evolutionary analysis of the β-adrenoreceptors delivers new insights about the diversity of these receptors in vertebrates, the evolution of the expression patterns and a comparative perspective regarding the polymorphisms that in humans are linked to pathological conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Localization and expression of peroxiredoxin II in the mouse ovary, oviduct, uterus, and preimplantation embryo.

    PubMed

    Wang, Shie; Huang, Weiquan; Shi, Hexiu; Lin, Cuiying; Xie, Meirong; Wang, Jianxin

    2010-02-01

    Peroxiredoxin (Prx) II belongs to a recently discovered family of peroxidases that play important roles in antioxidation and signal transduction. In this study, we aimed to study the localization and expression of Prx II in the mouse ovary, oviduct, and uterus, and preimplantation embryos. Immunohistochemical staining analysis showed that, in the ovary, Prx II was expressed in the oocyte cytoplasm of the primary follicle, the secondary follicle, and the premature follicle; Prx II was expressed in germinal vesicle-intact oocytes (GV oocytes) and metaphase II eggs (MII eggs), as well as at various stages in early embryos. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that the Prx II mRNA was expressed at a high level in GV eggs, slightly lower levels in MII eggs, and had no detectable expression in four-cell embryos and early blastocysts. In the oviduct, Prx II was expressed in the epithelia, while in the uterus Prx II was mainly distributed in the endometrial stroma. Taken together, our results suggest that Prx II plays a key antioxidation role in the maturation of oocytes and development of early embryos, thus providing crucial experimental evidence for further exploring the function of Prx II in the development of oocytes and preimplantation embryos. 2009 Wiley-Liss, Inc.

  16. [Effect of TGF-beta1 on embryo implantation and development in mice in vitro].

    PubMed

    Luo, Shan; Yin, Hai-ning; Li, Shang-wei

    2010-03-01

    To investigate the role of TGF-beta1 in embryo implantation and development in vitro in mice. Mouse embryos at 2-cell stage were cultured in the media of M16 with exposure to different levels of TGF-beta1 (0, 1, 10 and 50 ng/mL). The percentage of embryos reaching fixed stages (early blastocyst, expanding blastocyst and hatched blastocyst) was monitored 68 h and 92 h after the culture. The expanding blastocys cultured for 68 h in M16 without TGF-beta1 and those with 10 ng/mL of TGF-beta1 were transferred to pseudopregnant mice. On the 6th day post transfer, the successful rates of implantation were counted. The level of IL-10/IFN-gamma in the serum and maternal-fetus interface of the mice was detected by ELISA on the 6th day post transfer. TGF-beta1 improved embryo growth in vitro. TGF-beta1 at a level of 10 ng/mL had the maximum impact, with 15.6%, 68.09%, 1.42% of embryos reaching early, expanding, and hatched stage, respectively, 68 h after culture, and 6.38%, 28.37%, 53.19% of embryos reaching early, expanding, and hatched stage, respectively, 92 h after culture. The promoting effect declined when TGF-beta1 reached 50 ng/mL. The successful rate of implantation of embryos cultured in M16 with TGF-beta1 was significantly higher than those cultured in M16 without TGF-beta1 (35. 2% vs. 17.19%, P < 0.05). The embryos cultured in M16 with TGF-beta1 had significantly lower level of IFN-gamma in the maternal-fetus interface than those cultured in M16 without TGF-beta1 [(30.89 +/- 11.31) pg/mL vs. (43.23 +/- 18. 09) pg/mL, P < 0.053. TGF-beta1 at an appropriate dose improves embryo implantation in mice in vitro. The mechanism may involve the improvement of the quality of embryos and their development, and decrease of IFN-gamma synthesis in maternal-fetal interface, a chemical that could cause Th2 bias.

  17. Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos.

    PubMed

    Liu, Chengdong; Luan, Jing; Bai, Yan; Li, Yun; Lu, Ling; Liu, Yunzhang; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Duan, Cunming; Zhou, Jianfeng

    2014-02-01

    The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Time to take human embryo culture seriously.

    PubMed

    Sunde, Arne; Brison, Daniel; Dumoulin, John; Harper, Joyce; Lundin, Kersti; Magli, M Cristina; Van den Abbeel, Etienne; Veiga, Anna

    2016-10-01

    Is it important that end-users know the composition of human embryo culture media? We argue that there is as strong case for full transparency concerning the composition of embryo culture media intended for human use. Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring. A review of the literature was carried out. Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made. The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations. There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring. Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the

  19. The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo.

    PubMed

    Haillot, Emmanuel; Molina, Maria Dolores; Lapraz, François; Lepage, Thierry

    2015-01-01

    Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1

  20. The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

    PubMed Central

    Haillot, Emmanuel; Molina, Maria Dolores; Lapraz, François; Lepage, Thierry

    2015-01-01

    Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1

  1. Immunolocalization and expression of Na(+)/K(+) -ATPase in embryos, early larval stages and adults of the freshwater shrimp Palaemonetes argentinus (Decapoda, Caridea, Palaemonidae).

    PubMed

    Ituarte, Romina Belén; Lignot, Jehan-Hervé; Charmantier, Guy; Spivak, Eduardo; Lorin-Nebel, Catherine

    2016-06-01

    The euryhaline shrimp Palaemonetes argentinus exemplifies an evolutionary transition from brackish to freshwater habitats that requires adequate osmoregulatory capacities. Hyperosmoregulation is functional at hatching and it likely begins during the embryonic phase allowing this species to develop entirely in fresh water. Here, we investigated the Na(+)/K(+)-ATPase α-subunit gene (nka-α) expression using quantitative real-time PCR and localized Na(+)/K(+)-ATPase (NKA) in ion-transporting epithelia through immunofluorescence microscopy. We reared shrimps from spawning to juvenile stages at two salinities (1, 15 ‰) and maintained adults for 3 weeks at three salinity treatments (1, 15, 25 ‰). nka-α gene expression was measured in: (1) embryos at an early (SI), intermediate (SII) and late (SIII) stage of embryonic development; (2) newly hatched larvae (Zoea I, ZI); and (3) isolated gill tissue of adults. The nka-α expression was low in SI and SII embryos and reached maximum levels prior to hatching (SIII), which were similar to expression levels detected in the ZI. The nka-α expression in SIII and ZI was highest at 15 ‰, whereas salinity did not affect expression in earlier embryos. In SIII, in ZI and in a later zoeal stage ZIV, NKA was localized in epithelial cells of pleurae, in the inner-side epithelium of branchiostegite and in the antennal glands. Gills appeared in the ZIV but NKA immunolabeling of the cells of the gill shaft occurred in a subsequent developmental larval stage, the decapodid. Extrabranchial organs constitute the main site of osmoregulation in early ontogenetic stages of this freshwater shrimp.

  2. Transcriptome analysis of vertebral bone in the flounder, Paralichthys olivaceus (Teleostei, Pleuronectiformes), using Illumina sequencing.

    PubMed

    Ibaraki, Harumi; Wu, Xiaoming; Uji, Susumu; Yokoi, Hayato; Sakai, Yoshifumi; Suzuki, Tohru

    2015-12-01

    The processes underlying vertebral development in teleosts and tetrapods differ markedly in a variety of ways. At present, the molecular basis of teleost vertebral development and growth is poorly understood. Understanding vertebral development at the molecular level is important for aquaculture to prevent vertebral anomalies that can arise from a variety of factors, including excess vitamin A (all-trans retinol, VA) in the diet. To facilitate studies on teloest vertebral development, we performed transcriptome analysis of four month old flounder, Paralichthys olivaceus, vertebrae using next-generation sequencing. Expression profile obtained demonstrates that some members of the hh, bmp, fgf, wnt gene families, and their receptors, hox, pax, sox, dlx and tbx gene families and ntl, which are known to function in notochord and somite development in embryos, are expressed in the vertebrae. It was also showed that in addition to the retinoic acid receptor (Rar), the vertebrae express alcohol dehydrogenase 1 and retinal dehydrogenase 2 which convert VA to all-trans-retinoic acid (RA). The assembled contigs also included cytochrome p450 family members, which inactivate RA, as well as phosphatidylcholine-retinol O-acetyltransferase, which converts VA to all-trans-retinyl ester, a stock form of VA. These data suggest that in teleost vertebrae, expression of various signals and transcription factors which function in the notochord and somite development is maintained until adult stage, and RA metabolism and signaling are active to regulate transcription of RA-responsible genes, such as hedgehog and hox genes. This is the first transcriptome analysis of teleost fish vertebrae. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. The largest Silurian vertebrate and its palaeoecological implications

    PubMed Central

    Choo, Brian; Zhu, Min; Zhao, Wenjin; Jia, Liaotao; Zhu, You'an

    2014-01-01

    An apparent absence of Silurian fishes more than half-a-metre in length has been viewed as evidence that gnathostomes were restricted in size and diversity prior to the Devonian. Here we describe the largest pre-Devonian vertebrate (Megamastax amblyodus gen. et sp. nov.), a predatory marine osteichthyan from the Silurian Kuanti Formation (late Ludlow, ~423 million years ago) of Yunnan, China, with an estimated length of about 1 meter. The unusual dentition of the new form suggests a durophagous diet which, combined with its large size, indicates a considerable degree of trophic specialisation among early osteichthyans. The lack of large Silurian vertebrates has recently been used as constraint in palaeoatmospheric modelling, with purported lower oxygen levels imposing a physiological size limit. Regardless of the exact causal relationship between oxygen availability and evolutionary success, this finding refutes the assumption that pre-Emsian vertebrates were restricted to small body sizes. PMID:24921626

  4. Stronger net posterior cortical forces and asymmetric microtubule arrays produce simultaneous centration and rotation of the pronuclear complex in the early Caenorhabditis elegans embryo

    PubMed Central

    Coffman, Valerie C.; McDermott, Matthew B. A.; Shtylla, Blerta; Dawes, Adriana T.

    2016-01-01

    Positioning of microtubule-organizing centers (MTOCs) incorporates biochemical and mechanical cues for proper alignment of the mitotic spindle and cell division site. Current experimental and theoretical studies in the early Caenorhabditis elegans embryo assume remarkable changes in the origin and polarity of forces acting on the MTOCs. These changes must occur over a few minutes, between initial centration and rotation of the pronuclear complex and entry into mitosis, and the models do not replicate in vivo timing of centration and rotation. Here we propose a model that incorporates asymmetry in the microtubule arrays generated by each MTOC, which we demonstrate with in vivo measurements, and a similar asymmetric force profile to that required for posterior-directed spindle displacement during mitosis. We find that these asymmetries are capable of and important for recapitulating the simultaneous centration and rotation of the pronuclear complex observed in vivo. The combination of theoretical and experimental evidence provided here offers a unified framework for the spatial organization and forces needed for pronuclear centration, rotation, and spindle displacement in the early C. elegans embryo. PMID:27733624

  5. Brachyury expression in tailless Molgulid ascidian embryos.

    PubMed

    Takada, Norio; York, Jonathan; Davis, J Muse; Schumpert, Brenda; Yasuo, Hitoyoshi; Satoh, Nori; Swalla, Billie J

    2002-01-01

    The T-box transcription factor gene Brachyury is important for the differentiation of notochord in all chordates, including the ascidians Halocynthia roretzi and Ciona intestinalis. We isolated Brachyury from molgulid ascidians, which have evolved tailless larvae multiple times independently, and found the genes appear functional by cDNA sequence analyses. We then compared the expression of Mocu-Bra in tailed Molgula oculata embryos to two tailless species, Molgula occulta (Mocc-Bra) and Molgula tectiformis (Mt-Bra). Here we show that both tailless species express Brachyury in the notochord lineage during embryogenesis. Initial expression of Mocu-Bra is normal in tailed M. oculata embryos; 10 precursor notochord cells divide twice to result in 40 notochord cells that converge and extend to make a notochord down the center of the tail. In contrast, in tailless Molgula occulta, Mocc-Bra expression disappears prematurely, and there is only one round of division, resulting in 20 cells in the final notochord lineage that never converge or extend. In M. occulta x M. oculata hybrid embryos, expression of Mocu-Bra is prolonged, and the embryos form a tail with 20 notochord cells that converge and extend normally. However, in Molgula tectiformis, a different tailless ascidian, Mt-Bra was expressed only in the 10 notochord precursor cells, which never divide, converge, or extend. In summary, neither Brachyury function nor the early establishment of the notochord lineage appears to be impaired in tailless embryos. In light of these results, we are continuing to investigate how and why notochord development is lost in tailless molgulid ascidian embryos.

  6. Critical role of mTOR, PPARγ and PPARδ signaling in regulating early pregnancy decidual function, embryo viability and feto-placental growth.

    PubMed

    Roberti, Sabrina L; Higa, Romina; White, Verónica; Powell, Theresa L; Jansson, Thomas; Jawerbaum, Alicia

    2018-06-01

    What are the consequences of inhibiting mTOR, the mechanistic target of rapamycin (mTOR), and the peroxisome proliferator activated receptor gamma (PPARγ) and PPARδ pathways in the early post-implantation period on decidual function, embryo viability and feto-placental growth in the rat? mTOR inhibition from Days 7 to 9 of pregnancy in rats caused decidual PPARγ and PPARδ upregulation on Day 9 of pregnancy and resulted in embryo resorption by Day 14 of pregnancy. PPARγ and PPARδ inhibition differentially affected decidual mTOR signaling and levels of target proteins relevant to lipid histotrophic nutrition and led to reduced feto-placental weights on Day 14 of pregnancy. Although mTOR, PPARγ and PPARδ are nutrient sensors important during implantation, the role of these signaling pathways in decidual function and how they interact in the early post-implantation period are unknown. Perilipin 2 (PLIN2) and fatty acid binding protein 4 (FABP4), two adipogenic proteins involved in lipid histotrophic nutrition, are targets of mTOR and PPAR signaling pathways in a variety of tissues. Rapamycin (mTOR inhibitor, 0.75 mg/kg, sc), T0070907 (PPARγ inhibitor, 0.001 mg/kg, sc), GSK0660 (PPARδ inhibitor, 0.1 mg/kg, sc) or vehicle was injected daily to pregnant rats from Days 7 to 9 of pregnancy and the studies were performed on Day 9 of pregnancy (n = 7 per group) or Day 14 of pregnancy (n = 7 per group). On Day 9 of pregnancy, rat decidua were collected and prepared for western blot and immunohistochemical studies. On Day 14 of pregnancy, the resorption rate, number of viable fetuses, crown-rump length and placental and decidual weights were determined. Inhibition of mTOR in the early post-implantation period led to a reduction in FABP4 protein levels, an increase in PLIN2 levels and an upregulation of PPARγ and PPARδ in 9-day-pregnant rat decidua. Most embryos were viable on Day 9 of pregnancy but had resorbed by Day 14 of pregnancy. This denotes a key function of

  7. Loss of maternal CTCF is associated with peri-implantation lethality of Ctcf null embryos.

    PubMed

    Moore, James M; Rabaia, Natalia A; Smith, Leslie E; Fagerlie, Sara; Gurley, Kay; Loukinov, Dmitry; Disteche, Christine M; Collins, Steven J; Kemp, Christopher J; Lobanenkov, Victor V; Filippova, Galina N

    2012-01-01

    CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

  8. Entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kao, K.R.; Elinson, R.P.

    1988-05-01

    The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland. In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted intomore » ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise.« less

  9. The characters of Palaeozoic jawed vertebrates

    PubMed Central

    Brazeau, Martin D; Friedman, Matt

    2014-01-01

    Newly discovered fossils from the Silurian and Devonian periods are beginning to challenge embedded perceptions about the origin and early diversification of jawed vertebrates (gnathostomes). Nevertheless, an explicit cladistic framework for the relationships of these fossils relative to the principal crown lineages of the jawed vertebrates (osteichthyans: bony fishes and tetrapods; chondrichthyans: sharks, batoids, and chimaeras) remains elusive. We critically review the systematics and character distributions of early gnathostomes and provide a clearly stated hierarchy of synapomorphies covering the jaw-bearing stem gnathostomes and osteichthyan and chondrichthyan stem groups. We show that character lists, designed to support the monophyly of putative groups, tend to overstate their strength and lack cladistic corroboration. By contrast, synapomorphic hierarchies are more open to refutation and must explicitly confront conflicting evidence. Our proposed synapomorphy scheme is used to evaluate the status of the problematic fossil groups Acanthodii and Placodermi, and suggest profitable avenues for future research. We interpret placoderms as a paraphyletic array of stem-group gnathostomes, and suggest what we regard as two equally plausible placements of acanthodians: exclusively on the chondrichthyan stem, or distributed on both the chondrichthyan and osteichthyan stems. PMID:25750460

  10. A history of normal plates, tables and stages in vertebrate embryology

    PubMed Central

    HOPWOOD, NICK

    2006-01-01

    Developmental biology is today unimaginable without the normal stages that define standard divisions of development. This history of normal stages, and the related normal plates and normal tables, shows how these standards have shaped and been shaped by disciplinary change in vertebrate embryology. The article highlights the Normal Plates of the Development of the Vertebrates edited by the German anatomist Franz Keibel (16 volumes, 1897–1938). These were a major response to problems in the relations between ontogeny and phylogeny that amounted in practical terms to a crisis in staging embryos, not just between, but (for some) also within species. Keibel’s design adapted a plate by Wilhelm His and tables by Albert Oppel in order to go beyond the already controversial comparative plates of the Darwinist propagandist Ernst Haeckel. The project responded to local pressures, including intense concern with individual variation, but recruited internationally and mapped an embryological empire. Though theoretically inconclusive, the plates became standard laboratory tools and forged a network within which the Institut International d’Embryologie (today the International Society of Developmental Biologists) was founded in 1911. After World War I, experimentalists, led by Ross Harrison and Viktor Hamburger, and human embryologists, especially George Streeter at the Carnegie Department of Embryology, transformed Keibel’s complex, bulky tomes to suit their own contrasting demands. In developmental biology after World War II, normal stages—reduced to a few journal pages—helped domesticate model organisms. Staging systems had emerged from discussions that questioned the very possibility of assigning an embryo to a stage. The historical issues resonate today as developmental biologists work to improve and extend stage series, to make results from different laboratories easier to compare and to take individual variation into account. PMID:17183461

  11. A history of normal plates, tables and stages in vertebrate embryology.

    PubMed

    Hopwood, Nick

    2007-01-01

    Developmental biology is today unimaginable without the normal stages that define standard divisions of development. This history of normal stages, and the related normal plates and normal tables, shows how these standards have shaped and been shaped by disciplinary change in vertebrate embryology. The article highlights the Normal Plates of the Development of the Vertebrates edited by the German anatomist Franz Keibel (16 volumes, 1897-1938). These were a major response to problems in the relations between ontogeny and phylogeny that amounted in practical terms to a crisis in staging embryos, not just between, but (for some) also within species. Keibel's design adapted a plate by Wilhelm His and tables by Albert Oppel in order to go beyond the already controversial comparative plates of the Darwinist propagandist Ernst Haeckel. The project responded to local pressures, including intense concern with individual variation, but recruited internationally and mapped an embryological empire. Though theoretically inconclusive, the plates became standard laboratory tools and forged a network within which the Institut International d'Embryologie (today the International Society of Developmental Biologists) was founded in 1911. After World War I, experimentalists, led by Ross Harrison and Viktor Hamburger, and human embryologists, especially George Streeter at the Carnegie Department of Embryology, transformed Keibel's complex, bulky tomes to suit their own contrasting demands. In developmental biology after World War II, normal stages-reduced to a few journal pages-helped domesticate model organisms. Staging systems had emerged from discussions that questioned the very possibility of assigning an embryo to a stage. The historical issues resonate today as developmental biologists work to improve and extend stage series, to make results from different laboratories easier to compare and to take individual variation into account.

  12. Evolution and the origin of the visual retinoid cycle in vertebrates.

    PubMed

    Kusakabe, Takehiro G; Takimoto, Noriko; Jin, Minghao; Tsuda, Motoyuki

    2009-10-12

    Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called 'visual cycle'. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.

  13. EVALUATING THE EFFECTS OF FLY ASH EXPOSURE ON FISH EARLY LIFE STAGES: FATHEAD MINNOW EMBRYO-LARVAL TESTS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Greeley Jr, Mark Stephen; Elmore, Logan R; McCracken, Kitty

    2012-05-01

    On December 22, 2008, a dike containing fly ash and bottom ash in an 84-acre complex of the Tennessee Valley Authority's (TVA) Kingston Steam Plant in East Tennessee failed and released a large quantity of ash into the adjacent Emory River. Ash deposits extended as far as 4 miles upstream (Emory River mile 6) of the Plant, and some ash was carried as far downstream as Tennessee River mile 564 ({approx}4 miles downstream of the Tennessee River confluence with the Clinch River). A byproduct of coal burning power plants, fly ash contains a variety of metals and other elements which,more » at sufficient concentrations and in specific forms, can be toxic to biological systems. The effects of fly ash contamination on exposed fish populations depend on the magnitude and duration of exposure, with the most significant risk considered to be the effects of specific ash constituents, especially selenium, on fish early life stages. Uptake by adult female fish of fly ash constituents through the food chain and subsequent maternal transfer of contaminants to the developing eggs is thought to be the primary route of selenium exposure to larval fish (Woock and others 1987, Coyle and others 1993, Lemly 1999, Moscatello and others 2006), but direct contact of the fertilized eggs and developing embryos to ash constituents in river water and sediments is also a potential risk factor (Woock and others 1987, Coyle and others 1993, Jezierska and others 2009). To address the risk of fly ash from the Kingston spill to the reproductive health of downstream fish populations, ORNL has undertaken a series of studies in collaboration with TVA including: (1) a field study of the bioaccumulation of fly ash constituents in fish ovaries and the reproductive condition of sentinel fish species in reaches of the Emory and Clinch Rivers affected by the fly ash spill; (2) laboratory tests of the potential toxicity of fly ash from the spill area on fish embryonic and larval development (reported in the

  14. [A comparative analysis of notochord formation in amphibian embryos].

    PubMed

    Novoselov, V V

    1992-01-01

    We studied the origin, structure, and development of the notochord in Pleurodeles waltlii (Urodela) and Xenopus laevis (Anura) embryos. The notochord rudiment is formed in both species at the early gastrula stage as a cluster of polarized chorda-mesoderm cells located along the sagittal plane of the embryo. In Pl. waltlii the notochord rudiment is separated from the gastrocoele roof as a result of contraction of apical cell surfaces. The contraction wave spreads forward and backward along craniocaudal axis, i.e., segregation of the notochord rudiment progresses in two directions simultaneously. Similar process takes place in X. laevis embryos; however, propagation of the contraction wave in the anterior part of the body somewhat differs from that in the posterior part. While the "anterior" contraction wave resembles that in Pl. waltlii embryos, progression of the wave in the posterior part of the body is distinguished by a closer association of the notochord rudiment with ectoderm and the presence of its delamination boundaries with the somite mesoderm.

  15. Perturbation of metabonome of embryo/larvae zebrafish after exposure to fipronil.

    PubMed

    Yan, Lu; Gong, Chenxue; Zhang, Xiaofeng; Zhang, Quan; Zhao, Meirong; Wang, Cui

    2016-12-01

    The escalating demand for fipronil by the increasing insects' resistance to synthetic pyrethroids placed a burden on aquatic vertebrates. Although awareness regarding the toxicity of fipronil to fish is arising, the integral alteration caused by fipronil remains unexplored. Here, we investigated on the development toxicity of fipronil and the metabolic physiology perturbation at 120h post fertilization through GC-MS metabolomics on zebrafish embryo. We observed that fipronil dose-dependently induced malformations including uninflated swim bladder and bent spine. Further, the "omic" technique hit 26 differential metabolites after exposure to fipronil and five significant signaling pathways. We speculated that changes in primary bile acid synthesis pathway and the content of saturated fatty acid in the chemical-related group indicated the liver toxicity. Pathway of Aminoacyl-tRNA biosynthesis changed by fipronil may relate to the macromolecular synthesis. Concurrently, methane metabolism pathway was also identified while the role in zebrafish needs further determination. Overall, this study revealed several new signaling pathways in fipronil-treated zebrafish embryo/larval. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Early results after vertebral body stenting for fractures of the anterior column of the thoracolumbar spine.

    PubMed

    Klezl, Zdenek; Majeed, Haroon; Bommireddy, Rajendranath; John, Joby

    2011-10-01

    Vertebroplasty and balloon kyphoplasty have shown to improve pain and functional outcome in cases with symptomatic vertebral fractures. Although restoration of the vertebral body height and kyphosis seemed to be easier with balloon kyphoplasty, it became clear that some of the correction achieved by the balloon is lost once it was deflated. Vertebral body stent was developed to eliminate this phenomenon. To our knowledge this is the first study in describing this technique in clinical settings. Seventeen patients with 20 fractured vertebral bodies were included. All fractures were Type A1.3 or A3.1 (incomplete burst). Information about pain (visual analogue scale-VAS) and function (Oswestry disability index-ODI) and vertebral body deformity (vertebral angle-VA) was recorded in a prospective way at regular intervals. Patients were classified into osteoporotic group (7 patients) and traumatic groups (10 patients, younger than 60 years). There were 6 male and 11 female patients with mean age of 58.1 years (31-88 years). Mean follow up was 12 months. The preoperative pain level showed a mean VAS score of 8.9 in osteoporotic group and 9.7 in traumatic group. Postoperatively, in osteoporotic group, mean VAS was 4.8 at 6 weeks, 4.0 at 6 months and 2.5 at 12 months compared with traumatic fracture group where it was 2.7 at 6 weeks, 2.2 at 6 months and 1.6 at 12 months. Mean ODI in osteoporotic group was 41.7% (14-58%) and in traumatic group it was 20.4% (6-33%). Mean vertebral body angle prior to surgery in osteoporotic group was 9.7 whilst postoperatively it was 5.2°; so the mean correction achieved was 4.5°. In traumatic group preoperative VA was 13° whilst postoperatively it was 5.7°; therefore the mean correction achieved was 7.3°. None of the patients lost reduction at their last follow up. Vertebral body stenting leads to satisfactory improvement in pain, function and kyphosis correction in the treatment of osteoporotic and traumatic fractures. Anterior spinal

  17. Fish embryo toxicity of carbamazepine, diclofenac and metoprolol.

    PubMed

    van den Brandhof, Evert-Jan; Montforts, Mark

    2010-11-01

    Frequently measured pharmaceuticals in environmental samples were tested in fish embryo toxicity (FET) tests with Danio rerio, based on the draft OECD test protocol. In this FET test 2-h-old zebrafish embryos were exposed for 72 h to carbamazepine, diclofenac and metoprolol to observe effects on embryo mortality, gastrulation, somite formation, tail movement and detachment, pigmentation, heartbeat, malformation of head, otoliths and heart, scoliosis, deformity of yolk, and hatching success at 24, 48 and 72 h. We found specific effects on growth retardation above 30.6 mg/l for carbamazepine, on hatching, yolk sac and tail deformation above 1.5mg/l for diclofenac, and on scoliosis and growth retardation above 12.6 mg/l for metoprolol. Scoring all effect parameters, the 72-h-EC(50) values were: for carbamazepine 86.5mg/l, for diclofenac 5.3mg/l and for metoprolol 31.0mg/l (mean measured concentrations). In conclusion, our results for carbamazepine and metoprolol are in agreement with other findings for aquatic toxicity, and also fish embryos responded in much the same way as rat embryos did. For diclofenac, the FET test performs comparably to Early Life Stage testing. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. Ectodermal Wnt6 is an early negative regulator of limb chondrogenesis in the chicken embryo

    PubMed Central

    2010-01-01

    Background Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis. Results In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II. Conclusion Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm. PMID:20334703

  19. A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG.

    PubMed

    Rumfelt, L L; Avila, D; Diaz, M; Bartl, S; McKinney, E C; Flajnik, M F

    2001-02-13

    In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM(1gj), from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells ("germline-joined"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H(1gj) in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H(1gj) chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM(1gj). Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

  20. A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG

    PubMed Central

    Rumfelt, Lynn L.; Avila, David; Diaz, Marilyn; Bartl, Simona; McKinney, E. Churchill; Flajnik, Martin F.

    2001-01-01

    In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM1gj, from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells (“germline-joined”). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H1gj in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H1gj chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM1gj. Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system. PMID:11172027

  1. Juxtaposed Polycomb complexes co-regulate vertebral identity.

    PubMed

    Kim, Se Young; Paylor, Suzanne W; Magnuson, Terry; Schumacher, Armin

    2006-12-01

    Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 and EED, pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity.

  2. Forskolin improves the cryosurvival of in vivo-derived porcine embryos at very early stages using two vitrification methods.

    PubMed

    Gomis, J; Cuello, C; Sanchez-Osorio, J; Gil, M A; Parrilla, I; Angel, M A; Vazquez, J M; Roca, J; Martinez, E A

    2013-04-01

    This study was aimed to determine the effect of forskolin on the viability of in vivo-derived porcine embryos vitrified by the superfine open pulled straw (SOPS) or solid surface vitrification (SSV) methods at the 2-cell, 4-cell, and blastocyst stages. Zygotes, 2- to 4-cell embryos, and morulae were obtained from superovulated sows. After collection, embryos were cultured for 24h with 0 or 10 μM forskolin and then vitrified using the SOPS and SSV method, or not vitrified (fresh controls). Fresh and vitrified-warmed 2-cells, 4-cells, and blastocysts were cultured for additional 96 h, 72 h and 24 h, respectively. At the end of the culture, embryos were evaluated for progression to the blastocyst stage and total cell number. The vitrification method did not affect any of the parameters evaluated for any embryo stage. Forskolin increased (P<0.01) the blastocyst formation and the final developmental stage of vitrified 2- and 4-cell embryos. However, these embryos exhibited lower (P<0.003) blastocyst formation rates than their fresh counterparts. The total cell number and hatching rate were similar in both groups (vitrified and fresh) of 2- and 4-cell embryos. Vitrified blastocysts exhibited viabilities, final developmental stages, hatching rates, and total cell numbers that were similar to those of their fresh counterparts, regardless of the addition of forskolin. In conclusion, the SOPS and SSV methods are suitable for the cryopreservation of in vivo-derived 2- to 4-cell porcine embryos. Pre-treatment with forskolin for 24h before vitrification improves the cryotolerance of 2- and 4-cell porcine embryos. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Tracing the evolutionary origin of vertebrate skeletal tissues: insights from cephalochordate amphioxus.

    PubMed

    Yong, Luok Wen; Yu, Jr-Kai

    2016-08-01

    Vertebrate mineralized skeletal tissues are widely considered as an evolutionary novelty. Despite the importance of these tissues to the adaptation and radiation of vertebrate animals, the evolutionary origin of vertebrate skeletal tissues remains largely unclear. Cephalochordates (Amphioxus) occupy a key phylogenetic position and can serve as a valuable model for studying the evolution of vertebrate skeletal tissues. Here we summarize recent advances in amphioxus developmental biology and comparative genomics that can help to elucidate the evolutionary origins of the vertebrate skeletal tissues and their underlying developmental gene regulatory networks (GRN). By making comparisons to the developmental studies in vertebrate models and recent discoveries in paleontology and genomics, it becomes evident that the collagen matrix-based connective tissues secreted by the somite-derived cells in amphioxus likely represent the rudimentary skeletal tissues in chordates. We propose that upon the foundation of this collagenous precursor, novel tissue mineralization genes that arose from gene duplications were incorporated into an ancestral mesodermal GRN that makes connective and supporting tissues, leading to the emergence of highly-mineralized skeletal tissues in early vertebrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Role of glucose in mouse preimplantation embryo development.

    PubMed

    Martin, K L; Leese, H J

    1995-04-01

    Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca x C57BL/6) were cultured from the one- and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D + L-lactate, in the presence and absence of 1 mM glucose (M16 + G and M16 - G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16 - G were transferred to M16 + G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16 + G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16 - G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 +/- 2.5 [28] vs. 105 +/- 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16 - G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16 - G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16 + G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Patterning in time and space: HoxB cluster gene expression in the developing chick embryo.

    PubMed

    Gouveia, Analuce; Marcelino, Hugo M; Gonçalves, Lisa; Palmeirim, Isabel; Andrade, Raquel P

    2015-01-01

    The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space.

  6. Patterning in time and space: HoxB cluster gene expression in the developing chick embryo

    PubMed Central

    Gouveia, Analuce; Marcelino, Hugo M; Gonçalves, Lisa; Palmeirim, Isabel; Andrade, Raquel P

    2015-01-01

    The developing embryo is a paradigmatic model to study molecular mechanisms of time control in Biology. Hox genes are key players in the specification of tissue identity during embryo development and their expression is under strict temporal regulation. However, the molecular mechanisms underlying timely Hox activation in the early embryo remain unknown. This is hindered by the lack of a rigorous temporal framework of sequential Hox expression within a single cluster. Herein, a thorough characterization of HoxB cluster gene expression was performed over time and space in the early chick embryo. Clear temporal collinearity of HoxB cluster gene expression activation was observed. Spatial collinearity of HoxB expression was evidenced in different stages of development and in multiple tissues. Using embryo explant cultures we showed that HoxB2 is cyclically expressed in the rostral presomitic mesoderm with the same periodicity as somite formation, suggesting a link between timely tissue specification and somite formation. We foresee that the molecular framework herein provided will facilitate experimental approaches aimed at identifying the regulatory mechanisms underlying Hox expression in Time and Space. PMID:25602523

  7. Pleistocene vertebrates of the Yukon Territory

    NASA Astrophysics Data System (ADS)

    Harington, C. R.

    2011-08-01

    Unglaciated parts of the Yukon constitute one of the most important areas in North America for yielding Pleistocene vertebrate fossils. Nearly 30 vertebrate faunal localities are reviewed spanning a period of about 1.6 Ma (million years ago) to the close of the Pleistocene some 10 000 BP (radiocarbon years before present, taken as 1950). The vertebrate fossils represent at least 8 species of fishes, 1 amphibian, 41 species of birds and 83 species of mammals. Dominant among the large mammals are: steppe bison ( Bison priscus), horse ( Equus sp.), woolly mammoth ( Mammuthus primigenius), and caribou ( Rangifer tarandus) - signature species of the Mammoth Steppe fauna ( Fig. 1), which was widespread from the British Isles, through northern Europe, and Siberia to Alaska, Yukon and adjacent Northwest Territories. The Yukon faunas extend from Herschel Island in the north to Revenue Creek in the south and from the Alaskan border in the west to Ketza River in the east. The Yukon holds evidence of the earliest-known people in North America. Artifacts made from bison, mammoth and caribou bones from Bluefish Caves, Old Crow Basin and Dawson City areas show that people had a substantial knowledge of making and using bone tools at least by 25 000 BP, and possibly as early as 40 000 BP. A suggested chronological sequence of Yukon Pleistocene vertebrates ( Table 1) facilitates comparison of selected faunas and indicates the known duration of various taxa.

  8. Using Ambystoma mexicanum (Mexican Axolotl) Embryos, Chemical Genetics, and Microarray Analysis to Identify Signaling Pathways Associated with Tissue Regeneration

    PubMed Central

    Ponomareva, Larissa V.; Athippozhy, Antony; Thorson, Jon S.; Voss, S. Randal

    2015-01-01

    Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7 days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. PMID:26092703

  9. Evolution of developmental regulation in the vertebrate FgfD subfamily.

    PubMed

    Jovelin, Richard; Yan, Yi-Lin; He, Xinjun; Catchen, Julian; Amores, Angel; Canestro, Cristian; Yokoi, Hayato; Postlethwait, John H

    2010-01-15

    Fibroblast growth factors (Fgfs) encode small signaling proteins that help regulate embryo patterning. Fgfs fall into seven families, including FgfD. Nonvertebrate chordates have a single FgfD gene; mammals have three (Fgf8, Fgf17, and Fgf18); and teleosts have six (fgf8a, fgf8b, fgf17, fgf18a, fgf18b, and fgf24). What are the evolutionary processes that led to the structural duplication and functional diversification of FgfD genes during vertebrate phylogeny? To study this question, we investigated conserved syntenies, patterns of gene expression, and the distribution of conserved noncoding elements (CNEs) in FgfD genes of stickleback and zebrafish, and compared them with data from cephalochordates, urochordates, and mammals. Genomic analysis suggests that Fgf8, Fgf17, Fgf18, and Fgf24 arose in two rounds of whole genome duplication at the base of the vertebrate radiation; that fgf8 and fgf18 duplications occurred at the base of the teleost radiation; and that Fgf24 is an ohnolog that was lost in the mammalian lineage. Expression analysis suggests that ancestral subfunctions partitioned between gene duplicates and points to the evolution of novel expression domains. Analysis of CNEs, at least some of which are candidate regulatory elements, suggests that ancestral CNEs partitioned between gene duplicates. These results help explain the evolutionary pathways by which the developmentally important family of FgfD molecules arose and the deduced principles that guided FgfD evolution are likely applicable to the evolution of developmental regulation in many vertebrate multigene families. (c) 2009 Wiley-Liss, Inc.

  10. Developmental imaging: the avian embryo hatches to the challenge.

    PubMed

    Kulesa, Paul M; McKinney, Mary C; McLennan, Rebecca

    2013-06-01

    The avian embryo provides a multifaceted model to study developmental mechanisms because of its accessibility to microsurgery, fluorescence cell labeling, in vivo imaging, and molecular manipulation. Early two-dimensional planar growth of the avian embryo mimics human development and provides unique access to complex cell migration patterns using light microscopy. Later developmental events continue to permit access to both light and other imaging modalities, making the avian embryo an excellent model for developmental imaging. For example, significant insights into cell and tissue behaviors within the primitive streak, craniofacial region, and cardiovascular and peripheral nervous systems have come from avian embryo studies. In this review, we provide an update to recent advances in embryo and tissue slice culture and imaging, fluorescence cell labeling, and gene profiling. We focus on how technical advances in the chick and quail provide a clearer understanding of how embryonic cell dynamics are beautifully choreographed in space and time to sculpt cells into functioning structures. We summarize how these technical advances help us to better understand basic developmental mechanisms that may lead to clinical research into human birth defects and tissue repair. Copyright © 2013 Wiley Periodicals, Inc.

  11. Pregnancy rates for embryos transferred from early postpartum beef cows into recipients with normal estrous cycles.

    PubMed

    Schrick, F N; Inskeep, E K; Butcher, R L

    1993-09-01

    Survival rate of embryos from first ovulations of postpartum cows with SHORT (6.9 +/- 0.3 days; n = 35) or NORMAL (17.1 +/- 0.3 days; n = 42) luteal phases and quality of the embryos on Day 6 were compared. At 19 to 23 days postpartum, cows were allotted to receive a norgestomet implant for 9 days (normal luteal phase) or to serve as untreated controls (short luteal phase). Calves were weaned 7 days after initiation of treatment to induce behavioral estrus in cows for mating. In 25 cows, growth of the ovulatory follicle was monitored by ultrasonography. On Day 6 after estrus, embryos were recovered nonsurgically, and live embryos were transferred into recipient cows exhibiting normal estrous cycles. The medium used to flush the embryos from the uterus of each donor cow was assayed for prostaglandin F2 alpha (PGF2 alpha). Days from calf removal to estrus and size of ovulatory follicles at ovulation (4.1 +/- 0.3 days and 16.7 +/- 0.7 mm, respectively) did not differ between NORMAL and SHORT cows. Interval from detection of the ovulatory follicle to ovulation was longer in NORMAL (10 +/- 0.7 days) than in SHORT cows (8 +/- 0.6 days; p < 0.05). Rates of recovery of an embryo or ovum (64%), rates of fertilization (65%), and quality or stage of development of Day 6 embryos did not differ between SHORT and NORMAL cows. Overall pregnancy rate from recovered oocytes was 13% for SHORT and 32% for NORMAL cows (p = 0.06); survival of fertilized oocytes was 23% for SHORT and 47% for NORMAL cows (p = 0.08).(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Oldest known dinosaurian nesting site and reproductive biology of the Early Jurassic sauropodomorph Massospondylus

    PubMed Central

    Reisz, Robert R.; Evans, David C.; Roberts, Eric M.; Sues, Hans-Dieter; Yates, Adam M.

    2012-01-01

    The extensive Early Jurassic continental strata of southern Africa have yielded an exceptional record of dinosaurs that includes scores of partial to complete skeletons of the sauropodomorph Massospondylus, ranging from embryos to large adults. In 1976 an incomplete egg clutch including in ovo embryos of this dinosaur, the oldest known example in the fossil record, was collected from a road-cut talus, but its exact provenance was uncertain. An excavation program at the site started in 2006 has yielded multiple in situ egg clutches, documenting the oldest known dinosaurian nesting site, predating other similar sites by more than 100 million years. The presence of numerous clutches of eggs, some of which contain embryonic remains, in at least four distinct horizons within a small area, provides the earliest known evidence of complex reproductive behavior including site fidelity and colonial nesting in a terrestrial vertebrate. Thus, fossil and sedimentological evidence from this nesting site provides empirical data on reproductive strategies in early dinosaurs. A temporally calibrated optimization of dinosaurian reproductive biology not only demonstrates the primary significance of the Massospondylus nesting site, but also provides additional insights into the initial stages of the evolutionary history of dinosaurs, including evidence that deposition of eggs in a tightly organized single layer in a nest evolved independently from brooding. PMID:22308330

  13. [Vertebral fractures in children with Type I Osteogenesis imperfecta].

    PubMed

    Sepúlveda, Andrea M; Terrazas, Claudia V; Sáez, Josefina; Reyes, María L

    2017-06-01

    Osteogenesis imperfecta (OI) is an hereditary disease affecting conective tissue, mainly associated to growth retardation and pathological fractures. OI type I (OI type I), is the mildest, most often, and homogeneous in its fenotype. Vertebral fractures are the most significant complications, associated to skeletical and cardiopulmonary morbidity. To characterize clinically a cohort of children with OI type I. A cohort of OI type I children younger than 20 year old was evaluated. Demographic, clinical, biochemical and radiological data were registered. Sixty seven patients were included, 55% male, 69% resident in the Metropolitan Region. The mean age of diagnose was 2.9 years, 70% presented vertebral fractures on follow-up, mostly thoracic, and 50% before the age of 5 years. Fifty percentage presented vertebral fractures at diagnose, which was about the age of 5 years. Bone metabolic parameters were in the normal range, without significant change at the moment of vertebral fractures. Calcium intake was found to be below American Academy of Pediatrics recommendations at the time of the first fracture. In this study OI type I has an early diagnose, and vertebral fractures show a high incidence, mostly in toddlers. Calcium intake was found to be below reccomended values, and should be closely supervised in these patients.

  14. Haemoglobin function in vertebrates: evolutionary changes in cellular regulation in hypoxia.

    PubMed

    Nikinmaa, M

    2001-11-15

    The evolution of erythrocytic hypoxia responses is reviewed by comparing the cellular control of haemoglobin-oxygen affinity in agnathans, teleost fish and terrestrial vertebrates. The most ancient response to hypoxic conditions appears to be an increase in cell volume, which increases the haemoglobin-oxygen affinity in lampreys. In teleost fish, an increase of cell volume in hypoxic conditions is also evident. The volume increase is coupled to an increase in erythrocyte pH. These changes are caused by an adrenergic activation of sodium/proton exchange across the erythrocyte membrane. The mechanism is important in acute hypoxia and is followed by a decrease in cellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP) concentrations in continued hypoxia. In hypoxic bird embryos, the ATP levels are also reduced. The mechanisms by which hypoxia decreases cellular ATP and GTP concentrations remains unknown, although at least in bird embryos cAMP-dependent mechanisms have been implicated. In mammals, hypoxia responses appear to occur mainly via modulation of cellular organic phosphate concentrations. In moderate hypoxia, 2,3-diphosphoglycerate levels are increased as a result of alkalosis caused by increased ventilation.

  15. Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis.

    PubMed

    Kugler, Jamie E; Passamaneck, Yale J; Feldman, Taya G; Beh, Jeni; Regnier, Todd W; Di Gregorio, Anna

    2008-11-01

    To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes. For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20. The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins. We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels. We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription. Copyright 2008 Wiley-Liss, Inc.

  16. Novel embryo selection techniques to increase embryo implantation in IVF attempts.

    PubMed

    Sigalos, George Α; Triantafyllidou, Olga; Vlahos, Nikos F

    2016-11-01

    The final success of an IVF attempt depends on several steps and decisions taken during the ovarian stimulation, the oocyte retrieval, the embryo culture and the embryo transfer. The final selection of the embryos most likely to implant is the final step in this process and the responsibility of the lab. Apart from strict morphologic criteria that historically have been used in embryo selection, additional information on genetic, metabolomic and morphokinetic characteristics of the embryo is recently combined to morphology to select the embryo most likely to produce a pregnancy. In this manuscript, we review the most recent information on the current methods used for embryo selection presenting the predictive capability of each one. A literature search was performed on Pubmed, Medline and Cochrane Database of Systematic Reviews for published studies using appropriate key words and phrases with no limits placed on time. It seems that the combination of morphologic criteria in conjunction to embryo kinetics as documented by time-lapse technology provides the most reliable information on embryo quality. Blastocyst biopsy with subsequent comprehensive chromosome analysis allows the selection of the euploid embryos with the higher implantation potential. Embryo time-lapse imaging and blastocyst biopsy combined to comprehensive chromosome analysis are the most promising technologies to increase pregnancy rates and reduce the possibility of multiple pregnancies. However, further studies will demonstrate the capability of routinely using these technologies to significantly improve IVF outcomes.

  17. Death in the clinic: women's perceptions and experiences of discarding supernumerary IVF embryos.

    PubMed

    de Lacey, Sheryl

    2017-03-01

    Perspectives on the status of human embryos and whether they should be discarded differ globally. Some countries protect embryos in law while in other countries embryos 'die' or 'succumb' in assisted reproductive technology clinics on a daily basis. This study analyses interview data drawn from a larger qualitative study conducted in South Australia from 2004-2007. 21 women and 12 of 21 partners were interviewed about the decision they made to discard their embryos. The analysis reported here sought to examine the ways in which women constructed and experienced the decision to discard embryos. The article highlights the ways in which embryo discard is a contested discursive space. Embryo death is sequestered through their confinement in the laboratory and their invisibility to the naked eye. The clinic treated embryo discard as disposal of biological waste and failed to acknowledge the meaning of the event. By contrast women experienced emotional bereavement described as similar to early pregnancy loss, and described experiences of attachment and grief. For sensitive and compassionate care these differences in perceptions of embryo discard need to be addressed. © 2016 Foundation for the Sociology of Health & Illness.

  18. Culturing Chick Embryos--A Simplification of New's Method.

    ERIC Educational Resources Information Center

    Downie, J. R.

    1979-01-01

    Describes a simplified version of New's method for culturing early chick embryos. The technique allows continuous observation of the critical first three days of development and the conditions for setting up successful cultures are also presented to help both teachers and students. (HM)

  19. Morphological embryo selection: an elective single embryo transfer proposal.

    PubMed

    Déniz, Francisco Parera; Encinas, Carlos; Fuente, Jorge La

    2018-03-01

    To describe a patient selection method for elective single embryo transfer (eSET), emphasizing inclusion criteria and results. This retrospective study included all cases seen in a private clinic between June 2011 and December 2016, in La Paz, Bolivia (3600 meters above sea level). Elective single embryo transfer was the method of choice in 34 IVF/ICSI cycles, all in the blastocyst stage. Gardner's blastocyst classification criteria were used. Between the two stages of the study (July 2015), each embryo grade implantation rate was recalculated, which led to the expansion of the inclusion criteria. The clinical pregnancy rate of the 34 cases in the first transfer group was 55.9% (19/34). Twin or multiple pregnancies did not occur. The cumulative pregnancy rate to date is 64% [(19+3)/34]. The first stage comprised 2.56% (12/468) of the patients offered elective single embryo transfers; the implantation rate was 58.3% (7/12). In the second stage, 14.29% (22/154) of the patients were eligible, and the implantation rate was 54.55% (12/22). The implementation of an eSET program based on in-depth morphological embryo assessment combined with the calculation of the implantation potential of each embryo grade led to acceptable clinical outcomes and fewer multiple pregnancies in patients transferred two embryos. Each clinic should be aware of the implantation rates of each embryo grade in its own setting.

  20. Morphological embryo selection: an elective single embryo transfer proposal

    PubMed Central

    Déniz, Francisco Parera; Encinas, Carlos; Fuente, Jorge La

    2018-01-01

    Objective To describe a patient selection method for elective single embryo transfer (eSET), emphasizing inclusion criteria and results. Methods This retrospective study included all cases seen in a private clinic between June 2011 and December 2016, in La Paz, Bolivia (3600 meters above sea level). Elective single embryo transfer was the method of choice in 34 IVF/ICSI cycles, all in the blastocyst stage. Gardner's blastocyst classification criteria were used. Between the two stages of the study (July 2015), each embryo grade implantation rate was recalculated, which led to the expansion of the inclusion criteria. Results The clinical pregnancy rate of the 34 cases in the first transfer group was 55.9% (19/34). Twin or multiple pregnancies did not occur. The cumulative pregnancy rate to date is 64% [(19+3)/34]. The first stage comprised 2.56% (12/468) of the patients offered elective single embryo transfers; the implantation rate was 58.3% (7/12). In the second stage, 14.29% (22/154) of the patients were eligible, and the implantation rate was 54.55% (12/22). Conclusion The implementation of an eSET program based on in-depth morphological embryo assessment combined with the calculation of the implantation potential of each embryo grade led to acceptable clinical outcomes and fewer multiple pregnancies in patients transferred two embryos. Each clinic should be aware of the implantation rates of each embryo grade in its own setting. PMID:29338137

  1. Embryology of the lamprey and evolution of the vertebrate jaw: insights from molecular and developmental perspectives.

    PubMed Central

    Kuratani, S; Nobusada, Y; Horigome, N; Shigetani, Y

    2001-01-01

    Evolution of the vertebrate jaw has been reviewed and discussed based on the developmental pattern of the Japanese marine lamprey, Lampetra japonica. Though it never forms a jointed jaw apparatus, the L. japonica embryo exhibits the typical embryonic structure as well as the conserved regulatory gene expression patterns of vertebrates. The lamprey therefore shares the phylotype of vertebrates, the conserved embryonic pattern that appears at pharyngula stage, rather than representing an intermediate evolutionary state. Both gnathostomes and lampreys exhibit a tripartite configuration of the rostral-most crest-derived ectomesenchyme, each part occupying an anatomically equivalent site. Differentiated oral structure becomes apparent in post-pharyngula development. Due to the solid nasohypophyseal plate, the post-optic ectomesenchyme of the lamprey fails to grow rostromedially to form the medial nasal septum as in gnathostomes, but forms the upper lip instead. The gnathostome jaw may thus have arisen through a process of ontogenetic repatterning, in which a heterotopic shift of mesenchyme-epithelial relationships would have been involved. Further identification of shifts in tissue interaction and expression of regulatory genes are necessary to describe the evolution of the jaw fully from the standpoint of evolutionary developmental biology. PMID:11604127

  2. Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration

    PubMed Central

    Gerlach, Gary F.; Wingert, Rebecca A.

    2012-01-01

    Vertebrates form a progressive series of up to three kidney organs during development—the pronephros, mesonephros, and metanephros. Each kidney derives from the intermediate mesoderm and is comprised of conserved excretory units called nephrons. The zebrafish is a powerful model for vertebrate developmental genetics, and recent studies have illustrated that zebrafish and mammals share numerous similarities in nephron composition and physiology. The zebrafish embryo forms an architecturally simple pronephros that has two nephrons, and these eventually become a scaffold onto which a mesonephros of several hundred nephrons is constructed during larval stages. In adult zebrafish, the mesonephros exhibits ongoing nephrogenesis, generating new nephrons from a local pool of renal progenitors during periods of growth or following kidney injury. The characteristics of the zebrafish pronephros and mesonephros make them genetically tractable kidney systems in which to study the functions of renal genes and address outstanding questions about the mechanisms of nephrogenesis. Here, we provide an overview of the formation and composition of these zebrafish kidney organs, and discuss how various zebrafish mutants, gene knockdowns, and transgenic models have created frameworks in which to further delineate nephrogenesis pathways. PMID:24014448

  3. An approach to successful freezing of demi-embryos derived from day-7 bovine embryos.

    PubMed

    Niemann, H; Brem, G; Sacher, B; Smidt, D; Kräusslich, H

    1986-04-01

    The developmental capacity of frozen/thawed bisected embryos (n = 33) derived from day-7 bovine embryos was investigated and compared to ordinary embryos after freezing and thawing (n = 28) and to freshly bisected embryos (n = 19). The freezing and thawing protocol was identical for ordinary and demi-embryos. The percentage of intact embryos classified as excellent, good, or poor after thawing was 92.9 and 96.3% for ordinary and demi-embryos, respectively. Pregnancy rates of 53.8 (8 15 ), 46.2 (6 13 ), and 47.5% (9 19 ) were obtained when frozen/thawed ordinary embryos and frozen/thawed demi-embryos classified as excellent or good and sealed with an additional zona pellucida from hatched pig blastocysts or freshly bisected embryos were transferred. One pair of identical twins resulted from the transfer of frozen/thawed demi-embryos sealed with an additional zona pellucida. Transfer of four frozen/thawed demi-embryos without an additional zona pellucida led to one pregnancy. In contrast, demi-embryos derived from frozen/thawed ordinary embryos (n = 8) as well as frozen/thawed demi-embryos classified as poor (n = 6) did not result in any pregnancies although two halves were transferred per recipient. It is concluded that sealing the punctured zona pellucida improves the developmental capacity of frozen/thawed demi-embryos derived from day-7 bovine embryos, and freezing demi-embryos is more efficient compared to the splitting of frozen/thawed ordinary embryos.

  4. Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut.

    PubMed

    Essner, Jeffrey J; Amack, Jeffrey D; Nyholm, Molly K; Harris, Erin B; Yost, H Joseph

    2005-03-01

    Monocilia have been proposed to establish the left-right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. In zebrafish, dorsal forerunner cells (DFCs) express a conserved ciliary dynein gene (left-right dynein-related1, lrdr1) and form a ciliated epithelium inside a fluid-filled organ called Kupffer's vesicle (KV). Here, videomicroscopy demonstrates that cilia inside KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells. Laser ablation of DFCs and surgical disruption of KV provide direct evidence that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut. Antisense morpholinos against lrdr1 disrupt KV fluid flow and perturb LR development. Furthermore, lrdr1 morpholinos targeted to DFC/KV cells demonstrate that Lrdr1 functions in these ciliated cells to control LR patterning. This provides the first direct evidence, in any vertebrate, that impairing cilia function in derivatives of the dorsal organizer, and not in other cells that express ciliogenic genes, alters LR development. Finally, genetic analysis reveals novel roles for the T-box transcription factor no tail and the Nodal signaling pathway as upstream regulators of lrdr1 expression and KV morphogenesis. We propose that KV is a transient embryonic 'organ of asymmetry' that directs LR development by establishing a directional fluid flow. These results suggest that cilia are an essential component of a conserved mechanism that controls the transition from bilateral symmetry to LR asymmetry in vertebrates.

  5. Evolutionary Genomics and Adaptive Evolution of the Hedgehog Gene Family (Shh, Ihh and Dhh) in Vertebrates

    PubMed Central

    Pereira, Joana; Johnson, Warren E.; O’Brien, Stephen J.; Jarvis, Erich D.; Zhang, Guojie; Gilbert, M. Thomas P.; Vasconcelos, Vitor; Antunes, Agostinho

    2014-01-01

    The Hedgehog (Hh) gene family codes for a class of secreted proteins composed of two active domains that act as signalling molecules during embryo development, namely for the development of the nervous and skeletal systems and the formation of the testis cord. While only one Hh gene is found typically in invertebrate genomes, most vertebrates species have three (Sonic hedgehog – Shh; Indian hedgehog – Ihh; and Desert hedgehog – Dhh), each with different expression patterns and functions, which likely helped promote the increasing complexity of vertebrates and their successful diversification. In this study, we used comparative genomic and adaptive evolutionary analyses to characterize the evolution of the Hh genes in vertebrates following the two major whole genome duplication (WGD) events. To overcome the lack of Hh-coding sequences on avian publicly available databases, we used an extensive dataset of 45 avian and three non-avian reptilian genomes to show that birds have all three Hh paralogs. We find suggestions that following the WGD events, vertebrate Hh paralogous genes evolved independently within similar linkage groups and under different evolutionary rates, especially within the catalytic domain. The structural regions around the ion-binding site were identified to be under positive selection in the signaling domain. These findings contrast with those observed in invertebrates, where different lineages that experienced gene duplication retained similar selective constraints in the Hh orthologs. Our results provide new insights on the evolutionary history of the Hh gene family, the functional roles of these paralogs in vertebrate species, and on the location of mutational hotspots. PMID:25549322

  6. Early embryonic brain development in rats requires the trophic influence of cerebrospinal fluid.

    PubMed

    Martin, C; Alonso, M I; Santiago, C; Moro, J A; De la Mano, A; Carretero, R; Gato, A

    2009-11-01

    Cerebrospinal fluid has shown itself to be an essential brain component during development. This is particularly evident at the earliest stages of development where a lot of research, performed mainly in chick embryos, supports the evidence that cerebrospinal fluid is involved in different mechanisms controlling brain growth and morphogenesis, by exerting a trophic effect on neuroepithelial precursor cells (NPC) involved in controlling the behaviour of these cells. Despite it being known that cerebrospinal fluid in mammals is directly involved in corticogenesis at fetal stages, the influence of cerebrospinal fluid on the activity of NPC at the earliest stages of brain development has not been demonstrated. Here, using "in vitro" organotypic cultures of rat embryo brain neuroepithelium in order to expose NPC to or deprive them of cerebrospinal fluid, we show that the neuroepithelium needs the trophic influence of cerebrospinal fluid to undergo normal rates of cell survival, replication and neurogenesis, suggesting that NPC are not self-sufficient to induce their normal activity. This data shows that cerebrospinal fluid is an essential component in chick and rat early brain development, suggesting that its influence could be constant in higher vertebrates.

  7. A vesicular glutamate transporter in lampreys: cDNA cloning and early expression in the nervous system.

    PubMed

    Villar-Cerviño, Verona; Rocancourt, Claire; Menuet, Arnaud; Da Silva, Corinne; Wincker, Patrick; Anadón, Ramón; Mazan, Sylvie; Rodicio, Maria Celina

    2010-09-01

    Vesicular glutamate transporters (VGLUTs) accumulate glutamate into synaptic vesicles of glutamatergic neurons, and thus are considered to define the phenotype of these neurons. Glutamate also appears to play a role in the development of the nervous system of vertebrates. Here we report the characterization of a vesicular glutamate transporter of lamprey (lVGluT), a novel member of the VGluT gene family. Phylogenetic analysis indicates that lVGLUT cannot be assigned to any of the three VGLUT isoforms characterized in teleosts and mammals, suggesting that these classes may have been fixed after the splitting between cyclostomes and gnathostomes. Expression pattern analysis during lamprey embryogenesis and prolarval stages shows that lVGluT expression is restricted to the nervous system. The first structure to express lVGluT was the olfactory epithelium of late embryos. In the brain of early prolarvae, lVGluT was expressed in most of the neuronal populations that generate the early axonal scaffold. lVGluT expression was also observed in neuronal populations of the rhombencephalon and spinal cord and in ganglia of the branchiomeric, octaval and posterior lateral line nerves. In the rhombencephalon, lVGluT expression appears to be spatially restricted in dorsal and ventral longitudinal domains. Comparison of the early expression of VGluT genes between the lamprey and some anamniotan gnathostomes (frog, zebrafish) reveals a conserved expression pattern, likely to reflect ancestral vertebrate characteristics. 2010 Elsevier B.V. All rights reserved.

  8. Oxidative stress, endocrine disruption, and malformation of Bufo gargarizans embryo exposed to sub-lethal cadmium concentrations.

    PubMed

    Wu, Chao; Zhang, Yuhui; Chai, Lihong; Wang, Hongyuan

    2017-01-01

    Thyroid hormone (TH) is critical for vertebrate postembryonic development as well as embryonic development. Chinese toad (Bufo gargarizans) embryos were exposed to different concentrations of cadmium (5, 50, 100, 200 and 500μg Cd L -1 ) for 7days. Malformations were monitored daily, and growth and development of embryos were measured at day 4 and 7, and type 2 and 3 iodothyronine deiodinase (Dio2 and Dio3), thyroid hormone receptors (TRα and TRβ) mRNA levels were also measured to assess disruption of TH synthesis. In addition, superoxide dismutase (SOD), glutathione peroxidase (GPx) and heat shock proteins (HSPs) mRNA expression were examined to evaluate the ability of scavenging ROS. Our results demonstrated a bimodal inhibitory effect of Cd on the embryo growth and development of Bufo gargarizans. Reduced mean stage, total length and weight were observed at 5, 50, 200 and 500, but not at 100μg Cd L -1 . Embryos malformation occurred in all cadmium treatments. Morphological abnormalities of embryos are characterized by axial flexures, abdominal edema, stunted growth and fin flexure. Real-time PCR results show that exposure to cadmium down-regulated TRα and Dio3 mRNA expression and up-regulated Dio2 mRNA level. SOD and GPx mRNA expression was significantly up-regulated after cadmium exposure. We concluded that cadmium could change mRNA expression of TRα, Dio2 and Dio3 leading the inhibition of growth and development of B. gargarizans embryo, which suggests that cadmium might have the endocrine-disrupting effect in embryos. Moreover, the reduced ability of scavenging ROS induced by cadmium might be responsible for the teratogenic effects of cadmium. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Vertebral pneumatocysts.

    PubMed

    Arslan, G; Ceken, K; Cubuk, M; Ozkaynak, C; Lüleci, E

    2001-01-01

    To review the prevalence and location of vertebral pneumatocysts and evaluate the CT findings of these benign lesions. Retrospectively we reviewed CT images of 89 patients with suspected disc disease during a 6-month period. Distinctive CT pattern of intraosseous pneumatocysts involving the cervical, thoracic and lumbar spine was found. In 8 patients (9%), 10 vertebral pneumatocysts were detected. Five were located in the vertebral body and 4 of these were associated with vacuum phenomenon in adjacent intervertebral discs. Five were located near the facet joint and all were associated with vacuum phenomenon in adjacent facet joint. Intraosseous pneumatocyst is a benign lesion, therefore biopsy and follow-up are unnecessary. Although vertebral pneumatocysts seem to be uncommon with a few reported cases, this study shows them to be more frequent than previously thought.

  10. Initial specification of the epibranchial placode in zebrafish embryos depends on the fibroblast growth factor signal.

    PubMed

    Nikaido, Masataka; Doi, Kazunao; Shimizu, Takashi; Hibi, Masahiko; Kikuchi, Yutaka; Yamasu, Kyo

    2007-02-01

    In vertebrates, cranial sensory ganglia are mainly derived from ectodermal placodes, which are focal thickenings at characteristic positions in the embryonic head. Here, we provide the first description of the early development of the epibranchial placode in zebrafish embryos using sox3 as a molecular marker. By the one-somite stage, we saw a pair of single sox3-expressing domains appear lateral to the future hindbrain. The sox3 domain, which is referred to here as the early lateral placode, is segregated during the early phase of segmentation to form a pax2a-positive medial area and a pax2a-negative lateral area. The medial area subsequently developed to form the otic placode, while the lateral area was further segregated along the anteroposterior axis, giving rise to four sox3-positive subdomains by 26 hr postfertilization. Given their spatial relationship with the expression of the markers for the epibranchial ganglion, as well as their positions and temporal changes, we propose that these four domains correspond to the facial, glossopharyngeal, vagal, and posterior lateral line placodes in an anterior-to-posterior order. The expression of sox3 in the early lateral placode was absent in mutants lacking functional fgf8, while implantation of fibroblast growth factor (FGF) beads restored the sox3 expression. Using SU5402, which inhibits the FGF signal, we were able to demonstrate that formation of both the early lateral domains and later epibranchial placodes depends on the FGF signal operating at the beginning of somitogenesis. Together, these data provide evidence for the essential role of FGF signals in the development of the epibranchial placodes.

  11. Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos

    PubMed Central

    Hassoun, Romia; Schwartz, Peter; Rath, Detlef; Viebahn, Christoph; Männer, Jörg

    2010-01-01

    Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human-like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal–epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two-step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron-dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam-like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid-somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations. PMID:20874819

  12. Pre-hatching embryo-dependent and -independent programming of endometrial function in cattle

    PubMed Central

    Sponchiado, Mariana; Gomes, Nathália Souza; Fontes, Patrícia Kubo; Martins, Thiago; del Collado, Maite; Pastore, Athos de Assumpção; Pugliesi, Guilherme; Nogueira, Marcelo Fábio Gouveia

    2017-01-01

    The bovine pre-implantation embryo secretes bioactive molecules from early development stages, but effects on endometrial function are reported to start only after elongation. Here, we interrogated spatially defined regions of the endometrium transcriptome for responses to a day 7 embryo in vivo. We hypothesize that exposure to an embryo changes the abundance of specific transcripts in the cranial region of the pregnant uterine horn. Endometrium was collected from the uterotubal junction (UTJ), anterior (IA), medial (IM) and posterior (IP) regions of the uterine horn ipsilateral to the CL 7 days after estrus from sham-inseminated (Con) or artificially inseminated, confirmed pregnant (Preg) cows. Abundance of 86 transcripts was evaluated by qPCR using a microfluidic platform. Abundance of 12 transcripts was modulated in the Preg endometrium, including classical interferon-stimulated genes (ISG15, MX1, MX2 and OAS1Y), prostaglandin biosynthesis genes (PTGES, HPGD and AKR1C4), water channel (AQP4) and a solute transporter (SLC1A4) and this was in the UTJ and IA mainly. Additionally, for 71 transcripts, abundance varied according to region of the reproductive tract. Regulation included downregulation of genes associated with proliferation (IGF1, IGF2, IGF1R and IGF2R) and extracellular matrix remodeling (MMP14, MMP19 and MMP2) and upregulation of anti-adhesive genes (MUC1) in the cranial regions of uterine horn. Physical proximity to the embryo provides paracrine regulation of endometrial function. Embryo-independent regulation of the endometrial transcriptome may support subsequent stages of embryo development, such as elongation and implantation. We speculate that successful early embryo-dependent and -independent programming fine-tune endometrial functions that are important for maintenance of pregnancy in cattle. PMID:28423001

  13. V. Terrestrial vertebrates

    Treesearch

    Dean Pearson; Deborah Finch

    2011-01-01

    Within the Interior West, terrestrial vertebrates do not represent a large number of invasive species relative to invasive weeds, aquatic vertebrates, and invertebrates. However, several invasive terrestrial vertebrate species do cause substantial economic and ecological damage in the U.S. and in this region (Pimental 2000, 2007; Bergman and others 2002; Finch and...

  14. Iodine-enhanced micro-CT imaging: methodological refinements for the study of the soft-tissue anatomy of post-embryonic vertebrates.

    PubMed

    Gignac, Paul M; Kley, Nathan J

    2014-05-01

    The now widespread use of non-destructive X-ray computed tomography (CT) and micro-CT (µCT) has greatly augmented our ability to comprehensively detail and quantify the internal hard-tissue anatomy of vertebrates. However, the utility of X-ray imaging for gaining similar insights into vertebrate soft-tissue anatomy has yet to be fully realized due to the naturally low X-ray absorption of non-mineralized tissues. In this study, we show how a wide diversity of soft-tissue structures within the vertebrate head-including muscles, glands, fat deposits, perichondria, dural venous sinuses, white and gray matter of the brain, as well as cranial nerves and associated ganglia-can be rapidly visualized in their natural relationships with extraordinary levels of detail using iodine-enhanced (i-e) µCT imaging. To date, Lugol's iodine solution (I2 KI) has been used as a contrast agent for µCT imaging of small invertebrates, vertebrate embryos, and certain isolated parts of larger, post-embryonic vertebrates. These previous studies have all yielded promising results, but visualization of soft tissues in smaller invertebrate and embryonic vertebrate specimens has generally been more complete than that for larger, post-embryonic vertebrates. Our research builds on these previous studies by using high-energy µCT together with more highly concentrated I2 KI solutions and longer staining times to optimize the imaging and differentiation of soft tissues within the heads of post-embryonic archosaurs (Alligator mississippiensis and Dromaius novaehollandiae). We systematically quantify the intensities of tissue staining, demonstrate the range of anatomical structures that can be visualized, and generate a partial three-dimensional reconstruction of alligator cephalic soft-tissue anatomy. © 2014 Wiley Periodicals, Inc.

  15. Age distribution of human gene families shows significant roles of both large- and small-scale duplications in vertebrate evolution.

    PubMed

    Gu, Xun; Wang, Yufeng; Gu, Jianying

    2002-06-01

    The classical (two-round) hypothesis of vertebrate genome duplication proposes two successive whole-genome duplication(s) (polyploidizations) predating the origin of fishes, a view now being seriously challenged. As the debate largely concerns the relative merits of the 'big-bang mode' theory (large-scale duplication) and the 'continuous mode' theory (constant creation by small-scale duplications), we tested whether a significant proportion of paralogous genes in the contemporary human genome was indeed generated in the early stage of vertebrate evolution. After an extensive search of major databases, we dated 1,739 gene duplication events from the phylogenetic analysis of 749 vertebrate gene families. We found a pattern characterized by two waves (I, II) and an ancient component. Wave I represents a recent gene family expansion by tandem or segmental duplications, whereas wave II, a rapid paralogous gene increase in the early stage of vertebrate evolution, supports the idea of genome duplication(s) (the big-bang mode). Further analysis indicated that large- and small-scale gene duplications both make a significant contribution during the early stage of vertebrate evolution to build the current hierarchy of the human proteome.

  16. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    PubMed

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  17. Changes in the dielectric properties of medaka fish embryos during development, studied by electrorotation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shirakashi, Ryo, E-mail: aa21150@iis.u-tokyo.ac.jp; Mischke, Miriam; Fischer, Peter

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer Electrorotation offers a non-invasive tool for dielectric analysis of fish embryos. Black-Right-Pointing-Pointer The three-shell dielectric model matches the rotation spectra of medaka eggs. Black-Right-Pointing-Pointer The capacitance value suggests a double-membrane structure of yolk envelope. -- Abstract: The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during pre-hatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to earlymore » somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.« less

  18. Methods for imaging individual cilia in living echinoid embryos.

    PubMed

    Morris, Robert L; Pope, Hans W; Sholi, Adam N; Williams, Leah M; Ettinger, Chelsea R; Beacham, Gwendolyn M; Shintaku, Tatsushi; Abbott, Zachary D; Doherty, Elyse M

    2015-01-01

    The embryos of echinoids (sea urchins and sand dollars) serve as excellent models for studying cilia differentiation and stages of the cilia life cycle including ciliogenic initiation, growth, maintenance, and retraction. Early in echinoid development, uniform motile cilia form on all cells simultaneously but then rapidly differentiate into multiple cilia types that differ in morphology, motility, and signaling sensitivity. Metal ion treatments that shift germ layer boundaries and thereby "animalize" or "vegetalize" embryos can be used to enrich for low-abundance cilia types rendering those specialized cilia and the differentiation processes they exhibit much easier to study. The experimental advantages of having robust cilia growth and differentiation is tempered by the challenge of restraining ciliated embryos well enough to view the process of ciliogenesis live. We have developed four observation chambers as modifications of the Kiehart chamber for long-term light microscopic imaging of ciliated echinoid embryos. One of these systems employs paramagnetic beads to render ciliated larvae magnetic so they can be gently and reversibly trapped directly under the objective lens. With this magnetic trapping system, the larva can be positioned and repositioned until they achieve the orientation with the clearest view of any cilia of interest. These methods of gentle embryo restraint allow normal embryo development and the normal ciliogenic cycle and ciliary differentiation processes to continue in direct view. Sequential image series can then be collected and analyzed to quantitatively study the wide spectrum of cilia behaviors and properties that arise in developing echinoid embryos. Copyright © 2015. Published by Elsevier Inc.

  19. Clinical evaluation of frozen/thawed embryo transfer following transport of oocytes and embryos

    PubMed Central

    2004-01-01

    Background and Aims:  We evaluated the efficacy of the transport oocyte/embryo frozen/thawed embryo transfer method, in which oocytes or embryos were transported from satellite clinics to the main assisted reproductive technology (ART) center, and surplus embryos were placed in cryopreservation. Methods:  We evaluated 41 cycles in 34 patients in the transport oocyte group (TO group). In the TO group the oocytes were collected at the satellite clinics, transported to the main ART center and underwent in vitro fertilization or intracytoplasmic sperm injection. Surplus embryos were used for frozen/thawed embryo transfer. We also evaluated 17 cycles in 10 patients in the transport embryo group (TE group), where surplus embryos were transported to the main ART center and used for frozen/thawed embryo transfer; and 189 cycles in 134 patients in the center group (C group), where surplus embryos collected at the same time at the main ART center were used for frozen/thawed embryo transfer. Oocytes were transported from satellite clinics in HEPES buffered human tubal fluid (HTF) culture medium, and embryos in 30% synthetic serum substitute + HEPES buffered HTF, using a portable incubator we devised. Results:  The proportions of undamaged embryos after freeze/thawing were 47% for the C group, 46% for the TO group, and 46% for the TE group. The numbers of embryos transferred were 2.0 ± 0.7 for the C group, 2.0 ± 0.6 for the TO group, and 2.2 ± 0.4 for the TE group. The rate of embryo transfer was 63% for the C group, 68% for the TO group, and 76% for the TE group. Pregnancy rates per patient were 16% for the C group, 24% for the TO group, and 40% for the TE group. The embryo survival rates (number of embryos with ≥50% viable blastomeres/total number of embryos) were 55% for the C group, 60% for the TO group, and 54% for the TE group. No significant differences were seen between the C group and either the TO or TE groups in any of these parameters

  20. The fate of paternal mitochondria in marmoset pre-implantation embryos.

    PubMed

    Luetjens, C M; Wesselmann, R

    2008-06-01

    Sperm-derived mitochondria are integrated into the oocyte at fertilization but seem to vanish during the early cleavage phase. The developmental potential of pre-implantation embryos seems to be closely related to their ability to induce degeneration of these mitochondria, but the mechanisms underlying their loss of function are not yet understood. This study focuses on the fate of paternal mitochondria in pre-implantation embryos. Stimulation, collection and in vitro culture of oocytes from Callithrix jacchus, allows the study of the destiny of paternal mitochondria by utilizing immunostaining of pre-implantation embryos, fluorescence and laserscanning microscopy. Live pre-implantation embryos were stained with a fluorescence indicator reflecting mitochondrial membrane potential. Evidence indicating the loss of mitochondrial function was not found nor that apoptosis pathways were involved in the disappearance of paternally derived mitochondria. These findings may have implications for mitochondrially inherited diseases and could lead to new strategies for improving assisted reproduction.

  1. Parallel Expansions of Sox Transcription Factor Group B Predating the Diversifications of the Arthropods and Jawed Vertebrates

    PubMed Central

    Zhong, Lei; Wang, Dengqiang; Gan, Xiaoni; Yang, Tong; He, Shunping

    2011-01-01

    Group B of the Sox transcription factor family is crucial in embryo development in the insects and vertebrates. Sox group B, unlike the other Sox groups, has an unusually enlarged functional repertoire in insects, but the timing and mechanism of the expansion of this group were unclear. We collected and analyzed data for Sox group B from 36 species of 12 phyla representing the major metazoan clades, with an emphasis on arthropods, to reconstruct the evolutionary history of SoxB in bilaterians and to date the expansion of Sox group B in insects. We found that the genome of the bilaterian last common ancestor probably contained one SoxB1 and one SoxB2 gene only and that tandem duplications of SoxB2 occurred before the arthropod diversification but after the arthropod-nematode divergence, resulting in the basal repertoire of Sox group B in diverse arthropod lineages. The arthropod Sox group B repertoire expanded differently from the vertebrate repertoire, which resulted from genome duplications. The parallel increases in the Sox group B repertoires of the arthropods and vertebrates are consistent with the parallel increases in the complexity and diversification of these two important organismal groups. PMID:21305035

  2. Human cloning and embryo research: the 2003 John J. Conley Lecture on medical ethics.

    PubMed

    George, Robert P

    2004-01-01

    The author, a member of the U.S. President's Council on Bioethics, discusses ethical issues raised by human cloning, whether for purposes of bringing babies to birth or for research purposes. He first argues that every cloned human embryo is a new, distinct, and enduring organism, belonging to the species Homo sapiens, and directing its own development toward maturity. He then distinguishes between two types of capacities belonging to individual organisms belonging to this species, an immediately exerciseable capacity and a basic natural capacity that develops over time. He argues that it is the second type of capacity that is the ground for full moral respect, and that this capacity (and its concomitant degree of respect) belongs to cloned human embryos no less than to adult human beings. He then considers and rejects counter-arguments to his position, including the suggestion that the capacity of embryos is equivalent to the capacity of somatic cells, that full human rights are afforded only to human organisms with functioning brains, that the possibility of twinning diminishes the moral status of embryos, that the fact that people do not typically mourn the loss of early embryos implies that they have a diminished moral status, that the fact that early spontaneous abortions occur frequently diminishes the moral status of embryos, and that his arguments depend upon a concept of ensoulment. He concludes that if the moral status of cloned human embryos is equivalent to that of adults, then public policy should be based upon this assumption.

  3. Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6.

    PubMed

    Liu, Wan-Ju; Reece-Hoyes, John S; Walhout, Albertha J M; Eisenmann, David M

    2014-05-13

    Hox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, we aimed to identify transcription factors necessary for lin-39 expression in the VPCs, and in particular sought factors that initiate lin-39 expression in the embryo. We used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs. These results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a

  4. Oxidative Damage to Rhesus Macaque Spermatozoa Results in Mitotic Arrest and Transcript Abundance Changes in Early Embryos1

    PubMed Central

    Burruel, Victoria; Klooster, Katie L.; Chitwood, James; Ross, Pablo J.; Meyers, Stuart A.

    2013-01-01

    ABSTRACT Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development. PMID:23904511

  5. Comparative Physiology of Energy Metabolism: Fishing for Endocrine Signals in the Early Vertebrate Pool

    PubMed Central

    van de Pol, Iris; Flik, Gert; Gorissen, Marnix

    2017-01-01

    Energy is the common currency of life. To guarantee a homeostatic supply of energy, multiple neuro-endocrine systems have evolved in vertebrates; systems that regulate food intake, metabolism, and distribution of energy. Even subtle (lasting) dysregulation of the delicate balance of energy intake and expenditure may result in severe pathologies. Feeding-related pathologies have fueled research on mammals, including of course the human species. The mechanisms regulating food intake and body mass are well-characterized in these vertebrates. The majority of animal life is ectothermic, only birds and mammals are endotherms. What can we learn from a (comparative) study on energy homeostasis in teleostean fishes, ectotherms, with a very different energy budget and expenditure? We present several adaptation strategies in fish. In recent years, the components that regulate food intake in fishes have been identified. Although there is homology of the major genetic machinery with mammals (i.e., there is a vertebrate blueprint), in many cases this does not imply analogy. Although both mammals and fish must gain their energy from food, the expenditure of the energy obtained is different. Mammals need to spend vast amounts of energy to maintain body temperature; fishes seem to utilize a broader metabolic range to their advantage. In this review, we briefly discuss ecto- and endothermy and their consequences for energy balance. Next, we argue that the evolution of endothermy and its (dis-)advantages may explain very different strategies in endocrine regulation of energy homeostasis among vertebrates. We follow a comparative and evolutionary line of thought: we discuss similarities and differences between fish and mammals. Moreover, given the extraordinary radiation of teleostean fishes (with an estimated number of 33,400 contemporary species, or over 50% of vertebrate life forms), we also compare strategies in energy homeostasis between teleostean species. We present recent

  6. The role of eggshell and underlying vitelline membrane for normal pattern formation in the early C. elegans embryo.

    PubMed

    Schierenberg, Einhard; Junkersdorf, Bernd

    1992-12-01

    The embryo of the nematode Caenorhabditis elegans is surrounded by an inconspicuous inner vitelline membrane and a prominent outer chitinous eggshell proper. We demonstrate that the complete removal of the chitinous eggshell does not interfere with successful development to yield a normal worm. The same result can be obtained when the vitelline membrane is penetrated with laser microbeam irradiation of only the eggshell proper, gently enough to permit its resealing after a while. However, when large holes are made into the eggshell the concomitantly penetrated vitelline membrane does not reseal. While early development is quite normal under these conditions, gastrulation is defective in that gut precursor cells do not migrate in properly, eventually leading to embryonic arrest. This suggests a crucial role for pattern formation of the "micro-environment" around the embryo preserved by the intact vitelline membrane. Removing both eggshell and vitelline membrane results in a string-like arrangement of founder cells and subsequent grossly abnormal cell patterns. Our experiments support the idea that the prominent eggshell proper just functions as a mechanical protection while the thin vitelline membrane directly or indirectly serves as a necessary control element affecting the positions of cells which to begin with are determined by the orientation of the cleavage spindle.

  7. Evaluation of treatments with hCG and carprofen at embryo transfer in a demi-embryo and recipient virgin heifer model.

    PubMed

    Torres, A; Chagas E Silva, J; Diniz, P; Lopes-da-Costa, L

    2013-08-01

    An in vivo model, combining a low developmental competence embryo (demi-embryo) and a high-fertility recipient (virgin dairy heifer) was used to evaluate the effects of treatment with human chorionic gonadotropin (hCG) and carprofen at embryo transfer (ET) on plasma progesterone (P₄) concentrations of recipients and on embryonic growth and survival. Embryos were bisected and each demi-embryo was transferred to a recipient on Day 7 of the estrous cycle. At ET, heifers (n = 163) were randomly allocated to treatment with hCG (2500 IU im), carprofen (500 mg iv), hCG plus carprofen or to untreated controls. Plasma P₄ concentrations were measured on Days 0, 7, 14 and 21 of all recipients plus on Days 28, 42 and 63 of pregnant recipients. Pregnancy was presumed to be present in recipients with luteal plasma P4 concentrations until Day 21 and confirmed by using transrectal ultrasonography on Days 28, 42 and 63. Embryonic measurements (crown-rump length and width) were obtained on Day 42. Treatment with hCG induced formation of secondary corpora lutea (CL) in 97% of heifers and increased (P < 0.01) mean plasma P₄ concentrations of non-pregnant recipients on Day 14 and of pregnant heifers on Days 14 to 63. This was associated to a significant decrease in early embryonic mortality. In contrast, subsequent embryonic losses resulted in a non-significant numerical increase by 8% of pregnancies maintained to Day 63. Therefore, treatment with hCG significantly rescued embryos through the maternal recognition of pregnancy window but was not able to support development thereafter. Treatment with carprofen at ET had no significant effects on plasma P₄ concentrations and rate of embryo mortality. Treatment with hCG plus carprofen at ET induced formation of secondary CL in 90% of heifers but decreased the luteotrophic effect of hCG, resulting in no effect on embryo survival. Low developmental competence embryos showed an intrinsic deficiency in overcoming the maternal

  8. Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

    PubMed

    Beck, Aaron P; Watt, Roland M; Bonner, Jennifer

    2014-02-28

    The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

  9. Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

    PubMed Central

    Beck, Aaron P.; Watt, Roland M.; Bonner, Jennifer

    2014-01-01

    The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue. PMID:24637734

  10. Aspergillus vertebral osteomyelitis and epidural abscess.

    PubMed

    Tew, C W; Han, F C; Jureen, R; Tey, B H

    2009-04-01

    We present the first reported case of Aspergillus vertebral osteomyelitis and epidural abscess in Singapore in a 50-year-old man with post-tuberculous bronchiectasis. The patient presented with acute urinary retention and flaccid paraplegia. Despite surgical debridement and treatment with voriconazole, the patient developed multiorgan failure and died two weeks after presentation. Early diagnosis and prompt initiation of treatment are emphasised in the hope of improving the outcome of this aggressive condition.

  11. Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells.

    PubMed

    Ramalho-Santos, João; Varum, Sandra; Amaral, Sandra; Mota, Paula C; Sousa, Ana Paula; Amaral, Alexandra

    2009-01-01

    Mitochondria are multitasking organelles involved in ATP synthesis, reactive oxygen species (ROS) production, calcium signalling and apoptosis; and mitochondrial defects are known to cause physiological dysfunction, including infertility. The goal of this review was to identify and discuss common themes in mitochondrial function related to mammalian reproduction. The scientific literature was searched for studies reporting on the several aspects of mitochondrial activity in mammalian testis, sperm, oocytes, early embryos and embryonic stem cells. ATP synthesis and ROS production are the most discussed aspects of mitochondrial function. Metabolic shifts from mitochondria-produced ATP to glycolysis occur at several stages, notably during gametogenesis and early embryo development, either reflecting developmental switches or substrate availability. The exact role of sperm mitochondria is especially controversial. Mitochondria-generated ROS function in signalling but are mostly described when produced under pathological conditions. Mitochondria-based calcium signalling is primarily important in embryo activation and embryonic stem cell differentiation. Besides pathologically triggered apoptosis, mitochondria participate in apoptotic events related to the regulation of spermatogonial cell number, as well as gamete, embryo and embryonic stem cell quality. Interestingly, data from knock-out (KO) mice is not always straightforward in terms of expected phenotypes. Finally, recent data suggests that mitochondrial activity can modulate embryonic stem cell pluripotency as well as differentiation into distinct cellular fates. Mitochondria-based events regulate different aspects of reproductive function, but these are not uniform throughout the several systems reviewed. Low mitochondrial activity seems a feature of 'stemness', being described in spermatogonia, early embryo, inner cell mass cells and embryonic stem cells.

  12. Ultrastructural dynamics of human reproduction, from ovulation to fertilization and early embryo development.

    PubMed

    Familiari, Giuseppe; Heyn, Rosemarie; Relucenti, Michela; Nottola, Stefania A; Sathananthan, A Henry

    2006-01-01

    This study describes the updated, fine structure of human gametes, the human fertilization process, and human embryos, mainly derived from assisted reproductive technology (ART). As clearly shown, the ultrastructure of human reproduction is a peculiar multistep process, which differs in part from that of other mammalian models, having some unique features. Particular attention has been devoted to the (1) sperm ultrastructure, likely "Tygerberg (Kruger) strict morphology criteria"; (2) mature oocyte, in which the MII spindle is barrel shaped, anastral, and lacking centrioles; (3) three-dimensional microarchitecture of the zona pellucida with its unique supramolecular filamentous organization; (4) sperm-egg interactions with the peculiarity of the sperm centrosome that activates the egg and organizes the sperm aster and mitotic spindles of the embryo; and (5) presence of viable cumulus cells whose metabolic activity is closely related to egg and embryo behavior in in vitro as well as in vivo conditions, in a sort of extraovarian "microfollicular unit." Even if the ultrastructural morphodynamic features of human fertilization are well understood, our knowledge about in vivo fertilization is still very limited and the complex sequence of in vivo biological steps involved in human reproduction is only partially reproduced in current ART procedures.

  13. Measuring the electric activity of chick embryos heart through 16 bit audio card monitored by the Goldwavetm software

    NASA Astrophysics Data System (ADS)

    Silva, Dilson; Cortez, Celia Martins

    2015-12-01

    In the present work we used a high-resolution, low-cost apparatus capable of detecting waves fit inside the sound bandwidth, and the software package GoldwaveTM for graphical display, processing and monitoring the signals, to study aspects of the electric heart activity of early avian embryos, specifically at the 18th Hamburger & Hamilton stage of the embryo development. The species used was the domestic chick (Gallus gallus), and we carried out 23 experiments in which cardiographic spectra of QRS complex waves representing the propagation of depolarization waves through ventricles was recorded using microprobes and reference electrodes directly on the embryos. The results show that technique using 16 bit audio card monitored by the GoldwaveTM software was efficient to study signal aspects of heart electric activity of early avian embryos.

  14. Esrrb-Cre Excises loxP-Flanked Alleles in Early Four-Cell Embryos

    PubMed Central

    Kim, Suyeon; Shaffer, Benjamin; Simerly, Calvin R.; Richard Chaillet, J.; Barak, Yaacov

    2015-01-01

    Among transgenic mice with ubiquitous Cre recombinase activity, all strains to date excise loxP-flanked (floxed) alleles, either at or before the zygote stage or at nondescript stages of development. This manuscript describes a new mouse strain, in which Cre recombinase, integrated into the Esrrb locus, efficiently excises floxed alleles in pre-implantation embryos at the onset of the four-cell stage. By enabling inactivation of genes only after the embryo has undergone two cleavages, this strain should facilitate in vivo studies of genes with essential gametic or zygotic functions. In addition, this study describes a new, highly pluripotent hybrid C57BL/6J × 129S1/SvImJ mouse embryonic stem cell line, HYB12, in which this knock-in and additional targeted alleles have been generated. PMID:26663459

  15. Shock wave propagation in layered planetary embryos

    NASA Astrophysics Data System (ADS)

    Arkani-Hamed, Jafar; Ivanov, Boris A.

    2014-05-01

    The propagation of impact-induced shock wave inside a planetary embryo is investigated using the Hugoniot equations and a new scaling law, governing the particle velocity variations along a shock ray inside a spherical body. The scaling law is adopted to determine the impact heating of a growing embryo in its early stage when it is an undifferentiated and uniform body. The new scaling law, similar to other existing scaling laws, is not suitable for a large differentiated embryo consisting of a silicate mantle overlying an iron core. An algorithm is developed in this study on the basis of the ray theory in a spherically symmetric body which relates the shock parameters at the top of the core to those at the base of the mantle, thus enabling the adoption of scaling laws to estimate the impact heating of both the mantle and the core. The algorithm is applied to two embryo models: a simple two-layered model with a uniform mantle overlying a uniform core, and a model where the pre-shock density and acoustic velocity of the embryo are radially dependent. The former illustrates details of the particle velocity, shock pressure, and temperature increase behind the shock front in a 2D axisymmetric geometry. The latter provides a means to compare the results with those obtained by a hydrocode simulation. The agreement between the results of the two techniques in revealing the effects of the core-mantle boundary on the shock wave transmission across the boundary is encouraging.

  16. Progress Towards the Development of a Fathead Minnow Embryo Test and Comparison to the Zebrafish Embryo Test for Assessing Acute Fish Toxicity

    EPA Science Inventory

    The Zebrafish Embryo Test (ZFET) for acute fish toxicity is a well developed method nearing adoption as an OECD Test Guideline. Early drafts of the test guideline (TG) envisioned a suite of potential test species to be covered including zebrafish, fathead minnow, Japanese Medaka...

  17. Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration.

    PubMed

    Ponomareva, Larissa V; Athippozhy, Antony; Thorson, Jon S; Voss, S Randal

    2015-12-01

    Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. [Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

    PubMed

    Zhao, H; Teng, X M; Li, Y F

    2017-11-25

    Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all P< 0.05]. The copy number of mtDNA and the mitochondrial membrane potential in classⅠ frozen embryo group were significantly higher than those in classⅡ frozen embryo group (both P< 0.05). Conclusion: The mtDNA copy number and the mitochondrial membrane potential of embryos of the better quality embryo are higher.

  19. Asymmetric distribution of hypoxia-inducible factor α regulates dorsoventral axis establishment in the early sea urchin embryo.

    PubMed

    Chang, Wei-Lun; Chang, Yi-Cheng; Lin, Kuan-Ting; Li, Han-Ru; Pai, Chih-Yu; Chen, Jen-Hao; Su, Yi-Hsien

    2017-08-15

    Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs hypoxic acclimation and can result in various diseases, including cancers. The role of hypoxia signaling in early embryogenesis remains unclear. Here, we show that in the blastula of the sea urchin Strongylocentrotus purpuratus , hypoxia-inducible factor α (HIFα), the downstream transcription factor of the hypoxia pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricts the expression of nodal to the ventral side and activates several genes encoding transcription factors on the dorsal side. We also observed that intrinsic hypoxic signals in the early embryos formed a gradient, which was disrupted under hypoxic conditions. Our results reveal an unprecedented role of the hypoxia pathway in animal development. © 2017. Published by The Company of Biologists Ltd.

  20. TopBP1 deficiency causes an early embryonic lethality and induces cellular senescence in primary cells.

    PubMed

    Jeon, Yoon; Ko, Eun; Lee, Kyung Yong; Ko, Min Ji; Park, Seo Young; Kang, Jeeheon; Jeon, Chang Hwan; Lee, Ho; Hwang, Deog Su

    2011-02-18

    TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.