Muscle structure, sarcomere length and influences on meat quality: A review.

PubMed

Ertbjerg, Per; Puolanne, Eero

2017-10-01

The basic contractile unit of muscle, the sarcomere, will contract as the muscle goes into rigor post-mortem. Depending on the conditions, such as the rate of pH decline, the cooling rate and the mechanical restraints on the muscles, this longitudinal shortening will result in various post-mortem sarcomere lengths as well as lateral differences in the distances between the myosin and actin filaments. This shortening is underlying the phenomena described as rigor contraction, thaw rigor, cold shortening and heat shortening. The shortening in combination with the molecular architecture of the sarcomere as defined by the myosin filaments and their S-1 and S-2 units, the interaction with the actin filaments, and the boundaries formed by the Z-disks will subsequently influence basic meat quality traits including tenderness and water-holding capacity. Biochemical reactions from proteolysis and glycogen metabolism interrelate with the sarcomere length in a complex manner. The sarcomere length is also influencing the eating quality of cooked meat and the water-holding in meat products. Copyright © 2017 Elsevier Ltd. All rights reserved.

Eliminating Cancer Stem Cells by Targeting Embryonic Signaling Pathways.

PubMed

Oren, Ohad; Smith, B Douglas

2017-02-01

Dramatic advances have been made in the understanding of cancer over the past decade. Prime among those are better appreciation of the biology of cancer and the development of targeted therapies. Despite these improvements, however, most tumors remain refractory to anti-cancer medications and frequently recur. Cancer Stem Cells (CSCs), which in some cases express markers of pluripotency (e.g., Oct-4), share many of the molecular features of normal stem cells. These cells have been hypothesised to play a role in tumor resistance and relapse. They exhibit dependence on many primitive regulatory pathways and may be best viewed in the context of embryonic signaling pathways. In this article, we review important embryonic signaling cascades and their differential expression in CSCs. We also discuss these pathways as actionable targets for novel therapies in hopes that eliminating cancer stem cells will lead to an improvement in overall survival for patients.

In Vivo Imaging of Human Sarcomere Twitch Dynamics in Individual Motor Units

PubMed Central

Sanchez, Gabriel N.; Sinha, Supriyo; Liske, Holly; Chen, Xuefeng; Nguyen, Viet; Delp, Scott L.; Schnitzer, Mark J.

2017-01-01

SUMMARY Motor units comprise a pre-synaptic motor neuron and multiple post-synaptic muscle fibers. Many movement disorders disrupt motor unit contractile dynamics and the structure of sarcomeres, skeletal muscle’s contractile units. Despite the motor unit’s centrality to neuromuscular physiology, no extant technology can image sarcomere twitch dynamics in live humans. We created a wearable microscope equipped with a microendoscope for minimally invasive observation of sarcomere lengths and contractile dynamics in any major skeletal muscle. By electrically stimulating twitches via the microendoscope and visualizing the sarcomere displacements, we monitored single motor unit contractions in soleus and vastus lateralis muscles of healthy individuals. Control experiments verified that these evoked twitches involved neuromuscular transmission and faithfully reported muscle force generation. In post-stroke patients with spasticity of the biceps brachii, we found involuntary microscopic contractions and sarcomere length abnormalities. The wearable microscope facilitates exploration of many basic and disease-related neuromuscular phenomena never visualized before in live humans. PMID:26687220

In Vivo Imaging of Human Sarcomere Twitch Dynamics in Individual Motor Units.

PubMed

Sanchez, Gabriel N; Sinha, Supriyo; Liske, Holly; Chen, Xuefeng; Nguyen, Viet; Delp, Scott L; Schnitzer, Mark J

2015-12-16

Motor units comprise a pre-synaptic motor neuron and multiple post-synaptic muscle fibers. Many movement disorders disrupt motor unit contractile dynamics and the structure of sarcomeres, skeletal muscle's contractile units. Despite the motor unit's centrality to neuromuscular physiology, no extant technology can image sarcomere twitch dynamics in live humans. We created a wearable microscope equipped with a microendoscope for minimally invasive observation of sarcomere lengths and contractile dynamics in any major skeletal muscle. By electrically stimulating twitches via the microendoscope and visualizing the sarcomere displacements, we monitored single motor unit contractions in soleus and vastus lateralis muscles of healthy individuals. Control experiments verified that these evoked twitches involved neuromuscular transmission and faithfully reported muscle force generation. In post-stroke patients with spasticity of the biceps brachii, we found involuntary microscopic contractions and sarcomere length abnormalities. The wearable microscope facilitates exploration of many basic and disease-related neuromuscular phenomena never visualized before in live humans. Copyright © 2015 Elsevier Inc. All rights reserved.

High-frequency sarcomeric auto-oscillations induced by heating in living neonatal cardiomyocytes of the rat

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

Shintani, Seine A.; Oyama, Kotaro; Fukuda, Norio, E-mail: noriof@jikei.ac.jp

2015-02-06

Highlights: • We tested the effects of infra-red laser irradiation on cardiac sarcomere dynamics. • A rise in temperature (>∼38 °C) induced high-frequency sarcomeric auto-oscillations. • These oscillations occurred with and without blockade of intracellular Ca{sup 2+} stores. • Cardiac sarcomeres can play a role as a temperature-dependent rhythm generator. - Abstract: In the present study, we investigated the effects of infra-red laser irradiation on sarcomere dynamics in living neonatal cardiomyocytes of the rat. A rapid increase in temperature to >∼38 °C induced [Ca{sup 2+}]{sub i}-independent high-frequency (∼5–10 Hz) sarcomeric auto-oscillations (Hyperthermal Sarcomeric Oscillations; HSOs). In myocytes with the intactmore » sarcoplasmic reticular functions, HSOs coexisted with [Ca{sup 2+}]{sub i}-dependent spontaneous beating in the same sarcomeres, with markedly varying frequencies (∼10 and ∼1 Hz for the former and latter, respectively). HSOs likewise occurred following blockade of the sarcoplasmic reticular functions, with the amplitude becoming larger and the frequency lower in a time-dependent manner. The present findings suggest that in the mammalian heart, sarcomeres spontaneously oscillate at higher frequencies than the sinus rhythm at temperatures slightly above the physiologically relevant levels.« less

S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+ Sensitivity in Intact Cardiomyocytes

PubMed Central

Figueiredo-Freitas, Cícero; Dulce, Raul A.; Foster, Matthew W.; Liang, Jingsheng; Yamashita, Aline M.S.; Lima-Rosa, Frederico L.; Thompson, J. Will; Moseley, M. Arthur; Hare, Joshua M.; Nogueira, Leonardo; Sorenson, Martha M.

2015-01-01

Abstract Aims: The heart responds to physiological and pathophysiological stress factors by increasing its production of nitric oxide (NO), which reacts with intracellular glutathione to form S-nitrosoglutathione (GSNO), a protein S-nitrosylating agent. Although S-nitrosylation protects some cardiac proteins against oxidative stress, direct effects on myofilament performance are unknown. We hypothesize that S-nitrosylation of sarcomeric proteins will modulate the performance of cardiac myofilaments. Results: Incubation of intact mouse cardiomyocytes with S-nitrosocysteine (CysNO, a cell-permeable low-molecular-weight nitrosothiol) significantly decreased myofilament Ca2+ sensitivity. In demembranated (skinned) fibers, S-nitrosylation with 1 μM GSNO also decreased Ca2+ sensitivity of contraction and 10 μM reduced maximal isometric force, while inhibition of relaxation and myofibrillar ATPase required higher concentrations (≥100 μM). Reducing S-nitrosylation with ascorbate partially reversed the effects on Ca2+ sensitivity and ATPase activity. In live cardiomyocytes treated with CysNO, resin-assisted capture of S-nitrosylated protein thiols was combined with label-free liquid chromatography–tandem mass spectrometry to quantify S-nitrosylation and determine the susceptible cysteine sites on myosin, actin, myosin-binding protein C, troponin C and I, tropomyosin, and titin. The ability of sarcomere proteins to form S-NO from 10–500 μM CysNO in intact cardiomyocytes was further determined by immunoblot, with actin, myosin, myosin-binding protein C, and troponin C being the more susceptible sarcomeric proteins. Innovation and Conclusions: Thus, specific physiological effects are associated with S-nitrosylation of a limited number of cysteine residues in sarcomeric proteins, which also offer potential targets for interventions in pathophysiological situations. Antioxid. Redox Signal. 23, 1017–1034. PMID:26421519

Fucoidan promotes early step of cardiac differentiation from human embryonic stem cells and long-term maintenance of beating areas.

PubMed

Hamidi, Sofiane; Letourneur, Didier; Aid-Launais, Rachida; Di Stefano, Antonio; Vainchenker, William; Norol, Françoise; Le Visage, Catherine

2014-04-01

Somatic stem cells require specific niches and three-dimensional scaffolds provide ways to mimic this microenvironment. Here, we studied a scaffold based on Fucoidan, a sulfated polysaccharide known to influence morphogen gradients during embryonic development, to support human embryonic stem cells (hESCs) differentiation toward the cardiac lineage. A macroporous (pore 200 μm) Fucoidan scaffold was selected to support hESCs attachment and proliferation. Using a protocol based on the cardiogenic morphogen bone morphogenic protein 2 (BMP2) and transforming growth factor (TGFβ) followed by tumor necrosis factor (TNFα), an effector of cardiopoietic priming, we examined the cardiac differentiation in the scaffold compared to culture dishes and embryoid bodies (EBs). At day 8, Fucoidan scaffolds supported a significantly higher expression of the 3 genes encoding for transcription factors marking the early step of embryonic cardiac differentiation NKX2.5 (p<0.05), MEF2C (p<0.01), and GATA4 (p<0.01), confirmed by flow cytometry analysis for MEF2C and NKX2.5. The ability of Fucoidan scaffolds to locally concentrate and slowly release TGFβ and TNFα was confirmed by Luminex technology. We also found that Fucoidan scaffolds supported the late stage of embryonic cardiac differentiation marked by a significantly higher atrial natriuretic factor (ANF) expression (p<0.001), although only rare beating areas were observed. We postulated that absence of mechanical stress in the soft hydrogel impaired sarcomere formation, as confirmed by molecular analysis of the cardiac muscle myosin MYH6 and immunohistological staining of sarcomeric α-actinin. Nevertheless, Fucoidan scaffolds contributed to the development of thin filaments connecting beating areas through promotion of smooth muscle cells, thus enabling maintenance of beating areas for up to 6 months. In conclusion, Fucoidan scaffolds appear as a very promising biomaterial to control cardiac differentiation from hESCs that

Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes

PubMed Central

White, Jennifer; Barro, Marietta V.; Makarenkova, Helen P.; Sanger, Joseph W.; Sanger, Jean M.

2014-01-01

It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, non-muscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that non-muscle myosins II A and B are components of the Z-Bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-Bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. PMID:25125171

Sarcomere Length and Tension Changes in Tetanized Frog Muscle Fibers after Quick Stretches and Releases

NASA Astrophysics Data System (ADS)

Sugi, Haruo; Kobayashi, Takakazu

1983-10-01

The sarcomere length changes in tetanized frog muscle fibers in response to quick fiber length changes were examined along the fiber length with a high-sensitivity laser diffraction technique. The experiments were only performed with muscle fibers in which the uniform orientation and sarcomere length of the component myofibrils were well preserved during a tetanus. When the sarcomere length changes were recorded near the fixed fiber end, the delay of the onset of sarcomere length change in response to the applied fiber length change tended to be longer than that of the onset of tension changes recorded at the fixed fiber end. The magnitude of sarcomere length changes was larger near the moving fiber end than near the fixed fiber end. In the case of quick releases, the resulting sarcomere shortening tended to outlast the fiber shortening, so that the quick tension recovery started during the sarcomere shortening. These results indicate (i) that the tension changes in response to quick fiber length changes may not give direct information about the cross-bridge properties and (ii) that the viscoelastic multisegmental nature of muscle fibers should be taken into consideration in interpreting the tension responses to quick length changes.

T1 Measurements Identify Extracellular Volume Expansion in Hypertrophic Cardiomyopathy Sarcomere Mutation Carriers With and Without Left Ventricular Hypertrophy

PubMed Central

Ho, Carolyn Y.; Abbasi, Siddique A.; Neilan, Tomas G.; Shah, Ravi V.; Chen, Yucheng; Heydari, Bobak; Cirino, Allison L.; Lakdawala, Neal K.; Orav, E. John; González, Arantxa; López, Begoña; Díez, Javier; Jerosch-Herold, Michael; Kwong, Raymond Y.

2013-01-01

Background Myocardial fibrosis is a hallmark of hypertrophic cardiomyopathy (HCM) and a potential substrate for arrhythmias and heart failure. Sarcomere mutations appear to induce profibrotic changes before left ventricular hypertrophy (LVH) develops. To further evaluate these processes, we used cardiac magnetic resonance (CMR) with T1 measurements on a genotyped HCM population to quantify myocardial extracellular volume (ECV). Methods and Results Sarcomere mutation carriers with LVH (G+/LVH+, n = 37) and without LVH (G+/LVH−, n = 29); HCM patients without mutations (sarcomere-negative HCM, n = 11); and healthy controls (n = 11) underwent contrast CMR, measuring T1 times pre- and post-gadolinium infusion. Concurrent echocardiography and serum biomarkers of collagen synthesis, hemodynamic stress, and myocardial injury were also available in a subset. Compared to controls, ECV was increased in patients with overt HCM, as well as G+/LVH− mutation carriers (ECV= 0.36±0.01, 0.33±0.01, 0.27±0.01 in G+/LVH+, G+/LVH−, controls, respectively, P≤0.001 for all comparisons). ECV correlated with NT-proBNP levels (r = 0.58, P<0.001) and global E’ velocity (r = −0.48, P<0.001). Late gadolinium enhancement (LGE) was present in >60% of overt HCM patients but absent from G+/LVH− subjects. Both ECV and LGE were more extensive in sarcomeric HCM than sarcomere-negative HCM. Conclusions Myocardial ECV is increased in HCM sarcomere mutation carriers even in the absence of LVH. These data provide additional support that fibrotic remodeling is triggered early in disease pathogenesis. Quantifying ECV may help characterize the development myocardial fibrosis in HCM and ultimately assist in developing novel disease-modifying therapy, targeting interstitial fibrosis. PMID:23549607

Embryonic demise caused by targeted disruption of a cysteine protease Dub-2.

PubMed

Baek, Kwang-Hyun; Lee, Heyjin; Yang, Sunmee; Lim, Soo-Bin; Lee, Wonwoo; Lee, Jeoung Eun; Lim, Jung-Jin; Jun, Kisun; Lee, Dong-Ryul; Chung, Young

2012-01-01

A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.

Controversies in cancer stem cells: targeting embryonic signaling pathways.

PubMed

Takebe, Naoko; Ivy, S Percy

2010-06-15

Selectively targeting cancer stem cells (CSC) or tumor-initiating cells (TIC; from this point onward referred to as CSCs) with novel agents is a rapidly emerging field of oncology. Our knowledge of CSCs and their niche microenvironments remains a nascent field. CSC's critical dependence upon self-renewal makes these regulatory signaling pathways ripe for the development of experimental therapeutic agents. Investigational agents targeting the Notch, Hedgehog, and Wnt pathways are currently in late preclinical development stages, with some early phase 1-2 testing in human subjects. This series of articles will provide an overview and summary of the current state of knowledge of CSCs, their interactive microenvironment, and how they may serve as important targets for antitumor therapies. We also examine the scope and stage of development of early experimental agents that specifically target these highly conserved embryonic signaling pathways. (c) 2010 AACR.

Early results of sarcomeric gene screening from the Egyptian National BA-HCM Program.

PubMed

Kassem, Heba Sh; Azer, Remon S; Saber-Ayad, Maha; Ayad, Maha S; Moharem-Elgamal, Sarah; Magdy, Gehan; Elguindy, Ahmed; Cecchi, Franco; Olivotto, Iacopo; Yacoub, Magdi H

2013-02-01

The present study comprised sarcomeric genotyping of the three most commonly involved sarcomeric genes: MYBPC3, MYH7, and TNNT2 in 192 unrelated Egyptian hypertrophic cardiomyopathy (HCM) index patients. Mutations were detected in 40 % of cases. Presence of positive family history was significantly (p=0.002) associated with a higher genetic positive yield (49/78, 62.8 %). The majority of the detected mutations in the three sarcomeric genes were novel (40/62, 65 %) and mostly private (47/62, 77 %). Single nucleotide substitution was the most frequently detected mutation type (51/62, 82 %). Over three quarters of these substitutions (21/27, 78 %) involved CpG dinucleotide sites and resulted from C>T or G>A transition in the three analyzed genes, highlighting the significance of CpG high mutability within the sarcomeric genes examined. This study could aid in global comparative studies in different ethnic populations and constitutes an important step in the evolution of the integrated clinical, translational, and basic science HCM program.

Real-time determination of sarcomere length of a single cardiomyocyte during contraction

PubMed Central

Kalda, Mari; Vendelin, Marko

2013-01-01

Sarcomere length of a cardiomyocyte is an important control parameter for physiology studies on a single cell level; for instance, its accurate determination in real time is essential for performing single cardiomyocyte contraction experiments. The aim of this work is to develop an efficient and accurate method for estimating a mean sarcomere length of a contracting cardiomyocyte using microscopy images as an input. The novelty in developed method lies in 1) using unbiased measure of similarities to eliminate systematic errors from conventional autocorrelation function (ACF)-based methods when applied to region of interest of an image, 2) using a semianalytical, seminumerical approach for evaluating the similarity measure to take into account spatial dependence of neighboring image pixels, and 3) using a detrend algorithm to extract the sarcomere striation pattern content from the microscopy images. The developed sarcomere length estimation procedure has superior computational efficiency and estimation accuracy compared with the conventional ACF and spectral analysis-based methods using fast Fourier transform. As shown by analyzing synthetic images with the known periodicity, the estimates obtained by the developed method are more accurate at the subpixel level than ones obtained using ACF analysis. When applied in practice on rat cardiomyocytes, our method was found to be robust to the choice of the region of interest that may 1) include projections of carbon fibers and nucleus, 2) have uneven background, and 3) be slightly disoriented with respect to average direction of sarcomere striation pattern. The developed method is implemented in open-source software. PMID:23255581

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output

PubMed Central

Gresham, Kenneth S.; Mamidi, Ranganath; Li, Jiayang; Kwak, Hyerin

2017-01-01

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and β-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/dtmax and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca2+ activation compared with SAL-treated mice but unaltered myofilament Ca2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output. NEW & NOTEWORTHY Posttranslational modifications to sarcomeric

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output.

PubMed

Gresham, Kenneth S; Mamidi, Ranganath; Li, Jiayang; Kwak, Hyerin; Stelzer, Julian E

2017-03-01

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and β-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/d t max and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca 2+ activation compared with SAL-treated mice but unaltered myofilament Ca 2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output. NEW & NOTEWORTHY Posttranslational modifications to sarcomeric

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein-protein interactions.

PubMed

Lambert, Matthias; Richard, Elodie; Duban-Deweer, Sophie; Krzewinski, Frederic; Deracinois, Barbara; Dupont, Erwan; Bastide, Bruno; Cieniewski-Bernard, Caroline

2016-09-01

The sarcomere structure of skeletal muscle is determined through multiple protein-protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization. C2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level. Our data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin. For the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure. In the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Force Generation via β-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions.

PubMed

Chopra, Anant; Kutys, Matthew L; Zhang, Kehan; Polacheck, William J; Sheng, Calvin C; Luu, Rebeccah J; Eyckmans, Jeroen; Hinson, J Travis; Seidman, Jonathan G; Seidman, Christine E; Chen, Christopher S

2018-01-08

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that β-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from β-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Temporal changes in sarcomere lesions of rat adductor longus muscles during hindlimb reloading

NASA Technical Reports Server (NTRS)

Krippendorf, B. B.; Riley, D. A.

1994-01-01

Focal sarcomere disruptions were previously observed in adductor longus muscles of rats flown approximately two weeks aboard the Cosmos 1887 and 2044 biosatellite flights. These lesions, characterized by breakage and loss of myofilaments and Z-line streaming, resembled damage induced by unaccustomed exercise that includes eccentric contractions in which muscles lengthen as they develop tension. We hypothesized that sarcomere lesions in atrophied muscles of space flow rats were not produced in microgravity by muscle unloading but resulted from muscle reloading upon re-exposure to terrestrial gravity. To test this hypothesis, we examined temporal changes in sarcomere integrity of adductor longus muscles from rats subjected to 12.5 days of hindlimb suspension unloading and subsequent reloading by return to vivarium cages for 0, 6, 12, or 48 hours of normal weightbearing. Our ultrastructural observations suggested that muscle unloading (0 h reloading) induced myofibril misalignment associated with myofiber atrophy. Muscle reloading for 6 hours induced focal sarcomere lesions in which cross striations were abnormally widened. Such lesions were electron lucent due to extensive myofilament loss. Lesions in reloaded muscles showed rapid restructuring. By 12 hours of reloading, lesions were moderately stained foci and by 48 hours darkly stained foci in which the pattern of cross striations was indistinct at the light and electron microscopic levels. These lesions were spanned by Z-line-like electron dense filamentous material. Our findings suggest a new role for Z-line streaming in lesion restructuring: rather than an antecedent to damage, this type of Z-line streaming may be indicative of rapid, early sarcomere repair.

Breaking sarcomeres by in vitro exercise

PubMed Central

Orfanos, Zacharias; Gödderz, Markus P. O.; Soroka, Ekaterina; Gödderz, Tobias; Rumyantseva, Anastasia; van der Ven, Peter F. M.; Hawke, Thomas J.; Fürst, Dieter O.

2016-01-01

Eccentric exercise leads to focal disruptions in the myofibrils, referred to as “lesions”. These structures are thought to contribute to the post-exercise muscle weakness, and to represent areas of mechanical damage and/or remodelling. Lesions have been investigated in human biopsies and animal samples after exercise. However, this approach does not examine the mechanisms behind lesion formation, or their behaviour during contraction. To circumvent this, we used electrical pulse stimulation (EPS) to simulate exercise in C2C12 myotubes, combined with live microscopy. EPS application led to the formation of sarcomeric lesions in the myotubes, resembling those seen in exercised mice, increasing in number with the time of application or stimulation intensity. Furthermore, transfection with an EGFP-tagged version of the lesion and Z-disc marker filamin-C allowed us to observe the formation of lesions using live cell imaging. Finally, using the same technique we studied the behaviour of these structures during contraction, and observed them to be passively stretching. This passive behaviour supports the hypothesis that lesions contribute to the post-exercise muscle weakness, protecting against further damage. We conclude that EPS can be reliably used as a model for the induction and study of sarcomeric lesions in myotubes in vitro. PMID:26804343

Patterns of sarcomere activation, temperature dependence, and effect of ryanodine in chemically skinned cardiac fibers

PubMed Central

1986-01-01

Functionally skinned and electrochemically shunted myocytes were prepared by perfusing rat hearts with collagenase in order to obtain a technically improved measurement of sarcomere dynamics and to evaluate the role of sarcoplasmic reticulum in situ with respect to contractile activation. In the presence of micromolar calcium, the myocytes exhibited phasic and propagated contraction waves beginning at one end and proceeding along the myocyte. Beating rates, the propagation velocity of the activation wave, and single sarcomere shortening and relaxation velocities were obtained by manual or automated analysis of 16-mm film recorded at 170 frames/s from a camera attached to a microscope that was equipped with a temperature-controlled stage. In parallel experiments, calcium accumulation by the sarcoplasmic reticulum of the myocytes in situ was measured by direct isotopic tracer methods. The frequency (10-38 min-1) of spontaneous contractions, the velocity (1.9-7.4 microns . s-1) of sarcomere shortening, and the velocity (1.7-6.8 microns . s-1) of sarcomere relaxation displayed identical temperature dependences (Q10 = 2.2), which are similar to that of the calcium pump of sarcoplasmic reticulum and are consistent with a rate limit imposed by enzyme-catalyzed mechanisms on all these parameters. On the other hand, the velocity (77- 159 microns . s-1) of sequential sarcomere activation displayed a lower temperature dependence (Q10 = 1.5), which is consistent with a diffusion-limited and self-propagating release of calcium from one sarcomere to the other. The phasic contractile activity of the dissociated myocytes was inhibited by 10(-8)-10(6) M ryanodine (and not by myolemmal calcium blockers) under conditions in which calcium accumulation by sarcoplasmic reticulum in situ was demonstrated to proceed optimally. The effect of ryanodine is attributed to an interaction of this drug with sarcotubular structures, producing inhibition of calcium release from the sarcoplasmic

The conserved transmembrane proteoglycan Perdido/Kon-tiki is essential for myofibrillogenesis and sarcomeric structure in Drosophila

PubMed Central

Pérez-Moreno, Juan J.; Bischoff, Marcus; Martín-Bermudo, Maria D.; Estrada, Beatriz

2014-01-01

ABSTRACT Muscle differentiation requires the assembly of high-order structures called myofibrils, composed of sarcomeres. Even though the molecular organization of sarcomeres is well known, the mechanisms underlying myofibrillogenesis are poorly understood. It has been proposed that integrin-dependent adhesion nucleates myofibrils at the periphery of the muscle cell to sustain sarcomere assembly. Here, we report a role for the gene perdido (perd, also known as kon-tiki, a transmembrane chondroitin proteoglycan) in myofibrillogenesis. Expression of perd RNAi in muscles, prior to adult myogenesis, can induce misorientation and detachment of Drosophila adult abdominal muscles. In comparison to controls, perd-depleted muscles contain fewer myofibrils, which are localized at the cell periphery. These myofibrils are detached from each other and display a defective sarcomeric structure. Our results demonstrate that the extracellular matrix receptor Perd has a specific role in the assembly of myofibrils and in sarcomeric organization. We suggest that Perd acts downstream or in parallel to integrins to enable the connection of nascent myofibrils to the Z-bands. Our work identifies the Drosophila adult abdominal muscles as a model to investigate in vivo the mechanisms behind myofibrillogenesis. PMID:24794494

Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans.

PubMed

Hwang, Hyundoo; Barnes, Dawn E; Matsunaga, Yohei; Benian, Guy M; Ono, Shoichiro; Lu, Hang

2016-01-29

The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.

Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Hwang, Hyundoo; Barnes, Dawn E.; Matsunaga, Yohei; Benian, Guy M.; Ono, Shoichiro; Lu, Hang

2016-01-01

The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.

Hand-Held Model of a Sarcomere to Illustrate the Sliding Filament Mechanism in Muscle Contraction

ERIC Educational Resources Information Center

Jittivadhna, Karnyupha; Ruenwongsa, Pintip; Panijpan, Bhinyo

2009-01-01

From our teaching of the contractile unit of the striated muscle, we have found limitations in using textbook illustrations of sarcomere structure and its related dynamic molecular physiological details. A hand-held model of a striated muscle sarcomere made from common items has thus been made by us to enhance students' understanding of the…

MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

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

Gong, Xi; Zhang, Kunshan; Wang, Yanlu

2013-10-04

Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fatemore » specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.« less

The magnitude of muscle strain does not influence serial sarcomere number adaptations following eccentric exercise.

PubMed

Butterfield, Timothy A; Herzog, Walter

2006-02-01

It is generally accepted that eccentric exercise, when performed by a muscle that is unaccustomed to that type of contraction, results in a delayed onset of muscle soreness (DOMS). A prolonged exposure to eccentric exercise leads to the disappearance of the signs and symptoms associated with DOMS, which has been referred to as the repeated bout effect (RBE). Although the mechanisms underlying the RBE remain unclear, several mechanisms have been proposed, including the serial sarcomere number addition following exercise induced muscle damage. In the traditional DOMS and RBE protocols, muscle injury has been treated as a global parameter, with muscle force and strain assumed to be uniform throughout the muscle. To assess the effects of muscle-tendon unit strain, fiber strain, torque and injury on serial sarcomere number adaptations, three groups of New Zealand White (NZW) rabbits were subjected to chronic repetitive eccentric exercise bouts of the ankle dorsiflexors for 6 weeks. These eccentric exercise protocols consisted of identical muscle tendon unit (MTU) strain, but other mechanical factors were systematically altered. Following chronic eccentric exercise, serial sarcomere number adaptations were not identical between the three eccentric exercise protocols, and serial sarcomere number adaptations were not uniform across all regions of the muscle. Peak torque and relaxation fiber strain were the best predictors of serial sarcomere number across all three protocols. Therefore, MTU strain does not appear to be the primary cause for sarcomerogenesis, and differential adaptations within the muscle may be explained by the nonuniform architecture of the muscle, resulting in differential local fiber strains.

Cofilin-2 controls actin filament length in muscle sarcomeres

PubMed Central

Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka

2014-01-01

SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779

Sarcomere length-dependence of activity-dependent twitch potentiation in mouse skeletal muscle

PubMed Central

Rassier, Dilson E; MacIntosh, Brian R

2002-01-01

Background It has been reported that potentiation of a skeletal muscle twitch response is proportional to muscle length with a negative slope during staircase, and a positive slope during posttetanic potentiation. This study was done to directly compare staircase and posttetanic responses with measurement of sarcomere length to compare their length-dependence. Methods Mouse extensor digitorum longus (EDL) muscles were dissected to small bundles of fibers, which permit measurement of sarcomere length (SL), by laser diffraction. In vitro fixed-end contractions of EDL fiber bundles were elicited at 22°C and 35°C at sarcomere lengths ranging from 2.35 μm to 3.85 μm. Twitch contractions were assessed before and after 1.5 s of 75 Hz stimulation at 22°C or during 10 s of 10 Hz stimulation at 22°C or 35°C. Results Staircase potentiation was greater at 35°C than 22°C, and the relative magnitude of the twitch contraction (Pt*/Pt) was proportional to sarcomere length with a negative slope, over the range 2.3 μm – 3.7 μm. Linear regression yielded the following: Pt*/Pt = -0.59·SL+3.27 (r2 = 0.74); Pt*/Pt = -0.39·SL+2.34 (r2 = 0.48); and Pt*/Pt = -0.50·SL+2.45 (r2 = 0.80) for staircase at 35°C, and 22°C and posttetanic response respectively. Posttetanic depression rather than potentiation was present at long SL. This indicates that there may be two processes operating in these muscles to modulate the force: one that enhances and a second that depresses the force. Either or both of these processes may have a length-dependence of its mechanism. Conclusion There is no evidence that posttetanic potentiation is fundamentally different from staircase in these muscles. PMID:12475395

Pre-mRNA mis-splicing of sarcomeric genes in heart failure.

PubMed

Zhu, Chaoqun; Chen, Zhilong; Guo, Wei

2017-08-01

Pre-mRNA splicing is an important biological process that allows production of multiple proteins from a single gene in the genome, and mainly contributes to protein diversity in eukaryotic organisms. Alternative splicing is commonly governed by RNA binding proteins to meet the ever-changing demands of the cell. However, the mis-splicing may lead to human diseases. In the heart of human, mis-regulation of alternative splicing has been associated with heart failure. In this short review, we focus on alternative splicing of sarcomeric genes and review mis-splicing related heart failure with relatively well studied Sarcomeric genes and splicing mechanisms with identified regulatory factors. The perspective of alternative splicing based therapeutic strategies in heart failure has also been discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Sarcomeric hypertrophic cardiomyopathy: genetic profile in a Portuguese population.

PubMed

Brito, Dulce; Miltenberger-Miltenyi, Gabriel; Vale Pereira, Sónia; Silva, Doroteia; Diogo, António Nunes; Madeira, Hugo

2012-09-01

Sarcomeric hypertrophic cardiomyopathy has heterogeneous phenotypic expressions, of which sudden cardiac death is the most feared. A genetic diagnosis is essential to identify subjects at risk in each family. The spectrum of disease-causing mutations in the Portuguese population is unknown. Seventy-seven unrelated probands with hypertrophic cardiomyopathy were systematically screened for mutations by PCR and sequencing of five sarcomeric genes: MYBPC3, MYH7, TNNT2, TNNI3 and MYL2. Familial cosegregation analysis was performed in most patients. Thirty-four different mutations were identified in 41 (53%) index patients, 71% with familial hypertrophic cardiomyopathy. The most frequently involved gene was MYBPC3 (66%) with 22 different mutations (8 novel) in 27 patients, followed by MYH7 (22%), TNNT2 (12%) and TNNI3 (2.6%). In three patients (7%), two mutations were found in MYBPC3 and/or MYH7. Additionally, 276 relatives were screened, leading to the identification of a mean of three other affected relatives for each pedigree with the familial form of the disease. Disease-associated mutations were identified mostly in familial hypertrophic cardiomyopathy, corroborating the idea that rarely studied genes may be implicated in sporadic forms. Private mutations are the rule, MYBPC3 being the most commonly involved gene. Mutations in MYBPC3 and MYH7 accounted for most cases of sarcomere-related disease. Multiple mutations in these genes may occur, which highlights the importance of screening both. The detection of novel mutations strongly suggests that all coding regions should be systematically screened. Genotyping in hypertrophic cardiomyopathy enables a more precise diagnosis of the disease, with implications for risk stratification and genetic counseling. Copyright © 2011 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

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

Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.more » - Highlights: • Dexamethasone accelerates Ca{sup 2+} transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I{sub Ca,L} and action potential characteristics in hESC-CMs.« less

The role of sarcomere gene mutations in patients with idiopathic dilated cardiomyopathy

PubMed Central

Møller, Daniel Vega; Andersen, Paal Skytt; Hedley, Paula; Ersbøll, Mads Kristian; Bundgaard, Henning; Moolman-Smook, Johanna; Christiansen, Michael; Køber, Lars

2009-01-01

We investigated a Danish cohort of 31 unrelated patients with idiopathic dilated cardiomyopathy (IDC), to assess the role that mutations in sarcomere protein genes play in IDC. Patients were genetically screened by capillary electrophoresis single strand conformation polymorphism and subsequently by bidirectional DNA sequencing of conformers in the coding regions of MYH7, MYBPC3, TPM1, ACTC, MYL2, MYL3, TNNT2, CSRP3 and TNNI3. Eight probands carried disease-associated genetic variants (26%). In MYH7, three novel mutations were found; in MYBPC3, one novel variant and two known mutations were found; and in TNNT2, a known mutation was found. One proband was double heterozygous. We find evidence of phenotypic plasticity: three mutations described earlier as HCM causing were found in four cases of IDC, with no history of a hypertrophic phase. Furthermore, one pedigree presented with several cases of classic DCM as well as one case with left ventricular non-compaction. Disease-causing sarcomere gene mutations were found in about one-quarter of IDC patients, and seem to play an important role in the causation of the disease. The genetics is as complex as seen in HCM. Thus, our data suggest that a genetic work-up should include screening of the most prominent sarcomere genes even in the absence of a family history of the disease. PMID:19293840

Cardiac alpha-myosin (MYH6) is the predominant sarcomeric disease gene for familial atrial septal defects.

PubMed

Posch, Maximilian G; Waldmuller, Stephan; Müller, Melanie; Scheffold, Thomas; Fournier, David; Andrade-Navarro, Miguel A; De Geeter, Bernard; Guillaumont, Sophie; Dauphin, Claire; Yousseff, Dany; Schmitt, Katharina R; Perrot, Andreas; Berger, Felix; Hetzer, Roland; Bouvagnet, Patrice; Özcelik, Cemil

2011-01-01

Secundum-type atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD) and are associated with a familial risk. Mutations in transcription factors represent a genetic source for ASDII. Yet, little is known about the role of mutations in sarcomeric genes in ASDII etiology. To assess the role of sarcomeric genes in patients with inherited ASDII, we analyzed 13 sarcomeric genes (MYH7, MYBPC3, TNNT2, TCAP, TNNI3, MYH6, TPM1, MYL2, CSRP3, ACTC1, MYL3, TNNC1, and TTN kinase region) in 31 patients with familial ASDII using array-based resequencing. Genotyping of family relatives and control subjects as well as structural and homology analyses were used to evaluate the pathogenic impact of novel non-synonymous gene variants. Three novel missense mutations were found in the MYH6 gene encoding alpha-myosin heavy chain (R17H, C539R, and K543R). These mutations co-segregated with CHD in the families and were absent in 370 control alleles. Interestingly, all three MYH6 mutations are located in a highly conserved region of the alpha-myosin motor domain, which is involved in myosin-actin interaction. In addition, the cardiomyopathy related MYH6-A1004S and the MYBPC3-A833T mutations were also found in one and two unrelated subjects with ASDII, respectively. No mutations were found in the 11 other sarcomeric genes analyzed. The study indicates that sarcomeric gene mutations may represent a so far underestimated genetic source for familial recurrence of ASDII. In particular, perturbations in the MYH6 head domain seem to play a major role in the genetic origin of familial ASDII.

Differential Sarcomere and Electrophysiological Maturation of Human iPSC-Derived Cardiac Myocytes in Monolayer vs. Aggregation-Based Differentiation Protocols

PubMed Central

Jeziorowska, Dorota; Fontaine, Vincent; Jouve, Charlène; Villard, Eric; Dussaud, Sébastien; Akbar, David; Letang, Valérie; Cervello, Pauline; Itier, Jean-Michiel; Pruniaux, Marie-Pierre; Hulot, Jean-Sébastien

2017-01-01

Human induced pluripotent stem cells (iPSCs) represent a powerful human model to study cardiac disease in vitro, notably channelopathies and sarcomeric cardiomyopathies. Different protocols for cardiac differentiation of iPSCs have been proposed either based on embroid body formation (3D) or, more recently, on monolayer culture (2D). We performed a direct comparison of the characteristics of the derived cardiomyocytes (iPSC-CMs) on day 27 ± 2 of differentiation between 3D and 2D differentiation protocols with two different Wnt-inhibitors were compared: IWR1 (inhibitor of Wnt response) or IWP2 (inhibitor of Wnt production). We firstly found that the level of Troponin T (TNNT2) expression measured by FACS was significantly higher for both 2D protocols as compared to the 3D protocol. In the three methods, iPSC-CM show sarcomeric structures. However, iPSC-CM generated in 2D protocols constantly displayed larger sarcomere lengths as compared to the 3D protocol. In addition, mRNA and protein analyses reveal higher cTNi to ssTNi ratios in the 2D protocol using IWP2 as compared to both other protocols, indicating a higher sarcomeric maturation. Differentiation of cardiac myocytes with 2D monolayer-based protocols and the use of IWP2 allows the production of higher yield of cardiac myocytes that have more suitable characteristics to study sarcomeric cardiomyopathies. PMID:28587156

Tetragonal deformation of the hexagonal myofilament matrix in single skinned skeletal muscle fibres owing to change in sarcomere length.

PubMed

Schiereck, P; de Beer, E L; Grundeman, R L; Manussen, T; Kylstra, N; Bras, W

1992-10-01

Single skinned skeletal muscle fibres were immersed in solutions containing two different levels of activator calcium (pCa: 4.4; 6.0). Sarcomere length was varied from 1.6 to 3.5 microns and recorded by laser diffraction. Slack length was 2.0 microns. Small-angle equatorial X-ray diffraction patterns of relaxed and activated fibres at different sarcomere lengths were recorded using synchrotron radiation. The position and amplitude of the diffraction peaks were calculated from the spectra based on the hexagonal arrangement of the myofilament matrix, relating the position of the (1.0)- and (1.1)-diffraction peaks in this model by square root of 3. The diffraction peaks were fitted by five Gaussian functions (1.0, 1.1, 2.0, 2.1 and Z-line) and residual background was corrected by means of a hyperbola. The coupling of the position of the (1.0)- and (1.1)-peak was expressed as a factor: FAC = [d(1.0)/d(1.1)]/square root 3. In the relaxed state this coupling factor decreased at increasing sarcomere length (0.9880 +/- 0.002 at 2.0 microns; 0.900 +/- 0.01 at 3.5 microns). The coupling factor tends toward the one that will be obtained from the squared structure of actin filaments near the Z-discs. At shorter sarcomere lengths a decrease of the coupling factor has also been seen (0.9600 +/- 0.005 at 1.6 microns), giving rise to an increased uniform deformation of the hexagonal matrix, when sarcomere length is changed from slack length. From these experiments we conclude that a change in sarcomere length (from slack length) increases the deformation of the actin-myosin matrix to a tetragonal lattice.

Spontaneous oscillation of tension and sarcomere length in skeletal myofibrils. Microscopic measurement and analysis.

PubMed

Anazawa, T; Yasuda, K; Ishiwata, S

1992-05-01

We have devised a simple method for measuring tension development of single myofibrils by micromanipulation with a pair of glass micro-needles. The tension was estimated from the deflection of a flexible needle under an inverted phase-contrast microscope equipped with an image processor, so that the tension development is always accompanied by the shortening of the myofibril (auxotonic condition) in the present setup. The advantage of this method is that the measurement of tension (1/30 s for time resolution and about 0.05 micrograms for accuracy of tension measurement; 0.05 microns as a spatial resolution for displacement of the micro-needle) and the observation of sarcomere structure are possible at the same time, and the technique to hold myofibrils, even single myofibrils, is very simple. This method has been applied to study the tension development of glycerinated skeletal myofibrils under the condition where spontaneous oscillation of sarcomeres is induced, i.e., the coexistence of MgATP, MgADP and inorganic phosphate without free Ca2+. Under this condition, we found that the tension of myofibrils spontaneously oscillates accompanied by the oscillation of sarcomere length with a main period of a few seconds; the period was lengthened and shortened with stretch and release of myofibrils. A possible mechanism of the oscillation is discussed.

The interdependence of Ca2+ activation, sarcomere length, and power output in the heart.

PubMed

McDonald, Kerry S

2011-07-01

Myocardium generates power to perform external work on the circulation; yet, many questions regarding intermolecular mechanisms regulating power output remain unresolved. Power output equals force × shortening velocity, and some interesting new observations regarding control of these two factors have arisen. While it is well established that sarcomere length tightly controls myocyte force, sarcomere length-tension relationships also appear to be markedly modulated by PKA-mediated phosphorylation of myofibrillar proteins. Concerning loaded shortening, historical models predict independent cross-bridge mechanics; however, it seems that the mechanical state of one population of cross-bridges affects the activity of other cross-bridges by, for example, recruitment of cross-bridges from the non-cycling pool to the cycling force-generating pool during submaximal Ca(2+) activation. This is supported by the findings that Ca(2+) activation levels, myofilament phosphorylation, and sarcomere length are all modulators of loaded shortening and power output independent of their effects on force. This fine tuning of power output probably helps optimize myocardial energetics and to match ventricular supply with peripheral demand; yet, the discernment of the chemo-mechanical signals that modulate loaded shortening needs further clarification since power output may be a key convergent point and feedback regulator of cytoskeleton and cellular signals that control myocyte growth and survival.

New gene targets for glucagon-like peptide-1 during embryonic development and in undifferentiated pluripotent cells.

PubMed

Sanz, Carmen; Blázquez, Enrique

2011-09-01

In humans, glucagon-like peptide (GLP-1) functions during adult life as an incretin hormone with anorexigenic and antidiabetogenic properties. Also, the therapeutic potential of GLP-1 in preventing the adipocyte hyperplasia associated with obesity and in bolstering the maintenance of human mesenchymal stem cell (hMSC) stores by promoting the proliferation and cytoprotection of hMSC seems to be relevant. Since these observations suggest a role for GLP-1 during developmental processes, the aim of the present work was to characterize GLP-1 in early development as well as its gene targets in mouse embryonic stem (mES) cells. Mouse embryos E6, E8, and E10.5 and pluripotent mES were used for the inmunodetection of GLP-1 and GLP-1 receptor. Quantitative real-time PCR was used to determine the expression levels of GLP-1R in several tissues from E12.5 mouse embryos. Additionally, GLP-1 gene targets were studied in mES by multiple gene expression analyses. GLP-1 and its receptors were identified in mES and during embryonic development. In pluripotent mES, GLP-1 modified the expression of endodermal, ectodermal, and mesodermal gene markers as well as sonic hedgehog, noggin, members of the fibroblast and hepatic growth factor families, and others involved in pancreatic development. Additionally, GLP-1 promoted the expression of the antiapoptotic gene bcl2 and at the same time reduced proapoptotic caspase genes. Our results indicate that apart from the effects and therapeutic benefits of GLP-1 in adulthood, it may have additional gene targets in mES cells during embryonic life. Furthermore, the pathophysiological implications of GLP-1 imbalance in adulthood may have a counterpart during development.

Embryonic Stem Cell Therapy of Heart Failure in Genetic Cardiomyopathy

PubMed Central

Yamada, Satsuki; Nelson, Timothy J.; Crespo-Diaz, Ruben J.; Perez-Terzic, Carmen; Liu, Xiao-Ke; Miki, Takashi; Seino, Susumu; Behfar, Atta; Terzic, Andre

2009-01-01

Pathogenic causes underlying nonischemic cardiomyopathies are increasingly being resolved, yet repair therapies for these commonly heritable forms of heart failure are lacking. A case in point is human dilated cardiomyopathy 10 (CMD10; Online Mendelian Inheritance in Man #608569), a progressive organ dysfunction syndrome refractory to conventional therapies and linked to mutations in cardiac ATP-sensitive K+ (KATP) channel sub-units. Embryonic stem cell therapy demonstrates benefit in ischemic heart disease, but the reparative capacity of this allogeneic regenerative cell source has not been tested in inherited cardiomyopathy. Here, in a Kir6.2-knockout model lacking functional KATP channels, we recapitulated under the imposed stress of pressure overload the gene-environment substrate of CMD10. Salient features of the human malignant heart failure phenotype were reproduced, including compromised contractility, ventricular dilatation, and poor survival. Embryonic stem cells were delivered through the epicardial route into the left ventricular wall of cardiomyopathic stressed Kir6.2-null mutants. At 1 month of therapy, transplantation of 200,000 cells per heart achieved teratoma-free reversal of systolic dysfunction and electrical synchronization and halted maladaptive remodeling, thereby preventing end-stage organ failure. Tracked using the lacZ reporter transgene, stem cells engrafted into host heart. Beyond formation of cardiac tissue positive for Kir6.2, transplantation induced cell cycle activation and halved fibrotic zones, normalizing sarcomeric and gap junction organization within remuscularized hearts. Improved systemic function induced by stem cell therapy translated into increased stamina, absence of anasarca, and benefit to overall survivorship. Embryonic stem cells thus achieve functional repair in nonischemic genetic cardiomyopathy, expanding indications to the therapy of heritable heart failure. PMID:18669912

On high heels and short muscles: A multiscale model for sarcomere loss in the gastrocnemius muscle

PubMed Central

Zöllner, Alexander M.; Pok, Jacquelynn M.; McWalter, Emily J.; Gold, Garry E.; Kuhl, Ellen

2014-01-01

High heels are a major source of chronic lower limb pain. Yet, more than one third of all women compromise health for looks and wear high heels on a daily basis. Changing from flat footwear to high heels induces chronic muscle shortening associated with discomfort, fatigue, reduced shock absorption, and increased injury risk. However, the long-term effects of high-heeled footwear on the musculoskeletal kinematics of the lower extremities remain poorly understood. Here we create a multiscale computational model for chronic muscle adaptation to characterize the acute and chronic effects of global muscle shortening on local sarcomere lengths. We perform a case study of a healthy female subject and show that raising the heel by 13 cm shortens the gastrocnemius muscle by 5% while the Achilles tendon remains virtually unaffected. Our computational simulation indicates that muscle shortening displays significant regional variations with extreme values of 22% in the central gastrocnemius. Our model suggests that the muscle gradually adjusts to its new functional length by a chronic loss of sarcomeres in series. Sarcomere loss varies significantly across the muscle with an average loss of 9%, virtually no loss at the proximal and distal ends, and a maximum loss of 39% in the central region. These changes reposition the remaining sarcomeres back into their optimal operating regime. Computational modeling of chronic muscle shortening provides a valuable tool to shape our understanding of the underlying mechanisms of muscle adaptation. Our study could open new avenues in orthopedic surgery and enhance treatment for patients with muscle contracture caused by other conditions than high heel wear such as paralysis, muscular atrophy, and muscular dystrophy. PMID:25451524

Disorder profile of nebulin encodes a vernierlike position sensor for the sliding thin and thick filaments of the skeletal muscle sarcomere

NASA Astrophysics Data System (ADS)

Wu, Ming-Chya; Forbes, Jeffrey G.; Wang, Kuan

2016-06-01

Nebulin is an about 1 μ m long intrinsically disordered scaffold for the thin filaments of skeletal muscle sarcomere. It is a multifunctional elastic protein that wraps around actin filament, stabilizes thin filaments, and regulates Ca-dependent actomyosin interactions. This study investigates whether the disorder profile of nebulin might encode guidelines for thin and thick filament interactions in the sarcomere of the skeletal muscle. The question was addressed computationally by analyzing the predicted disorder profile of human nebulin (6669 residues, ˜200 actin-binding repeats) by pondr and the periodicity of the A-band stripes (reflecting the locations of myosin-associated proteins) in the electron micrographs of the sarcomere. Using the detrended fluctuation analysis, a scale factor for the A-band stripe image data with respect to the nebulin disorder profile was determined to make the thin and thick filaments aligned to have maximum correlation. The empirical mode decomposition method was then applied to identify hidden periodicities in both the nebulin disorder profile and the rescaled A-band data. The decomposition reveals three characteristic length scales (45 nm, 100 nm, and 200 nm) that are relevant for correlational analysis. The dynamical cross-correlation analyses with moving windows at various sarcomere lengths depict a vernierlike design for both periodicities, thus enabling nebulin to sense position and fine tune sarcomere overlap. This shows that the disorder profile of scaffolding proteins may encode a guideline for cellular architecture.

Hypertrophic Cardiomyopathy: A Vicious Cycle Triggered by Sarcomere Mutations and Secondary Disease Hits.

PubMed

Wijnker, Paul J M; Sequeira, Vasco; Kuster, Diederik W D; Velden, Jolanda van der

2018-04-11

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by left ventricular hypertrophy, diastolic dysfunction, and myocardial disarray. Disease onset occurs between 20 and 50 years of age, thus affecting patients in the prime of their life. HCM is caused by mutations in sarcomere proteins, the contractile building blocks of the heart. Despite increased knowledge of causal mutations, the exact path from genetic defect leading to cardiomyopathy is complex and involves additional disease hits. Recent Advances: Laboratory-based studies indicate that HCM development not only depends on the primary sarcomere impairment caused by the mutation but also on secondary disease-related alterations in the heart. Here we propose a vicious mutation-induced disease cycle, in which a mutation-induced energy depletion alters cellular metabolism with increased mitochondrial work, which triggers secondary disease modifiers that will worsen disease and ultimately lead to end-stage HCM. Evidence shows excessive cellular reactive oxygen species (ROS) in HCM patients and HCM animal models. Oxidative stress markers are increased in the heart (oxidized proteins, DNA, and lipids) and serum of HCM patients. In addition, increased mitochondrial ROS production and changes in endogenous antioxidants are reported in HCM. Mutant sarcomeric protein may drive excessive levels of cardiac ROS via changes in cardiac efficiency and metabolism, mitochondrial activation and/or dysfunction, impaired protein quality control, and microvascular dysfunction. Interventions restoring metabolism, mitochondrial function, and improved ROS balance may be promising therapeutic approaches. We discuss the effects of current HCM pharmacological therapies and potential future therapies to prevent and reverse HCM. Antioxid. Redox Signal. 00, 000-000.

Thin filament length in the cardiac sarcomere varies with sarcomere length but is independent of titin and nebulin.

PubMed

Kolb, Justin; Li, Frank; Methawasin, Mei; Adler, Maya; Escobar, Yael-Natalie; Nedrud, Joshua; Pappas, Christopher T; Harris, Samantha P; Granzier, Henk

2016-08-01

Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20(ΔRRM) mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20(ΔRRM) mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1μm per 0.35μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0μm where TFL was ~1.05μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin. Copyright © 2016. Published by Elsevier Ltd.

SarcOptiM for ImageJ: high-frequency online sarcomere length computing on stimulated cardiomyocytes.

PubMed

Pasqualin, Côme; Gannier, François; Yu, Angèle; Malécot, Claire O; Bredeloux, Pierre; Maupoil, Véronique

2016-08-01

Accurate measurement of cardiomyocyte contraction is a critical issue for scientists working on cardiac physiology and physiopathology of diseases implying contraction impairment. Cardiomyocytes contraction can be quantified by measuring sarcomere length, but few tools are available for this, and none is freely distributed. We developed a plug-in (SarcOptiM) for the ImageJ/Fiji image analysis platform developed by the National Institutes of Health. SarcOptiM computes sarcomere length via fast Fourier transform analysis of video frames captured or displayed in ImageJ and thus is not tied to a dedicated video camera. It can work in real time or offline, the latter overcoming rotating motion or displacement-related artifacts. SarcOptiM includes a simulator and video generator of cardiomyocyte contraction. Acquisition parameters, such as pixel size and camera frame rate, were tested with both experimental recordings of rat ventricular cardiomyocytes and synthetic videos. It is freely distributed, and its source code is available. It works under Windows, Mac, or Linux operating systems. The camera speed is the limiting factor, since the algorithm can compute online sarcomere shortening at frame rates >10 kHz. In conclusion, SarcOptiM is a free and validated user-friendly tool for studying cardiomyocyte contraction in all species, including human. Copyright © 2016 the American Physiological Society.

Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function

PubMed Central

Vandenboom, Rene; Herron, Todd; Favre, Elizabeth; Albayya, Faris P.

2011-01-01

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a “headless” myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC + endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P < 0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules. PMID:21112946

Embryonic stem cell therapy of heart failure in genetic cardiomyopathy.

PubMed

Yamada, Satsuki; Nelson, Timothy J; Crespo-Diaz, Ruben J; Perez-Terzic, Carmen; Liu, Xiao-Ke; Miki, Takashi; Seino, Susumu; Behfar, Atta; Terzic, Andre

2008-10-01

Pathogenic causes underlying nonischemic cardiomyopathies are increasingly being resolved, yet repair therapies for these commonly heritable forms of heart failure are lacking. A case in point is human dilated cardiomyopathy 10 (CMD10; Online Mendelian Inheritance in Man #608569), a progressive organ dysfunction syndrome refractory to conventional therapies and linked to mutations in cardiac ATP-sensitive K(+) (K(ATP)) channel subunits. Embryonic stem cell therapy demonstrates benefit in ischemic heart disease, but the reparative capacity of this allogeneic regenerative cell source has not been tested in inherited cardiomyopathy. Here, in a Kir6.2-knockout model lacking functional K(ATP) channels, we recapitulated under the imposed stress of pressure overload the gene-environment substrate of CMD10. Salient features of the human malignant heart failure phenotype were reproduced, including compromised contractility, ventricular dilatation, and poor survival. Embryonic stem cells were delivered through the epicardial route into the left ventricular wall of cardiomyopathic stressed Kir6.2-null mutants. At 1 month of therapy, transplantation of 200,000 cells per heart achieved teratoma-free reversal of systolic dysfunction and electrical synchronization and halted maladaptive remodeling, thereby preventing end-stage organ failure. Tracked using the lacZ reporter transgene, stem cells engrafted into host heart. Beyond formation of cardiac tissue positive for Kir6.2, transplantation induced cell cycle activation and halved fibrotic zones, normalizing sarcomeric and gap junction organization within remuscularized hearts. Improved systemic function induced by stem cell therapy translated into increased stamina, absence of anasarca, and benefit to overall survivorship. Embryonic stem cells thus achieve functional repair in nonischemic genetic cardiomyopathy, expanding indications to the therapy of heritable heart failure. Disclosure of potential conflicts of interest is

MicroRNA Signatures as Biomarkers and Therapeutic Target for CNS Embryonal Tumors: The Pros and the Cons

PubMed Central

Shalaby, Tarek; Fiaschetti, Giulio; Baumgartner, Martin; Grotzer, Michael A.

2014-01-01

Embryonal tumors of the central nervous system represent a heterogeneous group of childhood cancers with an unknown pathogenesis; diagnosis, on the basis of histological appearance alone, is controversial and patients’ response to therapy is difficult to predict. They encompass medulloblastoma, atypical teratoid/rhabdoid tumors and a group of primitive neuroectodermal tumors. All are aggressive tumors with the tendency to disseminate throughout the central nervous system. The large amount of genomic and molecular data generated over the last 5–10 years encourages optimism that new molecular targets will soon improve outcomes. Recent neurobiological studies have uncovered the key role of microRNAs (miRNAs) in embryonal tumors biology and their potential use as biomarkers is increasingly being recognized and investigated. However the successful use of microRNAs as reliable biomarkers for the detection and management of pediatric brain tumors represents a substantial challenge. This review debates the importance of miRNAs in the biology of central nervous systemembryonal tumors focusing on medulloblastoma and atypical teratoid/rhabdoid tumors and highlights the advantages as well as the limitations of their prospective application as biomarkers and candidates for molecular therapeutic targets. PMID:25421247

A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells.

PubMed

Xue, Haipeng; Wu, Sen; Papadeas, Sophia T; Spusta, Steve; Swistowska, Anna Maria; MacArthur, Chad C; Mattson, Mark P; Maragakis, Nicholas J; Capecchi, Mario R; Rao, Mahendra S; Zeng, Xianmin; Liu, Ying

2009-08-01

In this study, we targeted Olig2, a basic helix-loop-helix transcription factor that plays an important role in motoneuron and oligodendrocyte development, in human embryonic stem cell (hESC) line BG01 by homologous recombination. One allele of Olig2 locus was replaced by a green fluorescent protein (GFP) cassette with a targeting efficiency of 5.7%. Targeted clone R-Olig2 (like the other clones) retained pluripotency, typical hESC morphology, and a normal parental karyotype 46,XY. Most importantly, GFP expression recapitulated endogenous Olig2 expression when R-Olig2 was induced by sonic hedgehog and retinoic acid, and GFP-positive cells could be purified by fluorescence-activated cell sorting. Consistent with previous reports on rodents, early GFP-expressing cells appeared biased to a neuronal fate, whereas late GFP-expressing cells appeared biased to an oligodendrocytic fate. This was corroborated by myoblast coculture, transplantation into the rat spinal cords, and whole genome expression profiling. The present work reports an hESC reporter line generated by homologous recombination targeting a neural lineage-specific gene, which can be differentiated and sorted to obtain pure neural progenitor populations.

New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells.

PubMed

Denning, Chris; Priddle, Helen

2003-07-01

Until recently, precise modification of the animal genome by gene targeting was restricted to the mouse because germline competent embryonic stem cells are not available in any other mammalian species. Nuclear transfer (NT) technology now provides an alternative route for cell-based transgenesis in domestic species, offering new opportunities in genetic modification. Livestock that produce human therapeutic proteins in their milk, have organs suitable for xenotransplantation, or that could provide resistance to diseases such as spongiform encephalopathies have been produced by NT from engineered, cultured somatic cells. However, improvements in the efficiency of somatic cell gene targeting and a greater understanding of the reprogramming events that occur during NT are required for the routine application of what is currently an inefficient process. The ability to reprogramme and genetically manipulate cells will also be crucial for full exploitation of human embryonic stem (hES) cells, which offer unparalleled opportunities in human health and biotechnology. Particularly pertinent are directed differentiation of hES lines to specific cell lineages, production of cells that evade the patient's immune system and ensuring the safety of ensuing transplants. This review will discuss some of the successes, applications and challenges facing gene targeting in livestock and hES cells.

A novel optical imaging system for investigating sarcomere dynamics in single skeletal muscle fibers

NASA Astrophysics Data System (ADS)

Panchangam, Appaji; Witte, Russell S.; Claflin, Dennis R.; O'Donnell, Matthew; Faulkner, John A.

2006-02-01

The protein substructure of skeletal muscle fibers forms a diffraction grating with repeating units, termed 'sarcomeres'. A laser scanning system is described that maps the lengths of sarcomeres (SL) and the widths of the first-order diffraction lines (DLW) of permeabilized single fibers in real-time. The apparatus translates a laser beam (λ = 670 nm and w 0 = ~75 μm) along the length of a fiber segment through 20 contiguous regions per sweep at 500 sweeps/s. The fiber segments (~1 mm long) were obtained from vastus lateralis muscles of humans by needle biopsy. During both passive stretches and maximum fixed-end activations, the mappings of SL and DLW of the fibers were extracted from the diffraction spectra. Heterogeneity of SLs was evaluated by computing the standard deviation ( σ SL) of the 20 SLs measured during a single sweep. Compared with the σ SL before a passive stretch, the increase of 5+/-0.5% in σ SL after the passive stretch, indicated differences in passive length-tension relationships along the fiber. In contrast, no change, ~0.5+/-0.1%, was observed in DLW. Within 10s after the fiber was returned to its initial length, the shape of the SL profile returned close to pre-stretch conditions ( σ SL = 1+/- 0.2%). Following maximum Ca 2+ - activation of the fiber, the heterogeneity of the steady state SLs increased greatly (DLW up by ~300% and σ SL up by ~100%). The scanning system provided high resolution tracking of sarcomere behavior single muscle fibers. Potential applications are for studies of the mechanisms of muscle fiber injury and injury propagation.

Sarcomeric gene mutations in sudden infant death syndrome (SIDS).

PubMed

Brion, Maria; Allegue, Catarina; Santori, Montserrat; Gil, Rocio; Blanco-Verea, Alejandro; Haas, Cordula; Bartsch, Christine; Poster, Simone; Madea, Burkhard; Campuzano, Oscar; Brugada, Ramon; Carracedo, Angel

2012-06-10

In developed countries, sudden infant death syndrome (SIDS) represents the most prevalent cause of death in children between 1 month and 1 year of age. SIDS is a diagnosis of exclusion, a negative autopsy which requires the absence of structural organ disease. Although investigators have confirmed that a significant percentage of SIDS cases are actually channelopathies, no data have been made available as to whether other sudden cardiac death-associated diseases, such as hypertrophic cardiomyopathy (HCM), could be responsible for some cases of SIDS. The presence of a genetic mutation in the sarcomeric protein usually affects the force of contraction of the myocyte, whose weakness is compensated with progressive hypertrophy and disarray. However, it is unclear whether in the most incipient forms, that is, first years of life, the lack of these phenotypes still confers a risk of arrhythmogenesis. The main goal of the present study is to wonder whether genetic defects in the sarcomeric proteins, previously associated with HCM, could be responsible for SIDS. We have analysed 286 SIDS cases for the most common genes implicated in HCM in adults. A total of 680 mutations localised in 16 genes were analysed by semi-automated matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDITOF-MS) using the Sequenom MassARRAY(®) System. Ten subjects with completely normal hearts showed mutated alleles at nine of the genetic variants analysed, and one additional novel mutation was detected by conventional sequencing. Therefore, a genetic mutation associated with HCM may cause sudden cardiac death in the absence of an identifiable phenotype. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Titin Mutations in iPS cells Define Sarcomere Insufficiency as a Cause of Dilated Cardiomyopathy

PubMed Central

Hinson, John T.; Chopra, Anant; Nafissi, Navid; Polacheck, William J.; Benson, Craig C.; Swist, Sandra; Gorham, Joshua; Yang, Luhan; Schafer, Sebastian; Sheng, Calvin C.; Haghighi, Alireza; Homsy, Jason; Hubner, Norbert; Church, George; Cook, Stuart A.; Linke, Wolfgang A.; Chen, Christopher S.; Seidman, J. G.; Seidman, Christine E.

2015-01-01

Human mutations that truncate the massive sarcomere protein titin (TTNtv) are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtv, diminish contractile performance and are pathogenic. By combining functional analyses with RNAseq, we explain why truncations in the A-band domain of TTN cause DCM while truncations in the I-band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS-cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodelling. PMID:26315439

Coherent Raman Imaging of Live Muscle Sarcomeres Assisted by SFG Microscopy.

PubMed

Kim, Hyunmin; Kim, Do-Young; Joo, Kyung-Il; Kim, Jung-Hye; Jeong, Soon Moon; Lee, Eun Seong; Hahm, Jeong-Hoon; Kim, Kyuhyung; Moon, Dae Woon

2017-08-23

In this study, we used spectrally focused coherent anti-Stokes Raman scattering (spCARS) microscopy assisted by sum-frequency generation (SFG) to monitor the variations in the structural morphology and molecular vibrations of a live muscle of Caenorhabditis elegans. The subunits of the muscle sarcomeres, such as the M-line, myosin, dense body, and α-actinin, were alternatively observed using spCARS microscopy for different sample orientations, with the guidance of a myosin positional marker captured by SFG microscopy. Interestingly enough, the beam polarization dependence of the spCARS contrasts for two parallel subunits (dense body and myosin) showed a ~90° phase difference. The chemically sensitive spCARS spectra induced by the time-varying overlap of two pulses allowed (after a robust subtraction of the non-resonant background using a modified Kramers-Krönig transformation method) high-fidelity detection of various genetically modified muscle sarcomeres tuned to the C-H vibration (2800-3100 cm -1 ). Conversely, SFG image mapping assisted by phase-retrieved spCARS spectra also facilitated label-free monitoring of the changes in the muscle content of C. elegans that are associated with aging, based on the hypothesis that the C-H vibrational modes could serve as qualitative chemical markers sensitive to the amount and/or structural modulation of the muscle.

New TFII-I family target genes involved in embryonic development.

PubMed

Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

2009-09-04

Two members of the TFII-I family transcription factor genes, GTF2I and GTF2IRD1, are the prime candidates responsible for the craniofacial and cognitive abnormalities of Williams syndrome patients. We have previously generated mouse lines with targeted disruption of Gtf2i and Gtf2ird1. Microarray analysis revealed significant changes in the expression profile of mutant embryos. Here we described three unknown genes that were dramatically down-regulated in mutants. The 2410018M08Rik/Scand3 gene encodes a protein of unknown function with CHCH and hATC domains. Scand3 is down-regulated during mouse embryonic stem cell (ES) differentiation. 4933436H12Rik is a testis-specific gene, which encodes a protein with no known domains. It is expressed in mouse ES cells. 1110008P08Rik/Kbtbd7 encodes an adapter protein with BTB/POZ, BACK, and Kelch motifs, previously shown to recruit substrates to the enzymatic complexes of the histone modifying or E3 ubiquitin ligase activities. Based on its expression pattern Kbtbd7 may have a specific role in brain development and function. All three genes possess well-conserved TFII-I-binding consensus sites within proximal promoters. Therefore our analysis suggests that these genes can be direct targets of TFII-I proteins and their impaired expression, as a result of the GTF2I and GTF2IRD1 haploinsufficiency, could contribute to the etiology of Williams syndrome.

Gene targeting in embryonic stem cells, II: conditional technologies

USDA-ARS?s Scientific Manuscript database

Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

In vivo cardiac nano-imaging: A new technology for high-precision analyses of sarcomere dynamics in the heart.

PubMed

Shimozawa, Togo; Hirokawa, Erisa; Kobirumaki-Shimozawa, Fuyu; Oyama, Kotaro; Shintani, Seine A; Terui, Takako; Kushida, Yasuharu; Tsukamoto, Seiichi; Fujii, Teruyuki; Ishiwata, Shin'ichi; Fukuda, Norio

2017-03-01

The cardiac pump function is a result of a rise in intracellular Ca 2+ and the ensuing sarcomeric contractions [i.e., excitation-contraction (EC) coupling] in myocytes in various locations of the heart. In order to elucidate the heart's mechanical properties under various settings, cardiac imaging is widely performed in today's clinical as well as experimental cardiology by using echocardiogram, magnetic resonance imaging and computed tomography. However, because these common techniques detect local myocardial movements at a spatial resolution of ∼100 μm, our knowledge on the sub-cellular mechanisms of the physiology and pathophysiology of the heart in vivo is limited. This is because (1) EC coupling occurs in the μm partition in a myocyte and (2) cardiac sarcomeres generate active force upon a length change of ∼100 nm on a beat-to-beat basis. Recent advances in optical technologies have enabled measurements of intracellular Ca 2+ dynamics and sarcomere length displacements at high spatial and temporal resolution in the beating heart of living rodents. Future studies with these technologies are warranted to open a new era in cardiac research. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of anisosmotic stress on cardiac muscle cell length, diameter, area, and sarcomere length

NASA Technical Reports Server (NTRS)

Tanaka, R.; Barnes, M. A.; Cooper, G. 4th; Zile, M. R.

1996-01-01

The purpose of this study was to examine the effects of anisosmotic stress on adult mammalian cardiac muscle cell (cardiocyte) size. Cardiocyte size and sarcomere length were measured in cardiocytes isolated from 10 normal rats and 10 normal cats. Superfusate osmolarity was decreased from 300 +/- 6 to 130 +/- 5 mosM and increased to 630 +/- 8 mosM. Cardiocyte size and sarcomere length increased progressively when osmolarity was decreased, and there were no significant differences between cat and rat cardiocytes with respect to percent change in cardiocyte area or diameter; however, there were significant differences in cardiocyte length (2.8 +/- 0.3% in cat vs. 6.1 +/- 0.3% in rat, P < 0.05) and sarcomere length (3.3 +/- 0.3% in cat vs. 6.1 +/- 0.3% in rat, P < 0.05). To determine whether these species-dependent differences in length were related to diastolic interaction of the contractile elements or differences in relative passive stiffness, cardiocytes were subjected to the osmolarity gradient 1) during treatment with 7 mM 2,3-butanedione monoxime (BDM), which inhibits cross-bridge interaction, or 2) after pretreatment with 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA), a bivalent Ca2+ chelator. Treatment with EGTA or BDM abolished the differences between cat and rat cardiocytes. Species-dependent differences therefore appeared to be related to the degree of diastolic cross-bridge association and not differences in relative passive stiffness. In conclusion, the osmolarity vs. cell size relation is useful in assessing the cardiocyte response to anisosmotic stress and may in future studies be useful in assessing changes in relative passive cardiocyte stiffness produced by pathological processes.

MicroRNA-127 Promotes Mesendoderm Differentiation of Mouse Embryonic Stem Cells by Targeting Left-Right Determination Factor 2.

PubMed

Ma, Haixia; Lin, Yu; Zhao, Zhen-Ao; Lu, Xukun; Yu, Yang; Zhang, Xiaoxin; Wang, Qiang; Li, Lei

2016-06-03

Specification of the three germ layers is a fundamental process and is essential for the establishment of organ rudiments. Multiple genetic and epigenetic factors regulate this dynamic process; however, the function of specific microRNAs in germ layer differentiation remains unknown. In this study, we established that microRNA-127 (miR-127) is related to germ layer specification via microRNA array analysis of isolated three germ layers of E7.5 mouse embryos and was verified through differentiation of mouse embryonic stem cells. miR-127 is highly expressed in endoderm and primitive streak. Overexpression of miR-127 increases and inhibition of miR-127 decreases the expression of mesendoderm markers. We further show that miR-127 promotes mesendoderm differentiation through the nodal pathway, a determinative signaling pathway in early embryogenesis. Using luciferase reporter assay, left-right determination factor 2 (Lefty2), an antagonist of nodal, is identified to be a novel target of miR-127. Furthermore, the role of miR-127 in mesendoderm differentiation is attenuated by Lefty2 overexpression. Altogether, our results indicate that miR-127 accelerates mesendoderm differentiation of mouse embryonic stem cells through nodal signaling by targeting Lefty2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

AMP-activated protein kinase-mediated glucose transport as a novel target of tributyltin in human embryonic carcinoma cells.

PubMed

Yamada, Shigeru; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2013-05-01

Organotin compounds such as tributyltin (TBT) are known to cause various forms of cytotoxicity, including developmental toxicity and neurotoxicity. However, the molecular target of the toxicity induced by nanomolar levels of TBT has not been identified. In the present study, we found that exposure to 100 nM TBT induced growth arrest in human pluripotent embryonic carcinoma cell line NT2/D1. Since glucose provides metabolic energy, we focused on the glycolytic system. We found that exposure to TBT reduced the levels of both glucose-6-phosphate and fructose-6-phosphate. To investigate the effect of TBT exposure on glycolysis, we examined glucose transporter (GLUT) activity. TBT exposure inhibited glucose uptake via a decrease in the level of cell surface-bound GLUT1. Furthermore, we examined the effect of AMP-activated protein kinase (AMPK), which is known to regulate glucose transport by facilitating GLUT translocation. Treatment with the potent AMPK activator, AICAR, restored the TBT-induced reduction in cell surface-bound GLUT1 and glucose uptake. In conclusion, these results suggest that exposure to nanomolar levels of TBT causes growth arrest by targeting glycolytic systems in human embryonic carcinoma cells. Thus, understanding the energy metabolism may provide new insights into the mechanisms of metal-induced cytotoxicity.

Improved targeting and enhanced retention of the human, autologous, fibroblast-derived, induced, pluripotent stem cells to the sarcomeres of the infarcted myocardium with the aid of the bioengineered, heterospecific, tetravalent antibodies**

PubMed Central

Malecki, Marek

2013-01-01

Clinical trials, to regenerate the human heart injured by myocardial infarction, involve the delivery of stem cells to the site of the injury. However, only a small fraction of the introduced stem cells are detected at the site of the injury, merely two weeks after this therapeutic intervention. This significantly hampers the effectiveness of the stem cell therapy. To resolve the aforementioned problem, we genetically and molecularly bioengineered heterospecific, tetravalent antibodies (htAbs), which have both exquisite specificity and high affinity towards human, pluripotent, stem cells through the htAbs’ domains binding SSEA-4, SSEA-3, TRA-1-60, and TRA-1-81, as well as towards the injured cardiac muscle through the htAbs’ domains binding human cardiac myosin, α-actinin, actin, and titin. The cardiac tissue was acquired from the patients, who were receiving heart transplants. The autologous, human, induced, pluripotent stem cells (hiPSCs) were generated from the patients’ fibroblasts by non-viral delivery and transient expression of the DNA constructs for: Oct4, Nanog, Sox2, Lin28, Klf4, c-Myc. In the trials involving the htAbs, the human, induced, pluripotent stem cells anchored to the myocardial sarcomeres with the efficiency, statistically, significantly higher, than in the trials with non-specific or without antibodies (p < 0.0003). Moreover, application of the htAbs resulted in cross-linking of the sarcomeric proteins to create the stable scaffolds for anchoring of the stem cells. Thereafter, these human, induced pluripotent stem cells differentiated into cardiomyocytes at their anchorage sites. By bioengineering of these novel heterospecific, tetravalent antibodies and using them to guide and to anchor the stem cells specifically to the stabilized sarcomeric scaffolds, we demonstrated the proof of concept in vitro for improving effectiveness of regenerative therapy of myocardial infarction and created the foundations for the trials in vivo. PMID

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

PubMed Central

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-01-01

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action. PMID:25092173

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

NASA Astrophysics Data System (ADS)

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-08-01

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells.

PubMed

Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

2014-08-05

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.

PubMed

Andréasson, Claes; Schick, Anna J; Pfeiffer, Susanne M; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A

2013-01-01

Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors.

Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

PubMed

Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

2015-07-06

Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling.

Impaired Ca2+ cycling of nonischemic myocytes contributes to sarcomere dysfunction early after myocardial infarction.

PubMed

Kronenbitter, Annette; Funk, Florian; Hackert, Katarzyna; Gorreßen, Simone; Glaser, Dennis; Boknik, Peter; Poschmann, Gereon; Stühler, Kai; Isić, Malgorzata; Krüger, Martina; Schmitt, Joachim P

2018-06-01

Changes in the nonischemic remote myocardium of the heart contribute to left ventricular dysfunction after ischemia and reperfusion (I/R). Understanding the underlying mechanisms early after I/R is crucial to improve the adaptation of the viable myocardium to increased mechanical demands. Here, we investigated the role of myocyte Ca 2+ handling in the remote myocardium 24 h after 60 min LAD occlusion. Cardiomyocytes isolated from the basal noninfarct-related parts of wild type mouse hearts demonstrated depressed beat-to-beat Ca 2+ handling. The amplitude of the Ca 2+ transients as well as the kinetics of Ca 2+ transport were reduced by up to 25%. These changes were associated with impaired sarcomere contraction. While expression levels of Ca 2+ regulatory proteins were unchanged in remote myocardium compared to the corresponding regions of sham-operated hearts, mobility shift analyses of phosphorylated protein showed 2.9 ± 0.4-fold more unphosphorylated phospholamban (PLN) monomers, the PLN species that inhibits the Ca 2+ ATPase SERCA2a (P ≤ 0.001). Phospho-specific antibodies revealed normal phosphorylation of PLN at T17 in remote myocardium, but markedly reduced phosphorylation at its PKA-dependent phosphorylation site, S16 (P ≤ 0.01). The underlying cause involved enhanced activity of protein phosphatases, particularly PP2A (P ≤ 0.01). In contrast, overall PKA activity was normal. The PLN interactome, as determined by co-immunoprecipitation and mass spectrometry, and the phosphorylation state of PKA targets other than PLN were also unchanged. Isoproterenol enhanced cellular Ca 2+ cycling much stronger in remote myocytes than in healthy controls and improved sarcomere function. We conclude that the reduced phosphorylation state of PLN at S16 impairs myocyte Ca 2+ cycling in the remote myocardium 24 h after I/R and contributes to contractile dysfunction. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain*

PubMed Central

Scruggs, Sarah B.; Reisdorph, Rick; Armstrong, Mike L.; Warren, Chad M.; Reisdorph, Nichole; Solaro, R. John; Buttrick, Peter M.

2010-01-01

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were

An Inexpensive 2-D and 3-D Model of the Sarcomere as a Teaching Aid

ERIC Educational Resources Information Center

Rios, Vitor Passos; Bonfim, Vanessa Maria Gomes

2013-01-01

To address a common problem of teaching the sliding filament theory (that is, students have difficulty in visualizing how the component proteins of the sarcomere differ, how they organize themselves into a single working unit, and how they function in relation to each other), we have devised a simple model, with inexpensive materials, to be built…

A Role for the p38 Mitogen-activated Protein Kinase Pathway in Myocardial Cell Growth, Sarcomeric Organization, and Cardiac-specific Gene Expression

PubMed Central

Zechner, Dietmar; Thuerauf, Donna J.; Hanford, Deanna S.; McDonough, Patrick M.; Glembotski, Christopher C.

1997-01-01

Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by α1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal–regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the α-skeletal actin (α-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and α-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and α-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy. PMID:9314533

Explaining the variation in the shear force of lamb meat using sarcomere length, the rate of rigor onset and pH.

PubMed

Hopkins, D L; Toohey, E S; Lamb, T A; Kerr, M J; van de Ven, R; Refshauge, G

2011-08-01

The temperature when the pH=6.0 (temp@pH6) impacts on the tenderness and eating quality of sheep meat. Due to the expense, sarcomere length is not routinely measured as a variable to explain variation in shear force, but whether measures such as temp@pH6 are as useful a parameter needs to be established. Measures of rigor onset in 261 carcases, including the temp@pH6, were evaluated in this study for their ability to explain some of the variation in shear force. The results show that for 1 day aged product combinations of the temp@pH6, the pH at 18 °C and the pH at 24 h provided a larger reduction (almost double) in total shear force variation than sarcomere length alone, with pH at 24 h being the single best measure. For 5 day aged product, pH at 18 °C was the single best measure. Inclusion of sarcomere length did represent some improvement, but the marginal increase would not be cost effective. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

PubMed

Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

2009-06-17

P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

PubMed

Sangiuolo, Federica; Scaldaferri, Maria Lucia; Filareto, Antonio; Spitalieri, Paola; Guerra, Lorenzo; Favia, Maria; Caroppo, Rosa; Mango, Ruggiero; Bruscia, Emanuela; Gruenert, Dieter C; Casavola, Valeria; De Felici, Massimo; Novelli, Giuseppe

2008-01-01

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.

In Vivo Function of the Chaperonin TRiC in α-Actin Folding during Sarcomere Assembly.

PubMed

Berger, Joachim; Berger, Silke; Li, Mei; Jacoby, Arie S; Arner, Anders; Bavi, Navid; Stewart, Alastair G; Currie, Peter D

2018-01-09

The TCP-1 ring complex (TRiC) is a multi-subunit group II chaperonin that assists nascent or misfolded proteins to attain their native conformation in an ATP-dependent manner. Functional studies in yeast have suggested that TRiC is an essential and generalized component of the protein-folding machinery of eukaryotic cells. However, TRiC's involvement in specific cellular processes within multicellular organisms is largely unknown because little validation of TRiC function exists in animals. Our in vivo analysis reveals a surprisingly specific role of TRiC in the biogenesis of skeletal muscle α-actin during sarcomere assembly in myofibers. TRiC acts at the sarcomere's Z-disk, where it is required for efficient assembly of actin thin filaments. Binding of ATP specifically by the TRiC subunit Cct5 is required for efficient actin folding in vivo. Furthermore, mutant α-actin isoforms that result in nemaline myopathy in patients obtain their pathogenic conformation via this function of TRiC. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Differentiation and sarcomere formation in skeletal myocytes directly prepared from human induced pluripotent stem cells using a sphere-based culture.

PubMed

Jiwlawat, Saowanee; Lynch, Eileen; Glaser, Jennifer; Smit-Oistad, Ivy; Jeffrey, Jeremy; Van Dyke, Jonathan M; Suzuki, Masatoshi

Human induced-pluripotent stem cells (iPSCs) are a promising resource for propagation of myogenic progenitors. Our group recently reported a unique protocol for the derivation of myogenic progenitors directly (without genetic modification) from human pluripotent cells using free-floating spherical culture. Here we expand our previous efforts and attempt to determine how differentiation duration, culture surface coatings, and nutrient supplements in the medium influence progenitor differentiation and formation of skeletal myotubes containing sarcomeric structures. A long differentiation period (over 6 weeks) promoted the differentiation of iPSC-derived myogenic progenitors and subsequent myotube formation. These iPSC-derived myotubes contained representative sarcomeric structures, consisting of organized myosin and actin filaments, and could spontaneously contract. We also found that a bioengineering approach using three-dimensional (3D) artificial muscle constructs could facilitate the formation of elongated myotubes. Lastly, we determined how culture surface coating matrices and different supplements would influence terminal differentiation. While both Matrigel and laminin coatings showed comparable effects on muscle differentiation, B27 serum-free supplement in the differentiation medium significantly enhanced myogenesis compared to horse serum. Our findings support the possibility to create an in vitro model of contractile sarcomeric myofibrils for disease modeling and drug screening to study neuromuscular diseases. Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

A microinjection technique for targeting regions of embryonic and neonatal mouse brain in vivo

PubMed Central

Davidson, Steve; Truong, Hai; Nakagawa, Yasushi; Giesler, Glenn J

2009-01-01

A simple pressure injection technique was developed to deliver substances into specific regions of the embryonic and neonatal mouse brain in vivo. The retrograde tracers Fluorogold and cholera toxin B subunit were used to test the validity of the technique. Injected animals survived the duration of transport (24–48 hrs) and then were sacrificed and perfused with fixative. Small injections (≤ 50 nL) were contained within targeted structures of the perinatal brain and labeled distant cells of origin in several model neural pathways. Traced neural pathways in the perinatal mouse were further examined with immunohistochemical methods to test the feasibility of double labeling experiments during development. Several experimental situations in which this technique would be useful are discussed, for example, to label projection neurons in slice or culture preparations of mouse embryos and neonates. The administration of pharmacological or genetic vectors directly into specific neural targets during development should also be feasible. An examination of the form of neural pathways during early stages of life may lead to insights regarding the functional changes that occur during critical periods of development and provide an anatomic basis for some neurodevelopmental disorders. PMID:19840780

Cardiac Development in Zebrafish and Human Embryonic Stem Cells Is Inhibited by Exposure to Tobacco Cigarettes and E-Cigarettes

PubMed Central

Palpant, Nathan J.; Hofsteen, Peter; Pabon, Lil; Reinecke, Hans; Murry, Charles E.

2015-01-01

Background Maternal smoking is a risk factor for low birth weight and other adverse developmental outcomes. Objective We sought to determine the impact of standard tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Methods Zebrafish (Danio rerio) were used to assess developmental effects in vivo and cardiac differentiation of human embryonic stem cells (hESCs) was used as a model for in vitro cardiac development. Results In zebrafish, exposure to both types of cigarettes results in broad, dose-dependent developmental defects coupled with severe heart malformation, pericardial edema and reduced heart function. Tobacco cigarettes are more toxic than e-cigarettes at comparable nicotine concentrations. During cardiac differentiation of hESCs, tobacco smoke exposure results in a delayed transition through mesoderm. Both types of cigarettes decrease expression of cardiac transcription factors in cardiac progenitor cells, suggesting a persistent delay in differentiation. In definitive human cardiomyocytes, both e-cigarette- and tobacco cigarette-treated samples showed reduced expression of sarcomeric genes such as MLC2v and MYL6. Furthermore, tobacco cigarette-treated samples had delayed onset of beating and showed low levels and aberrant localization of N-cadherin, reduced myofilament content with significantly reduced sarcomere length, and increased expression of the immature cardiac marker smooth muscle alpha-actin. Conclusion These data indicate a negative effect of both tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Tobacco cigarettes are more toxic than E-cigarettes and exhibit a broader spectrum of cardiac developmental defects. PMID:25978043

Diversity and Complexity in Chromatin Recognition by TFII-I Transcription Factors in Pluripotent Embryonic Stem Cells and Embryonic Tissues

PubMed Central

Makeyev, Aleksandr V.; Enkhmandakh, Badam; Hong, Seung-Hyun; Joshi, Pujan; Shin, Dong-Guk; Bayarsaihan, Dashzeveg

2012-01-01

GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a “feed-forward model” of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells. PMID:22970219

Diversity and complexity in chromatin recognition by TFII-I transcription factors in pluripotent embryonic stem cells and embryonic tissues.

PubMed

Makeyev, Aleksandr V; Enkhmandakh, Badam; Hong, Seung-Hyun; Joshi, Pujan; Shin, Dong-Guk; Bayarsaihan, Dashzeveg

2012-01-01

GTF2I and GTF2IRD1 encode a family of closely related transcription factors TFII-I and BEN critical in embryonic development. Both genes are deleted in Williams-Beuren syndrome, a complex genetic disorder associated with neurocognitive, craniofacial, dental and skeletal abnormalities. Although genome-wide promoter analysis has revealed the existence of multiple TFII-I binding sites in embryonic stem cells (ESCs), there was no correlation between TFII-I occupancy and gene expression. Surprisingly, TFII-I recognizes the promoter sequences enriched for H3K4me3/K27me3 bivalent domain, an epigenetic signature of developmentally important genes. Moreover, we discovered significant differences in the association between TFII-I and BEN with the cis-regulatory elements in ESCs and embryonic craniofacial tissues. Our data indicate that in embryonic tissues BEN, but not the highly homologous TFII-I, is primarily recruited to target gene promoters. We propose a "feed-forward model" of gene regulation to explain the specificity of promoter recognition by TFII-I factors in eukaryotic cells.

Sarcomere mechanics in uniform and non-uniform cardiac muscle: a link between pump function and arrhythmias.

PubMed

ter Keurs, Henk E D J; Shinozaki, Tsuyoshi; Zhang, Ying Ming; Zhang, Mei Luo; Wakayama, Yuji; Sugai, Yoshinao; Kagaya, Yutaka; Miura, Masahito; Boyden, Penelope A; Stuyvers, Bruno D M; Landesberg, Amir

2008-01-01

Starling's Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac

Sarcomere length dependence of rat skinned cardiac myocyte mechanical properties: dependence on myosin heavy chain

PubMed Central

Korte, F Steven; McDonald, Kerry S

2007-01-01

The effects of sarcomere length (SL) on sarcomeric loaded shortening velocity, power output and rates of force development were examined in rat skinned cardiac myocytes that contained either α-myosin heavy chain (α-MyHC) or β-MyHC at 12 ± 1°C. When SL was decreased from 2.3 μm to 2.0 μm submaximal isometric force decreased ∼40% in both α-MyHC and β-MyHC myocytes while peak absolute power output decreased 55% in α-MyHC myocytes and 70% in β-MyHC myocytes. After normalization for the fall in force, peak power output decreased about twice as much in β-MyHC as in α-MyHC myocytes (41%versus 20%). To determine whether the fall in normalized power was due to the lower force levels, [Ca2+] was increased at short SL to match force at long SL. Surprisingly, this led to a 32% greater peak normalized power output at short SL compared to long SL in α-MyHC myocytes, whereas in β-MyHC myocytes peak normalized power output remained depressed at short SL. The role that interfilament spacing plays in determining SL dependence of power was tested by myocyte compression at short SL. Addition of 2% dextran at short SL decreased myocyte width and increased force to levels obtained at long SL, and increased peak normalized power output to values greater than at long SL in both α-MyHC and β-MyHC myocytes. The rate constant of force development (ktr) was also measured and was not different between long and short SL at the same [Ca2+] in α-MyHC myocytes but was greater at short SL in β-MyHC myocytes. At short SL with matched force by either dextran or [Ca2+], ktr was greater than at long SL in both α-MyHC and β-MyHC myocytes. Overall, these results are consistent with the idea that an intrinsic length component increases loaded crossbridge cycling rates at short SL and β-MyHC myocytes exhibit a greater sarcomere length dependence of power output. PMID:17347271

Persistent fibrosis, hypertrophy and sarcomere disorganisation after endoscopy-guided heart resection in adult Xenopus

PubMed Central

Girardot, Fabrice; Péricard, Louise; Demeneix, Barbara A.; Coen, Laurent; Chai, Norin

2017-01-01

Models of cardiac repair are needed to understand mechanisms underlying failure to regenerate in human cardiac tissue. Such studies are currently dominated by the use of zebrafish and mice. Remarkably, it is between these two evolutionary separated species that the adult cardiac regenerative capacity is thought to be lost, but causes of this difference remain largely unknown. Amphibians, evolutionary positioned between these two models, are of particular interest to help fill this lack of knowledge. We thus developed an endoscopy-based resection method to explore the consequences of cardiac injury in adult Xenopus laevis. This method allowed in situ live heart observation, standardised tissue amputation size and reproducibility. During the first week following amputation, gene expression of cell proliferation markers remained unchanged, whereas those relating to sarcomere organisation decreased and markers of inflammation, fibrosis and hypertrophy increased. One-month post-amputation, fibrosis and hypertrophy were evident at the injury site, persisting through 11 months. Moreover, cardiomyocyte sarcomere organisation deteriorated early following amputation, and was not completely recovered as far as 11 months later. We conclude that the adult Xenopus heart is unable to regenerate, displaying cellular and molecular marks of scarring. Our work suggests that, contrary to urodeles and teleosts, with the exception of medaka, adult anurans share a cardiac injury outcome similar to adult mammals. This observation is at odds with current hypotheses that link loss of cardiac regenerative capacity with acquisition of homeothermy. PMID:28278282

Role of dystroglycan in limiting contraction-induced injury to the sarcomeric cytoskeleton of mature skeletal muscle.

PubMed

Rader, Erik P; Turk, Rolf; Willer, Tobias; Beltrán, Daniel; Inamori, Kei-Ichiro; Peterson, Taylor A; Engle, Jeffrey; Prouty, Sally; Matsumura, Kiichiro; Saito, Fumiaki; Anderson, Mary E; Campbell, Kevin P

2016-09-27

Dystroglycan (DG) is a highly expressed extracellular matrix receptor that is linked to the cytoskeleton in skeletal muscle. DG is critical for the function of skeletal muscle, and muscle with primary defects in the expression and/or function of DG throughout development has many pathological features and a severe muscular dystrophy phenotype. In addition, reduction in DG at the sarcolemma is a common feature in muscle biopsies from patients with various types of muscular dystrophy. However, the consequence of disrupting DG in mature muscle is not known. Here, we investigated muscles of transgenic mice several months after genetic knockdown of DG at maturity. In our study, an increase in susceptibility to contraction-induced injury was the first pathological feature observed after the levels of DG at the sarcolemma were reduced. The contraction-induced injury was not accompanied by increased necrosis, excitation-contraction uncoupling, or fragility of the sarcolemma. Rather, disruption of the sarcomeric cytoskeleton was evident as reduced passive tension and decreased titin immunostaining. These results reveal a role for DG in maintaining the stability of the sarcomeric cytoskeleton during contraction and provide mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy after lengthening contractions.

Muscle enzyme and fiber type-specific sarcomere protein increases in serum after inertial concentric-eccentric exercise.

PubMed

Carmona, G; Guerrero, M; Cussó, R; Padullés, J M; Moras, G; Lloret, M; Bedini, J L; Cadefau, J A

2015-12-01

Muscle damage induced by inertial exercise performed on a flywheel device was assessed through the serum evolution of muscle enzymes, interleukin 6, and fiber type-specific sarcomere proteins such as fast myosin (FM) and slow myosin (SM). We hypothesized that a model of muscle damage could be constructed by measuring the evolution of serum concentration of muscle proteins following inertial exercise, according to their molecular weight and the fiber compartment in which they are located. Moreover, by measuring FM and SM, the type of fibers that are affected could be assessed. Serum profiles were registered before and 24, 48, and 144 h after exercise in 10 healthy and recreationally active young men. Creatine kinase (CK) and CK-myocardial band isoenzyme increased in serum early (24 h) and returned to baseline values after 48 h. FM increased in serum late (48 h) and remained elevated 144 h post-exercise. The increase in serum muscle enzymes suggests increased membrane permeability of both fast and slow fibers, and the increase in FM reveals sarcomere disruption as well as increased membrane permeability of fast fibers. Consequently, FM could be adopted as a fiber type-specific biomarker of muscle damage. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Rhythmic beating cardiomyocytes derived from human embryonic germ (EG) cells in vitro].

PubMed

Hua, Jinlian; Xu, Xiaoming; Dou, Zhongying

2006-10-01

Embryonic germ (EG) cells are pluripotent cells derived from primordial germ cells (PGCs) of gonads, gonadal ridges and mesenteries, analogies of fetuses,with the ability to undergo both highly self-renewal and multiple differentiation. These cells in vitro can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cells of the body. The aim of this study is to investigate the potentiality of human EG cells differentiation into cardiomyocytes. Inducing human EG cells with the method of murine ES cells differentiation into cardiomyocytes, supplemented with 0.75%-1% DMSO, 20% NBS, 10(-7) mM RA and 20% cardiomyocytes conditioned medium. 20 heart-like (rhythmic beating cell masses were observed in vitro culture and delayed human EG cells, which beat spontaneously from 20-120 times per minute and maintained beating for 2-15 days, periodic acid's staining (PAS), Myoglobin and a-actin immunological histology positive were all positive and reacted with K+, Ca2+ and adrenalin. Relatively unorganized myofibrillar bundles or more organized sarcomeres, z-bands or a gap junction, the presence of desmosomes in a few cells of the cell masses was observed with transmision electron microscope, which initially demonstrated that these cells were cardiomyocytes. We could not get rhythmly beating cardiomyocytes with 0.75%-1% DMSO, 10-7 mM RA and 20% cardiomyocytes conditioned medium,but in which the percentage of cardiac alpha-actin immunostaining positive cells were increased. The results first demonstrated that human EG cells can differentiate into rhythmic beating cardiomyocytes in vitro and suggests that human EG cells may represent a new potent resource for cardiomyocytes transplantation therapy for myocardium infarction.

Skeletal and cardiac myogenesis accompany adipogenesis in P19 embryonal stem cells.

PubMed

Bouchard, Frédéric; Paquin, Joanne

2009-09-01

P19 embryonic carcinoma cells resemble normal embryonic stem (ES) cells. They generate cardiac and skeletal myocytes in response to retinoic acid (RA) or oxytocin (OT). RA treatment followed by exposure to triiodothyronine (T3) and insulin induces ES cells differentiation into adipocytes and skeletomyocytes. On the other hand, OT (10(-7) M) was reported to inhibit 3T3 preadipocyte maturation. The present work was undertaken to determine whether P19 cells have an adipogenic potential that could be affected by OT. Cells were treated with RA (10(-6) M)/T3+insulin (adipogenic protocol) or 10(-7) M OT (cardiomyogenic protocol), and analyzed by polymerase chain reaction, immunotechniques, and cytochemistry. Oil-Red-O staining and expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) and aP2 indicated the generation of adipocytes in cultures submitted to the adipogenic protocol. Contracting cells were also generated. Cells positive for sarcomeric actinin and negative for cardiac troponin inhibitor (cTpnI) indicated generation of skeletomyocytes, and cTpnI positive cells revealed generation of cardiomyocytes. Levels of cTpnI and of the skeletal marker MyoD were almost similar in both protocols, whereas no Oil-Red-O staining was associated with the cardiomyogenic protocol. Addition of 10(-7) M OT to the adipogenic protocol did not affect Oil-Red-O staining and PPARgamma expression. Interestingly, Oct3/4 pluripotency marker disappeared in the adipogenic protocol but remained expressed in the cardiomyogenic one. P19 cells thus have an adipogenic potential non affected by 10(-7) M OT. RA/T3+insulin combination generates a larger spectrum of mesodermal cell derivatives and is a more potent morphogenic treatment than OT. P19 cells could help investigating mechanisms of cell fate decision during development.

Beyond the binding site: in vivo identification of tbx2, smarca5 and wnt5b as molecular targets of CNBP during embryonic development.

PubMed

Armas, Pablo; Margarit, Ezequiel; Mouguelar, Valeria S; Allende, Miguel L; Calcaterra, Nora B

2013-01-01

CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.

Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

PubMed Central

Mouguelar, Valeria S.; Allende, Miguel L.; Calcaterra, Nora B.

2013-01-01

CNBP is a nucleic acid chaperone implicated in vertebrate craniofacial development, as well as in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human muscle diseases. CNBP is highly conserved among vertebrates and has been implicated in transcriptional regulation; however, its DNA binding sites and molecular targets remain elusive. The main goal of this work was to identify CNBP DNA binding sites that might reveal target genes involved in vertebrate embryonic development. To accomplish this, we used a recently described yeast one-hybrid assay to identify DNA sequences bound in vivo by CNBP. Bioinformatic analyses revealed that these sequences are G-enriched and show high frequency of putative G-quadruplex DNA secondary structure. Moreover, an in silico approach enabled us to establish the CNBP DNA-binding site and to predict CNBP putative targets based on gene ontology terms and synexpression with CNBP. The direct interaction between CNBP and candidate genes was proved by EMSA and ChIP assays. Besides, the role of CNBP upon the identified genes was validated in loss-of-function experiments in developing zebrafish. We successfully confirmed that CNBP up-regulates tbx2b and smarca5, and down-regulates wnt5b gene expression. The highly stringent strategy used in this work allowed us to identify new CNBP target genes functionally important in different contexts of vertebrate embryonic development. Furthermore, it represents a novel approach toward understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates. PMID:23667590

Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

EPA Science Inventory

Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 μM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post...

Adult Olfactory Bulb Interneuron Phenotypes Identified by Targeting Embryonic and Postnatal Neural Progenitors

PubMed Central

Figueres-Oñate, Maria; López-Mascaraque, Laura

2016-01-01

Neurons are generated during embryonic development and in adulthood, although adult neurogenesis is restricted to two main brain regions, the hippocampus and olfactory bulb. The subventricular zone (SVZ) of the lateral ventricles generates neural stem/progenitor cells that continually provide the olfactory bulb (OB) with new granule or periglomerular neurons, cells that arrive from the SVZ via the rostral migratory stream. The continued neurogenesis and the adequate integration of these newly generated interneurons is essential to maintain homeostasis in the olfactory bulb, where the differentiation of these cells into specific neural cell types is strongly influenced by temporal cues. Therefore, identifying the critical features that control the generation of adult OB interneurons at either pre- or post-natal stages is important to understand the dynamic contribution of neural stem cells. Here, we used in utero and neonatal SVZ electroporation along with a transposase-mediated stable integration plasmid, in order to track interneurons and glial lineages in the OB. These plasmids are valuable tools to study the development of OB interneurons from embryonic and post-natal SVZ progenitors. Accordingly, we examined the location and identity of the adult progeny of embryonic and post-natally transfected progenitors by examining neurochemical markers in the adult OB. These data reveal the different cell types in the olfactory bulb that are generated in function of age and different electroporation conditions. PMID:27242400

Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

NASA Astrophysics Data System (ADS)

Salick, Max R.

The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog.

PubMed

Reconditi, Massimo; Brunello, Elisabetta; Fusi, Luca; Linari, Marco; Martinez, Manuel Fernandez; Lombardi, Vincenzo; Irving, Malcolm; Piazzesi, Gabriella

2014-03-01

X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6-3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments.

Sarcomere-length dependence of myosin filament structure in skeletal muscle fibres of the frog

PubMed Central

Reconditi, Massimo; Brunello, Elisabetta; Fusi, Luca; Linari, Marco; Martinez, Manuel Fernandez; Lombardi, Vincenzo; Irving, Malcolm; Piazzesi, Gabriella

2014-01-01

X-ray diffraction patterns were recorded at beamline ID02 of the European Synchrotron Radiation Facility from small bundles of skeletal muscle fibres from Rana esculenta at sarcomere lengths between 2.1 and 3.5 μm at 4°C. The intensities of the X-ray reflections from resting fibres associated with the quasi-helical order of the myosin heads and myosin binding protein C (MyBP-C) decreased in the sarcomere length range 2.6–3.0 μm but were constant outside it, suggesting that an OFF conformation of the thick filament is maintained by an interaction between MyBP-C and the thin filaments. During active isometric contraction the intensity of the M3 reflection from the regular repeat of the myosin heads along the filaments decreased in proportion to the overlap between thick and thin filaments, with no change in its interference fine structure. Thus, myosin heads in the regions of the thick filaments that do not overlap with thin filaments are highly disordered during isometric contraction, in contrast to their quasi-helical order at rest. Heads in the overlap region that belong to two-headed myosin molecules that are fully detached from actin are also highly disordered, in contrast to the detached partners of actin-attached heads. These results provide strong support for the concept of a regulatory structural transition in the thick filament involving changes in both the organisation of the myosin heads on its surface and the axial periodicity of the myosin tails in its backbone, mediated by an interaction between MyBP-C and the thin filaments. PMID:24344169

Childhood Central Nervous System Embryonal Tumors Treatment (PDQ®)—Patient Version

Cancer.gov

Childhood central nervous system embryonal tumors and pineal tumors are treated with surgery, radiation therapy, chemotherapy, high-dose chemotherapy with stem cell rescue and targeted therapy. Learn more in this expert-reviewed summary.

Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells

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

Koller, B.H.; Hyungsuk Kim; Latour, A.M.

1991-12-01

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistantmore » cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possible passage number, influences the recovery of CFTR-targeted cells.« less

Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.

PubMed

Marian, A J; Yu, Q T; Mann, D L; Graham, F L; Roberts, R

1995-07-01

Mutations in the beta-myosin heavy chain (beta MyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant beta MyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A high rigor temperature, not sarcomere length, determines light scattering properties and muscle colour in beef M. sternomandibularis meat and muscle fibres.

PubMed

Hughes, J; Clarke, F; Purslow, P; Warner, R

2018-05-18

Beef meat colour is impacted by both myoglobin status and the light scattering properties of the muscle, and the specific causative scattering elements of the latter are still unknown. We hypothesize that stretching muscles during rigor will generate a structure which favours light scattering, by increasing the length of the I-band (longer sarcomeres) and that a high rigor temperature will cause protein reconfiguration, changing the muscle structure and promoting light scattering. Muscle fibre fragments were isolated from four beef M. sternomandibularis and subjected to stretching (plus, minus) and three incubation temperatures (5, 15, 35 °C). Reflectance confocal laser scanning microscopy (rCLSM) revealed sarcomere stretching alone was not solely responsible for light scattering development. A high rigor temperature (35 °C) was more favourable for light scattering. Stretching and taking muscle into rigor at 35 °C promoted transverse shrinkage of muscle fibres and increased light scattering and could be applied post-mortem (PM) to reduce the occurrence of problematic dark meat. Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.

The gene for creatine kinase, mitochondrial 2 (sarcomeric; CKMT2), maps to chromosome 5q13. 3

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

Richard, I.; Devaud, C.; Cherif, D.

1993-10-01

YAC clones for the creatine kinase, mitochrondial 2 (sarcomeric; CKMT2), gene were isolated. One of these YACs was localized on chromosome 5q13.3 by fluorescence in situ hybridization. A polymorphic dinucleotide repeat (heterozygosity 0.77) was identified within the seventh intron of the CKMT2 gene. Genotyping of CEPH families allowed positioning of CKMT2 on the multipoint map of chromosome 5 between D5S424 and D5S428, distal to spinal muscular atrophy (SMA) (5q12-q14). 8 refs., 1 fig., 2 tabs.

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells

PubMed Central

West, Michael D.; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B.; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-01

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1, encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro-derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition. PMID:29487692

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells.

PubMed

West, Michael D; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-30

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1 , encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro -derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition.

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.

Reprogramming multipotent tumor cells with the embryonic neural crest microenvironment

PubMed Central

Kasemeier-Kulesa, Jennifer C.; Teddy, Jessica M.; Postovit, Lynne-Marie; Seftor, Elisabeth A.; Seftor, Richard E.B.; Hendrix, Mary J.C.; Kulesa, Paul M.

2008-01-01

The embryonic microenvironment is an important source of signals that program multipotent cells to adopt a particular fate and migratory path, yet its potential to reprogram and restrict multipotent tumor cell fate and invasion is unrealized. Aggressive tumor cells share many characteristics with multipotent, invasive embryonic progenitors, contributing to the paradigm of tumour cell plasticity. In the vertebrate embryo, multiple cell types originate from a highly invasive cell population called the neural crest. The neural crest and the embryonic microenvironments they migrate through represent an excellent model system to study cell diversification during embryogenesis and phenotype determination. Recent exciting studies of tumor cells transplanted into various embryo models, including the neural crest rich chick microenvironment, have revealed the potential to control and revert the metastatic phenotype, suggesting further work may help to identify new targets for therapeutic intervention derived from a convergence of tumorigenic and embryonic signals. In this mini-review, we summarize markers that are common to the neural crest and highly aggressive human melanoma cells. We highlight advances in our understanding of tumor cell behaviors and plasticity studied within the chick neural crest rich microenvironment. In so doing, we honor the tremendous contributions of Professor Elizabeth D. Hay towards this important interface of developmental and cancer biology. PMID:18629870

Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer's disease.

PubMed

Yamamoto, Satoshi; Ooshima, Yuki; Nakata, Mitsugu; Yano, Takashi; Matsuoka, Kunio; Watanabe, Sayuri; Maeda, Ryouta; Takahashi, Hideki; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

2013-06-01

Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells.

PubMed

Barbet, Romain; Peiffer, Isabelle; Hatzfeld, Antoinette; Charbord, Pierre; Hatzfeld, Jacques A

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1(low), BMPR1B(low), FLT4(low), LRRC32(low), and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

Maternal embryonic leucine zipper kinase is a novel target for proliferation-associated high-risk myeloma

PubMed Central

Bolomsky, Arnold; Heusschen, Roy; Schlangen, Karin; Stangelberger, Kathrin; Muller, Joséphine; Schreiner, Wolfgang; Zojer, Niklas; Caers, Jo; Ludwig, Heinz

2018-01-01

Treatment of high-risk patients is a major challenge in multiple myeloma. This is especially true for patients assigned to the gene expression profiling-defined proliferation subgroup. Although recent efforts have identified some key players of proliferative myeloma, genetic interactions and players that can be targeted with clinically effective drugs have to be identified in order to overcome the poor prognosis of these patients. We therefore examined maternal embryonic leucine zipper kinase (MELK) for its implications in hyper-proliferative myeloma and analyzed the activity of the MELK inhibitor OTSSP167 both in vitro and in vivo. MELK was found to be significantly overexpressed in the proliferative subgroup of myeloma. This finding translated into poor overall survival in patients with high vs. low MELK expression. Enrichment analysis of upregulated genes in myeloma cells of MELKhigh patients confirmed the strong implications in myeloma cell proliferation. Targeting MELK with OTSSP167 impaired the growth and survival of myeloma cells, thereby affecting central survival factors such as MCL-1 and IRF4. This activity was also observed in the 5TGM.1 murine model of myeloma. OTSSP167 reduced bone marrow infiltration and serum paraprotein levels in a dose-dependent manner. In addition, we revealed a strong link between MELK and other proliferation-associated high-risk genes (PLK-1, EZH2, FOXM1, DEPDC1) and MELK inhibition also impaired the expression of those genes. We therefore conclude that MELK is an essential component of a proliferative gene signature and that pharmacological inhibition of MELK represents an attractive novel approach to overcome the poor prognosis of high-risk patients with a proliferative expression pattern. PMID:29122991

The titin A-band rod domain is dispensable for initial thick filament assembly in zebrafish.

PubMed

Myhre, J Layne; Hills, Jordan A; Prill, Kendal; Wohlgemuth, Serene L; Pilgrim, David B

2014-03-01

The sarcomeres of skeletal and cardiac muscle are highly structured protein arrays, consisting of thick and thin filaments aligned precisely to one another and to their surrounding matrix. The contractile mechanisms of sarcomeres are generally well understood, but how the patterning of sarcomeres is initiated during early skeletal muscle and cardiac development remains uncertain. Two of the most widely accepted hypotheses for this process include the "molecular ruler" model, in which the massive protein titin defines the length of the sarcomere and provides a scaffold along which the myosin thick filament is assembled, and the "premyofibril" model, which proposes that thick filament formation does not require titin, but that a "premyofibril" consisting of non-muscle myosin, α-actinin and cytoskeletal actin is used as a template. Each model posits a different order of necessity of the various components, but these have been difficult to test in vivo. Zebrafish motility mutants with developmental defects in sarcomere patterning are useful for the elucidation of such mechanisms, and here we report the analysis of the herzschlag mutant, which shows deficits in both cardiac and skeletal muscle. The herzschlag mutant produces a truncated titin protein, lacking the C-terminal rod domain that is proposed to act as a thick filament scaffold, yet muscle patterning is still initiated, with grossly normal thick and thin filament assembly. Only after embryonic muscle contraction begins is breakdown of sarcomeric myosin patterning observed, consistent with the previously noted role of titin in maintaining the contractile integrity of mature sarcomeres. This conflicts with the "molecular ruler" model of early sarcomere patterning and supports a titin-independent model of thick filament organization during sarcomerogenesis. These findings are also consistent with the symptoms of human titin myopathies that exhibit a late onset, such as tibial muscular dystrophy. Copyright © 2013

A trade-off between embryonic development rate and immune function of avian offspring is concealed by embryonic temperature

USGS Publications Warehouse

Martin, Thomas E.; Arriero, Elena; Majewska, Ania

2011-01-01

Long embryonic periods are assumed to reflect slower intrinsic development that are thought to trade off to allow enhanced physiological systems, such as immune function. Yet, the relatively rare studies of this trade-off in avian offspring have not found the expected trade-off. Theory and tests have not taken into account the strong extrinsic effects of temperature on embryonic periods of birds. Here, we show that length of the embryonic period did not explain variation in two measures of immune function when temperature was ignored, based on studies of 34 Passerine species in tropical Venezuela (23 species) and north temperate Arizona (11 species). Variation in immune function was explained when embryonic periods were corrected for average embryonic temperature, in order to better estimate intrinsic rates of development. Immune function of offspring trades off with intrinsic rates of embryonic development once the extrinsic effects of embryonic temperatures are taken into account.

The sarcomeric Z-disc component myopodin is a multiadapter protein that interacts with filamin and alpha-actinin.

PubMed

Linnemann, Anja; van der Ven, Peter F M; Vakeel, Padmanabhan; Albinus, Britta; Simonis, Dirk; Bendas, Gerd; Schenk, Jörg A; Micheel, Burkhard; Kley, Rudolf A; Fürst, Dieter O

2010-09-01

Here we introduce myopodin as a novel filamin C binding partner. Corroborative yeast two-hybrid and biochemical analyses indicate that the central part of myopodin that shows high homology to the closely related protein synaptopodin and that is common to all its currently known or predicted variants interacts with filamin C immunoglobulin-like domains 20-21. A detailed characterization of the previously described interaction between myopodin and alpha-actinin demonstrates for the first time that myopodin contains three independent alpha-actinin-binding sites. Newly developed myopodin-specific antibodies reveal expression at the earliest stages of in vitro differentiation of human skeletal muscle cells preceding the expression of sarcomeric alpha-actinin. Myopodin colocalizes with filamin and alpha-actinin during all stages of muscle development. By contrast, colocalization with its previously identified binding partner zyxin is restricted to early developmental stages. Genetic and cellular analyses of skeletal muscle provided direct evidence for an alternative transcriptional start site in exon three, corroborating the expression of a myopodin variant lacking the PDZ domain encoded by exons 1 and 2 in skeletal muscle. We conclude that myopodin is a multiadapter protein of the sarcomeric Z-disc that links nascent myofibrils to the sarcolemma via zyxin, and might play a role in early assembly and stabilization of the Z-disc. Mutations in FLNC, ACTN2 and several other genes encoding Z-disc-related proteins cause myopathy and cardiomyopathy. Its localization and its association with the myopathy-associated proteins filamin C and alpha-actinin make myopodin an interesting candidate for a muscle disease gene. Copyright 2010 Elsevier GmbH. All rights reserved.

Large-scale production of embryonic red blood cells from human embryonic stem cells.

PubMed

Olivier, Emmanuel N; Qiu, Caihong; Velho, Michelle; Hirsch, Rhoda Elison; Bouhassira, Eric E

2006-12-01

To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.

Radial stiffness characteristics of the overlap regions of sarcomeres in isolated skeletal myofibrils in pre-force generating state.

PubMed

Miyashiro, Daisuke; Ohtsuki, Misato; Shimamoto, Yuta; Wakayama, Jun'ichi; Kunioka, Yuki; Kobayashi, Takakazu; Ishiwata, Shin'ichi; Yamada, Takenori

2017-01-01

We have studied the stiffness of myofilament lattice in sarcomeres in the pre-force generating state, which was realized by a relaxing reagent, BDM (butane dione monoxime). First, the radial stiffness for the overlap regions of sarcomeres of isolated single myofibrils was estimated from the resulting decreases in diameter by osmotic pressure applied with the addition of Dextran. Then, the radial stiffness was also estimated from force-distance curve measurements with AFM technology. The radial stiffness for the overlap regions thus obtained was composed of a soft and a rigid component. The soft component visco-elastically changed in a characteristic fashion depending on the physiological conditions of myofibrils, suggesting that it comes from cross-bridge structures. BDM treatments significantly affected the soft radial component of contracting myofibrils depending on the approach velocity of cantilever: It was nearly equal to that in the contracting state at high approach velocity, whereas as low as that in the relaxing state at low approach velocity. However, comparable BDM treatments greatly suppressed the force production and the axial stiffness in contracting glycerinated muscle fibers and also the sliding velocity of actin filaments in the in vitro motility assay. Considering that BDM shifts the cross-bridge population from force generating to pre-force generating states in contracting muscle, the obtained results strongly suggest that cross-bridges in the pre-force generating state are visco-elastically attached to the thin filaments in such a binding manner that the axial stiffness is low but the radial stiffness significantly high similar to that in force generating state.

I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

PubMed

Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A; Panthier, Jean-Jacques

2012-01-01

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

Cardiomyopathy mutations in the tail of β-cardiac myosin modify the coiled-coil structure and affect integration into thick filaments in muscle sarcomeres in adult cardiomyocytes.

PubMed

Wolny, Marcin; Colegrave, Melanie; Colman, Lucy; White, Ed; Knight, Peter J; Peckham, Michelle

2013-11-01

It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301-1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy.

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis)

PubMed Central

Tate, Kevin B.; Rhen, Turk; Eme, John; Kohl, Zachary F.; Crossley, Janna; Elsey, Ruth M.

2016-01-01

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50–70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70–90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered β-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator. PMID:27101296

Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in 3D collagen matrix: Effects of niche cell supplementation and mechanical stimulation.

PubMed

Zhang, W; Kong, C W; Tong, M H; Chooi, W H; Huang, N; Li, R A; Chan, B P

2017-02-01

Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as a promising source for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. Here, we fabricate cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials. Supplementation of niche cells at 3% to the number of hESC-CMs enhance the maturation of the hESC-CMs in 3D tissue matrix. The benefits of adding mesenchymal stem cells (MSCs) are comparable to that of adding fibroblasts. These two cell types demonstrate similar effects in promoting the compaction and cell spreading, as well as expression of maturation markers at both gene and protein levels. Mechanical loading, particularly cyclic stretch, produces engineered cardiac tissues with higher maturity in terms of twitch force, elastic modulus, sarcomere length and molecular signature, when comparing to static stretch or non-stretched controls. The current study demonstrates that the application of niche cells and mechanical stretch both stimulate the maturation of hESC-CMs in 3D architecture. Our results therefore suggest that this 3D model can be used for in vitro cardiac maturation study. Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) are regarded as being a promising source of cells for regenerative medicine, drug testing and disease modeling. Nevertheless, cardiomyocytes are immature in terms of their contractile structure, metabolism and electrophysiological properties. In the current study, we have fabricated cardiac muscle strips by encapsulating hESC-CMs in collagen-based biomaterials and demonstrated that supplementation of mesenchymal niche cells as well as provision of mechanical loading particularly stretching have significantly promoted the maturation of the cardiomyocytes and hence improved the mechanical functional characteristics of the tissue strips

Embryonic death and the creation of human embryonic stem cells.

PubMed

Landry, Donald W; Zucker, Howard A

2004-11-01

The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

PubMed

Wang, Dachun; Quan, Yuan; Yan, Qing; Morales, John E; Wetsel, Rick A

2015-10-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

PubMed Central

Quan, Yuan; Yan, Qing; Morales, John E.

2015-01-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells

Timing and Targeting of Treatment in Left Ventricular Hypertrophy.

PubMed

Nam, Deokhwa; Reineke, Erin L

2017-01-01

In most clinical cases, left ventricular hypertrophy (LVH) occurs over time from persistent cardiac stress. At the molecular level, this results in both transient and long-term changes to metabolic, sarcomeric, ion handling, and stress signaling pathways. Although this is initially an adaptive change, the mechanisms underlying LVH eventually lead to maladaptive changes including fibrosis, decreased cardiac function, and failure. Understanding the regulators of long-term changes, which are largely driven by transcriptional remodeling, is a crucial step in identifying novel therapeutic targets for preventing the downstream negative effects of LVH and treatments that could reverse or prevent it. The development of effective therapeutics, however, will require a critical understanding of what to target, how to modify important pathways, and how to identify the stage of pathology in which a specific treatment should be used.

Sarcomere protein gene mutations and inherited heart disease: a beta-cardiac myosin heavy chain mutation causing endocardial fibroelastosis and heart failure.

PubMed

Kamisago, Mitsuhiro; Schmitt, Joachim P; McNamara, Dennis; Seidman, Christine; Seidman, J G

2006-01-01

Inherited human cardiomyopathies often lead to heart failure. A common feature of these conditions is that affected individuals can express the disease causing mutations for many years without showing clinical signs of the disease. Previous studies have demonstrated that sarcomere protein gene mutations can cause either dilated cardiomyopathy or hypertrophic cardiomyopathy. Here we demonstrate that the Arg442His missense mutation in beta-cardiac myosin heavy chain (betaMHC) causes dilated cardiomyopathy, endocardial fibroelastosis and heart failure at a very early age. Using standard genetic engineering tools we and others have made murine models by introducing human disease causing mutations into mice. The central hypothesis of these studies has been that by identifying the pathophysiological pathways activated by these mutations we can define enzymatic activities that are modified during the disease process and which may be involved in pathways that involve more common forms of cardiac disease. Murine models bearing different mutant myosins are being used to address whether each disease causing mutant betaMHC activates the same or different cellular pathways. Dissecting the molecular pathways modulated by mutations in sarcomere protein genes as well as other genes has already demonstrated that there are multiple pathways leading to cardiac remodelling and heart failure. Defining the mechanisms by which mutations in the same genes activate different cellular pathways remains an important question.

Orientation of Myosin Binding Protein C in the Cardiac Muscle Sarcomere Determined by Domain-Specific Immuno-EM

PubMed Central

Lee, Kyounghwan; Harris, Samantha P.; Sadayappan, Sakthivel; Craig, Roger

2014-01-01

Myosin binding protein-C is a thick filament protein of vertebrate striated muscle. The cardiac isoform (cMyBP-C) is essential for normal cardiac function, and mutations in cMyBP-C cause cardiac muscle disease. The rod-shaped molecule is composed primarily of 11 immunoglobulin- or fibronectin-like domains, and is located at 9 sites, 43 nm apart, in each half of the A-band. To understand how cMyBP-C functions, it is important to know its structural organization in the sarcomere, as this will affect its ability to interact with other sarcomeric proteins. Several models have been proposed, in which cMyBP-C wraps around, extends radially from, or runs axially along the thick filament. Our goal was to define cMyBP-C orientation by determining the relative axial positions of different cMyBP-C domains. Immuno-electron microscopy was performed using mouse cardiac myofibrils labeled with antibodies specific to the N- and C-terminal domains and to the middle of cMyBP-C. Antibodies to all regions of the molecule, except the C-terminus, labeled at the same nine axial positions in each half A-band, consistent with a circumferential and/or radial rather than an axial orientation of the bulk of the molecule. The C-terminal antibody stripes were slightly displaced axially, demonstrating an axial orientation of the C-terminal 3 domains, with the C-terminus closer to the M-line. These results, combined with previous studies, suggest that the C-terminal domains of cMyBP-C run along the thick filament surface, while the N-terminus extends towards neighboring thin filaments. This organization provides a structural framework for understanding cMyBP-C’s modulation of cardiac muscle contraction. PMID:25451032

Microarray Analysis of LTR Retrotransposon Silencing Identifies Hdac1 as a Regulator of Retrotransposon Expression in Mouse Embryonic Stem Cells

PubMed Central

Madej, Monika J.; Taggart, Mary; Gautier, Philippe; Garcia-Perez, Jose Luis; Meehan, Richard R.; Adams, Ian R.

2012-01-01

Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells. PMID:22570599

Optical coherence tomography guided microinjections in live mouse embryos: high-resolution targeted manipulation for mouse embryonic research

PubMed Central

Syed, Saba H.; Coughlin, Andrew J.; Garcia, Monica D.; Wang, Shang; West, Jennifer L.; Larin, Kirill V.; Larina, Irina V.

2015-01-01

Abstract. The ability to conduct highly localized delivery of contrast agents, viral vectors, therapeutic or pharmacological agents, and signaling molecules or dyes to live mammalian embryos is greatly desired to enable a variety of studies in the field of developmental biology, such as investigating the molecular regulation of cardiovascular morphogenesis. To meet such a demand, we introduce, for the first time, the concept of employing optical coherence tomography (OCT)-guide microinjections in live mouse embryos, which provides precisely targeted manipulation with spatial resolution at the micrometer scale. The feasibility demonstration is performed with experimental studies on cultured live mouse embryos at E8.5 and E9.5. Additionally, we investigate the OCT-guided microinjection of gold–silica nanoshells to the yolk sac vasculature of live cultured mouse embryos at the stage when the heart just starts to beat, as a potential approach for dynamic assessment of cardiovascular form and function before the onset of blood cell circulation. Also, the capability of OCT to quantitatively monitor and measure injection volume is presented. Our results indicate that OCT-guided microinjection could be a useful tool for mouse embryonic research. PMID:25581495

Optical coherence tomography guided microinjections in live mouse embryos: high-resolution targeted manipulation for mouse embryonic research.

PubMed

Syed, Saba H; Coughlin, Andrew J; Garcia, Monica D; Wang, Shang; West, Jennifer L; Larin, Kirill V; Larina, Irina V

2015-05-01

The ability to conduct highly localized delivery of contrast agents, viral vectors, therapeutic or pharmacological agents, and signaling molecules or dyes to live mammalian embryos is greatly desired to enable a variety of studies in the field of developmental biology, such as investigating the molecular regulation of cardiovascular morphogenesis. To meet such a demand, we introduce, for the first time, the concept of employing optical coherence tomography (OCT)-guide microinjections in live mouse embryos, which provides precisely targeted manipulation with spatial resolution at the micrometer scale. The feasibility demonstration is performed with experimental studies on cultured live mouse embryos at E8.5 and E9.5. Additionally, we investigate the OCT-guided microinjection of gold–silica nanoshells to the yolk sac vasculature of live cultured mouse embryos at the stage when the heart just starts to beat, as a potential approach for dynamic assessment of cardiovascular form and function before the onset of blood cell circulation. Also, the capability of OCT to quantitatively monitor and measure injection volume is presented. Our results indicate that OCT-guided microinjection could be a useful tool for mouse embryonic research.

Characterization of muscle contraction with second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Prent, Nicole

Muscle cells have the ability to change length and generate force due to orchestrated action of myosin nanomotors that cause sliding of actin filaments along myosin filaments in the sarcomeres, the fundamental contractile units, of myocytes. The correlated action of hundreds of sarcomeres is needed to produce the myocyte contractions. This study probes the molecular structure of the myofilaments and investigates the movement correlations between sarcomeres during contraction. In this study, second harmonic generation (SHG) microscopy is employed for imaging striated myocytes. Myosin filaments in striated myocytes inherently have a nonzero second-order susceptibility, [special characters omitted] and therefore generate efficient SHG. Employing polarization-in polarization-out (PIPO) SHG microscopy allows for the accurate determination of the characteristic ratio, [special characters omitted] in birefringent myocytes, which describes the structure of the myosin filament. Analysis shows that the b value at the centre of the myosin filament, where the nonlinear dipoles are better aligned, is slightly lower than the value at the edges of the filament, where there is more disorder in orientation of the nonlinear dipoles from the myosin heads. Forced stretching of myocytes resulted in an SHG intensity increase with the elongation of the sarcomere. SHG microscopy captured individual sarcomeres during contraction, allowing for the measurement of sarcomere length (SL) and SHG intensity (SI) fluctuations. The fluctuations also revealed higher SHG intensity in elongated sarcomeres. The sarcomere synchronization model (SSM) for contracting and quiescent myocytes was developed, and experimentally verified for three cases (isolated cardiomyocyte, embryonic chicken cardiomyocyte, and larva myocyte). During contraction, the action of SLs and SIs between neighbouring sarcomeres partially correlated, whereas in quiescent myocytes the SLs show an anti-correlation and the SIs have no

MicroRNA-195 targets ADP-ribosylation factor-like protein 2 to induce apoptosis in human embryonic stem cell-derived neural progenitor cells

PubMed Central

Zhou, Y; Jiang, H; Gu, J; Tang, Y; Shen, N; Jin, Y

2013-01-01

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy, drug screening and toxicity testing of neural degenerative diseases. However, the molecular regulation of their proliferation and apoptosis, which needs to be revealed before clinical application, is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here, we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells, hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently, global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically, a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression, revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover, forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly, we found that paraquat, a neurotoxin, not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs, indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably, inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively, miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would

Microfluidic perfusion shows intersarcomere dynamics within single skeletal muscle myofibrils

PubMed Central

Minozzo, Fabio C.; Altman, David; Rassier, Dilson E.

2017-01-01

The sarcomere is the smallest functional unit of myofibrils in striated muscles. Sarcomeres are connected in series through a network of elastic and structural proteins. During myofibril activation, sarcomeres develop forces that are regulated through complex dynamics among their structures. The mechanisms that regulate intersarcomere dynamics are unclear, which limits our understanding of fundamental muscle features. Such dynamics are associated with the loss in forces caused by mechanical instability encountered in muscle diseases and cardiomyopathy and may underlie potential target treatments for such conditions. In this study, we developed a microfluidic perfusion system to control one sarcomere within a myofibril, while measuring the individual behavior of all sarcomeres. We found that the force from one sarcomere leads to adjustments of adjacent sarcomeres in a mechanism that is dependent on the sarcomere length and the myofibril stiffness. We concluded that the cooperative work of the contractile and the elastic elements within a myofibril rules the intersarcomere dynamics, with important consequences for muscle contraction. PMID:28765372

Targeted Gene Silencing of Tumor Necrosis Factor Attenuates the Negative Inotropic Effects of Lipopolysaccharide in Isolated Contracting Cardiac Myocytes

PubMed Central

Ramabadran, R. S.; Chancey, Amanda; Vallejo, Jesus G.; Barger, Philip M.; Sivasubramanian, Natarajan; Mann, Douglas L.

2008-01-01

Bacterial endotoxin (lipopolysaccharide) depresses cardiovascular function; however, the mediators and signaling pathways that are responsible for the negative inotropic effects of lipopolysaccharide are not fully known. We used RNA interference to determine the relative role of tumor necrosis factor with respect to mediating the negative inotropic effects of lipopolysaccharide in isolated cardiac myocytes. Cardiac myocyte cultures were treated with lipopolysaccharide in the presence or absence of small interfering RNAs (siRNA) for tumor necrosis factor. We examined the effects of tumor necrosis factor siRNA on lipopolysaccharide-induced tumor necrosis factor messenger RNA (mRNA) and protein biosynthesis, as well as the negative inotropic effects of lipopolysaccharide in isolated contracting cardiac myocytes. Treatment of adult cardiac myocyte cultures with tumor necrosis factor siRNA significantly attenuated lipopolysaccharide-induced tumor necrosis factor mRNA and protein biosynthesis, whereas transfection with a double-stranded RNA that does not target mammalian mRNA had no effect. Pretreatment with tumor necrosis factor siRNA significantly attenuated, but did not abrogate, the lipopolysaccharide-induced decrease in sarcomere shortening in isolated contracting cardiac myocytes. In contrast, tumor necrosis factor siRNA had a comparatively smaller effect on improving sarcomere shortening once the negative inotropic effects of lipopolysaccharide were fully established. These results suggest that tumor necrosis factor plays an important upstream role in lipopolysaccharide-induced negative inotropic effects in isolated contracting cardiac myocytes and that other molecular mechanisms are responsible for the decrease in sarcomere shortening after sustained lipopolysaccharide signaling. PMID:18427645

Altered glucose transport to utero-embryonic unit in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

PubMed

Arnab, Banerjee; Amitabh, Krishna

2011-02-10

The aim of this study was to compare the changes in concentration of glucose and glucose transporters (GLUTs) in the utero-embryonic unit, consisting of decidua, trophoblast and embryo, during delayed and non-delayed periods to understand the possible cause of delayed embryonic development in Cynopterus sphinx. The results showed a significantly decreased concentration of glucose in the utero-embryonic unit due to decline in the expression of insulin receptor (IR) and GLUT 3, 4 and 8 proteins in the utero-embryonic unit during delayed period. The in vitro study showed suppressive effect of insulin on expression of GLUTs 4 and 8 in the utero-embryonic unit and a significant positive correlation between the decreased amount of glucose consumed by the utero-embryonic unit and decreased expression of GLUTs 4 (r=0.99; p<0.05) and 8 (r=0.98; p<0.05). The in vivo study showed expression of IR and GLUT 4 proteins in adipose tissue during November suggesting increased transport of glucose to adipose tissue for adipogenesis. This study showed increased expression of HSL and OCTN2 and increased availability of l-carnitine to utero-embryonic unit suggesting increased transport of fatty acid to utero-embryonic unit during the period of delayed embryonic development. Hence it appears that due to increased transport of glucose for adipogenesis prior to winter, glucose utilization by utero-embryonic unit declines and this may be responsible for delayed embryonic development in C. sphinx. Increased supply of fatty acid to the delayed embryo may be responsible for its survival under low glucose condition but unable to promote embryonic development in C. sphinx. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

An integrated miRNA functional screening and target validation method for organ morphogenesis.

PubMed

Rebustini, Ivan T; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A; Maas, Richard L

2016-03-16

The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs.

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

PubMed Central

Sehgal, Poonam; Chaturvedi, Pankaj; Kumaran, R. Ileng; Kumar, Satish; Parnaik, Veena K.

2013-01-01

Background Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. Methodology and Principal Findings We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna+/− embryonic stem cells. Conclusions We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon

Embryonic Methamphetamine Exposure Inhibits Methamphetamine Cue Conditioning and Reduces Dopamine Concentrations in Adult N2 C. elegans

PubMed Central

Katner, S.N.; Neal-Beliveau, B.S.; Engleman, E.A.

2016-01-01

Methamphetamine (MAP) addiction is substantially prevalent in today's society, resulting in thousands of deaths and costing billions of dollars annually. Despite the potential deleterious consequences, few studies have examined the long-term effects of embryonic MAP exposure. Using the invertebrate nematode Caenorhabditis elegans (C. elegans) allows for a controlled analysis of behavioral and neurochemical changes due to early developmental drug exposure. The objective of the current studies was to determine the long-term behavioral and neurochemical effects of embryonic exposure to MAP in C. elegans. In addition, we sought to improve our conditioning and testing procedures by utilizing liquid filtration, as opposed to agar, and smaller, 6-well testing plates to increase throughput. Wild-type N2 C. elegans were embryonically exposed to 50 μM MAP. Using classical conditioning, adult-stage C. elegans were conditioned to MAP (17 and 500 μM) in the presence of either sodium ions (Na+) or chloride (Cl-) ions as conditioned stimuli (CS+/CS-). Following conditioning, a preference test was performed by placing worms in 6-well test plates spotted with the CS+ and CS- at opposite ends of each well. A preference index (PI) was determined by counting the number of worms in the CS+ target zone divided by the total number of worms in the CS+ and CS- target zones. A food conditioning experiment was also performed in order to determine if embryonic MAP exposure affected food conditioning behavior. For the neurochemical experiments, adult worms that were embryonically exposed to MAP were analyzed for (dopamine) DA content using high performance liquid chromatography (HPLC). The liquid filtration conditioning procedure employed here in combination with the use 6-well test plates significantly decreased the time required to perform these experiments and ultimately increased throughput. The MAP conditioning data found that pairing an ion with MAP at 17 or 500 μM significantly

Embryonic Methamphetamine Exposure Inhibits Methamphetamine Cue Conditioning and Reduces Dopamine Concentrations in Adult N2 Caenorhabditis elegans.

PubMed

Katner, Simon N; Neal-Beliveau, Bethany S; Engleman, Eric A

2016-01-01

Methamphetamine (MAP) addiction is substantially prevalent in today's society, resulting in thousands of deaths and costing billions of dollars annually. Despite the potential deleterious consequences, few studies have examined the long-term effects of embryonic MAP exposure. Using the invertebrate nematode Caenorhabditis elegans allows for a controlled analysis of behavioral and neurochemical changes due to early developmental drug exposure. The objective of the current study was to determine the long-term behavioral and neurochemical effects of embryonic exposure to MAP in C. elegans. In addition, we sought to improve our conditioning and testing procedures by utilizing liquid filtration, as opposed to agar, and smaller, 6-well testing plates to increase throughput. Wild-type N2 C. elegans were embryonically exposed to 50 μM MAP. Using classical conditioning, adult-stage C. elegans were conditioned to MAP (17 and 500 μM) in the presence of either sodium ions (Na+) or chloride ions (Cl-) as conditioned stimuli (CS+/CS-). Following conditioning, a preference test was performed by placing worms in 6-well test plates spotted with the CS+ and CS- at opposite ends of each well. A preference index was determined by counting the number of worms in the CS+ target zone divided by the total number of worms in the CS+ and CS- target zones. A food conditioning experiment was also performed in order to determine whether embryonic MAP exposure affected food conditioning behavior. For the neurochemical experiments, adult worms that were embryonically exposed to MAP were analyzed for dopamine (DA) content using high-performance liquid chromatography. The liquid filtration conditioning procedure employed here in combination with the use of 6-well test plates significantly decreased the time required to perform these experiments and ultimately increased throughput. The MAP conditioning data found that pairing an ion with MAP at 17 or 500 μM significantly increased the preference

Ultra-structural time-course study in the C. elegans model for Duchenne muscular dystrophy highlights a crucial role for sarcomere-anchoring structures and sarcolemma integrity in the earliest steps of the muscle degeneration process.

PubMed

Brouilly, Nicolas; Lecroisey, Claire; Martin, Edwige; Pierson, Laura; Mariol, Marie-Christine; Qadota, Hiroshi; Labouesse, Michel; Streichenberger, Nathalie; Mounier, Nicole; Gieseler, Kathrin

2015-11-15

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle degeneration due to mutations in the dystrophin gene. In spite of great advances in the design of curative treatments, most patients currently receive palliative therapies with steroid molecules such as prednisone or deflazacort thought to act through their immunosuppressive properties. These molecules only slightly slow down the progression of the disease and lead to severe side effects. Fundamental research is still needed to reveal the mechanisms involved in the disease that could be exploited as therapeutic targets. By studying a Caenorhabditis elegans model for DMD, we show here that dystrophin-dependent muscle degeneration is likely to be cell autonomous and affects the muscle cells the most involved in locomotion. We demonstrate that muscle degeneration is dependent on exercise and force production. Exhaustive studies by electron microscopy allowed establishing for the first time the chronology of subcellular events occurring during the entire process of muscle degeneration. This chronology highlighted the crucial role for dystrophin in stabilizing sarcomeric anchoring structures and the sarcolemma. Our results suggest that the disruption of sarcomeric anchoring structures and sarcolemma integrity, observed at the onset of the muscle degeneration process, triggers subcellular consequences that lead to muscle cell death. An ultra-structural analysis of muscle biopsies from DMD patients suggested that the chronology of subcellular events established in C. elegans models the pathogenesis in human. Finally, we found that the loss of sarcolemma integrity was greatly reduced after prednisone treatment suggesting a role for this molecule in plasma membrane stabilization. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

[Embryonic stem cells and therapeutic cloning].

PubMed

Sunde, A; Eftedal, I

2001-08-30

Increased interest in the therapeutic use of human stem cells has emerged following significant progress in ongoing research. The cloning of a sheep, the isolation of human embryonic stem cells, and the discovery that adult stem cells may be reprogrammed taken together give substance to hopes that novel principles of treatment may be developed for a variety of serious conditions. Embryonic stem cells are derived from pre-embryos at the blastocyst stage and may give rise to all bodily tissues and cells. Animal models have demonstrated that embryonic stem cells when transplanted into adult hosts may differentiate and develop into cells and tissues applicable for treatment of a variety of conditions, including Parkinson's disease, multiple sclerosis, spinal injuries, cardiac stroke and cancer. Transplanted embryonic stem cells are exposed to immune reactions similar to those acting on organ transplants, hence immunosuppression of the recipient is generally required. It is, however, possible to obtain embryonic stem cells that are genetically identical to the patient's own cells by means of therapeutic cloning techniques. The nucleus from a somatic cell is transferred into an egg after removal of the egg's own genetic material. Under specific condition the egg will use genetic information from the somatic cell in organising the formation of a blastocyst which in turn generates embryonic stem cells. These cells have a genetic composition identical to that of the patient and are suitable for stem cell therapy.

Lethal Dysregulation of Energy Metabolism During Embryonic Vitamin E Deficiency

PubMed Central

McDougall, Melissa; Choi, Jaewoo; Kim, Hye-Kyeong; Bobe, Gerd; Stevens, J. Frederik; Cadenas, Enrique; Tanguay, Robert; Traber, Maret G.

2017-01-01

Vitamin E (α-tocopherol, VitE) was discovered in 1922 for its role in preventing embryonic mortality. We investigated the underlying mechanisms causing lethality using targeted metabolomics analyses of zebrafish VitE-deficient embryos over five days of development, which coincided with their increased morbidity and mortality. VitE deficiency resulted in peroxidation of docosahexaenoic acid (DHA), depleting DHA-containing phospholipids, especially phosphatidylcholine, which also caused choline depletion. This increased lipid peroxidation also increased NADPH oxidation, which depleted glucose by shunting it to the pentose phosphate pathway. VitE deficiency was associated with mitochondrial dysfunction with concomitant impairment of energy homeostasis. The observed morbidity and mortality outcomes could be attenuated, but not fully reversed, by glucose injection into VitE-deficient embryos at developmental day one. Thus, embryonic VitE deficiency in vertebrates leads to a metabolic reprogramming that adversely affects methyl donor status and cellular energy homeostasis with lethal outcomes. PMID:28095320

Generating gene knockout rats by homologous recombination in embryonic stem cells

PubMed Central

Tong, Chang; Huang, Guanyi; Ashton, Charles; Li, Ping; Ying, Qi-Long

2013-01-01

We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats. PMID:21637202

A Mitochondria-Targeted Nitroxide/Hemigramicidin S Conjugate Protects Mouse Embryonic Cells Against Gamma Irradiation

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

Jiang Jianfei; Belikova, Natalia A.; Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA

2008-03-01

Purpose: To evaluate the in vitro radioprotective effect of the mitochondria-targeted hemigramicidin S-conjugated 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (hemi-GS-TEMPO) 5-125 in {gamma}-irradiated mouse embryonic cells and adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells BEAS-2B and explore the mechanisms involved in its radioprotective effect. Methods and Materials: Cells were incubated with 5-125 before (10 minutes) or after (1 hour) {gamma}-irradiation. Superoxide generation was determined by using dihydroethidium assay, and lipid oxidation was quantitated by using a fluorescence high-performance liquid chromatography-based Amplex Red assay. Apoptosis was characterized by evaluating the accumulation of cytochrome c in the cytosol and externalization of phosphatidylserine on the cellmore » surface. Cell survival was measured by means of a clonogenic assay. Results: Treatment (before and after irradiation) of cells with 5-125 at low concentrations (5, 10, and 20 {mu}M) effectively suppressed {gamma}-irradiation-induced superoxide generation, cardiolipin oxidation, and delayed irradiation-induced apoptosis, evaluated by using cytochrome c release and phosphatidylserine externalization. Importantly, treatment with 5-125 increased the clonogenic survival rate of {gamma}-irradiated cells. In addition, 5-125 enhanced and prolonged {gamma}-irradiation-induced G{sub 2}/M phase arrest. Conclusions: Radioprotection/mitigation by hemi-GS-TEMPO likely is caused by its ability to act as an electron scavenger and prevent superoxide generation, attenuate cardiolipin oxidation in mitochondria, and hence prevent the release of proapoptotic factors from mitochondria. Other mechanisms, including cell-cycle arrest at the G{sub 2}/M phase, may contribute to the protection.« less

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans.

PubMed

Martin, Emmanuel; Rocheleau-Leclair, Olivier; Jenna, Sarah

2016-03-28

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Brief Embryonic Strychnine Exposure in Zebrafish Causes Long-Term Adult Behavioral Impairment with Indications of Embryonic Synaptic Changes

PubMed Central

Roy, Nicole M.; Arpie, Brianna; Lugo, Joseph; Linney, Elwood; Levin, Edward D.; Cerutti, Daniel

2015-01-01

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5 mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior. PMID:23022260

Dual function of the UNC-45b chaperone with myosin and GATA4 in cardiac development

PubMed Central

Chen, Daisi; Li, Shumin; Singh, Ram; Spinette, Sarah; Sedlmeier, Reinhard; Epstein, Henry F.

2012-01-01

Summary Cardiac development requires interplay between the regulation of gene expression and the assembly of functional sarcomeric proteins. We report that UNC-45b recessive loss-of-function mutations in C3H and C57BL/6 inbred mouse strains cause arrest of cardiac morphogenesis at the formation of right heart structures and failure of contractile function. Wild-type C3H and C57BL/6 embryos at the same stage, E9.5, form actively contracting right and left atria and ventricles. The known interactions of UNC-45b as a molecular chaperone are consistent with diminished accumulation of the sarcomeric myosins, but not their mRNAs, and the resulting decreased contraction of homozygous mutant embryonic hearts. The novel finding that GATA4 accumulation is similarly decreased at the protein but not mRNA levels is also consistent with the function of UNC-45b as a chaperone. The mRNAs of known downstream targets of GATA4 during secondary cardiac field development, the cardiogenic factors Hand1, Hand2 and Nkx-2.5, are also decreased, consistent with the reduced GATA4 protein accumulation. Direct binding studies show that the UNC-45b chaperone forms physical complexes with both the alpha and beta cardiac myosins and the cardiogenic transcription factor GATA4. Co-expression of UNC-45b with GATA4 led to enhanced transcription from GATA promoters in naïve cells. These novel results suggest that the heart-specific UNC-45b isoform functions as a molecular chaperone mediating contractile function of the sarcomere and gene expression in cardiac development. PMID:22553207

Induction of murine embryonic stem cell differentiation by medicinal plant extracts

PubMed Central

Reynertson, Kurt A.; Charlson, Mary E.; Gudas, Lorraine J.

2010-01-01

Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly-cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from twelve species of ethnomedically utilized plants, we found fractions from three species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. PMID:20955699

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

Ectopic expression of Cripto-1 in transgenic mouse embryos causes hemorrhages, fatal cardiac defects and embryonic lethality

PubMed Central

Lin, Xiaolin; Zhao, Wentao; Jia, Junshuang; Lin, Taoyan; Xiao, Gaofang; Wang, Shengchun; Lin, Xia; Liu, Yu; Chen, Li; Qin, Yujuan; Li, Jing; Zhang, Tingting; Hao, Weichao; Chen, Bangzhu; Xie, Raoying; Cheng, Yushuang; Xu, Kang; Yao, Kaitai; Huang, Wenhua; Xiao, Dong; Sun, Yan

2016-01-01

Targeted disruption of Cripto-1 in mice caused embryonic lethality at E7.5, whereas we unexpectedly found that ectopic Cripto-1 expression in mouse embryos also led to embryonic lethality, which prompted us to characterize the causes and mechanisms underlying embryonic death due to ectopic Cripto-1 expression. RCLG/EIIa-Cre embryos displayed complex phenotypes between embryonic day 14.5 (E14.5) and E17.5, including fatal hemorrhages (E14.5-E15.5), embryo resorption (E14.5-E17.5), pale body surface (E14.5-E16.5) and no abnormal appearance (E14.5-E16.5). Macroscopic and histological examination revealed that ectopic expression of Cripto-1 transgene in RCLG/EIIa-Cre embryos resulted in lethal cardiac defects, as evidenced by cardiac malformations, myocardial thinning, failed assembly of striated myofibrils and lack of heartbeat. In addition, Cripto-1 transgene activation beginning after E8.5 also caused the aforementioned lethal cardiac defects in mouse embryos. Furthermore, ectopic Cripto-1 expression in embryonic hearts reduced the expression of cardiac transcription factors, which is at least partially responsible for the aforementioned lethal cardiac defects. Our results suggest that hemorrhages and cardiac abnormalities are two important lethal factors in Cripto-1 transgenic mice. Taken together, these findings are the first to demonstrate that sustained Cripto-1 transgene expression after E11.5 causes fatal hemorrhages and lethal cardiac defects, leading to embryonic death at E14.5-17.5. PMID:27687577

Diverse Neurotoxicants Target the Differentiation of Embryonic Neural Stem Cells into Neuronal and Glial Phenotypes

PubMed Central

Slotkin, Theodore A.; Skavicus, Samantha; Card, Jennifer; Levin, Edward D.; Seidler, Frederic J.

2016-01-01

The large number of compounds that need to be tested for developmental neurotoxicity drives the need to establish in vitro models to evaluate specific neurotoxic endpoints. We used neural stem cells derived from rat neuroepithelium on embryonic day 14 to evaluate the impact of diverse toxicants on their ability to differentiate into glia and neurons: a glucocorticoid (dexamethasone), organophosphate insecticides (chlorpyrifos, diazinon, parathion), insecticides targeting the GABAA receptor (dieldrin, fipronil), heavy metals (Ni2+, Ag+), nicotine and tobacco smoke extract. We found three broad groupings of effects. One diverse set of compounds, dexamethasone, the organophosphate pesticides, Ni2+ and nicotine, suppressed expression of the glial phenotype while having little or no effect on the neuronal phenotype. The second pattern was restricted to the pesticides acting on GABAA receptors. These compounds promoted the glial phenotype and suppressed the neuronal phenotype. Notably, the actions of compounds eliciting either of these differentiation patterns were clearly unrelated to deficits in cell numbers: dexamethasone, dieldrin and fipronil all reduced cell numbers, whereas organophosphates and Ni2+ had no effect. The third pattern, shared by Ag+ and tobacco smoke extract, clearly delineated cytotoxicity, characterized major cell loss with suppression of differentiation into both glial and neuronal phenotypes; but here again, there was some selectivity in that glia were suppressed more than neurons. Our results, from this survey with diverse compounds, point to convergence of neurotoxicant effects on a specific “decision node” that controls the emergence of neurons and glia from neural stem cells. PMID:27816694

Brief embryonic strychnine exposure in zebrafish causes long-term adult behavioral impairment with indications of embryonic synaptic changes.

PubMed

Roy, Nicole M; Arpie, Brianna; Lugo, Joseph; Linney, Elwood; Levin, Edward D; Cerutti, Daniel

2012-01-01

Zebrafish provide a powerful model of the impacts of embryonic toxicant exposure on neural development that may result in long-term behavioral dysfunction. In this study, zebrafish embryos were treated with 1.5mM strychnine for short embryonic time windows to induce transient changes in inhibitory neural signaling, and were subsequently raised in untreated water until adulthood. PCR analysis showed indications that strychnine exposure altered expression of some genes related to glycinergic, GABAergic and glutamatergic neuronal synapses during embryonic development. In adulthood, treated fish showed significant changes in swimming speed and tank diving behavior compared to controls. Taken together, these data show that a short embryonic exposure to a neurotoxicant can alter development of neural synapses and lead to changes in adult behavior. Copyright © 2012 Elsevier Inc. All rights reserved.

Influences of Reduced Expression of Maternal Bone Morphogenetic Protein 2 on Embryonic Development

PubMed Central

Singh, Ajeet P.; Castranio, Trisha; Scott, Greg; Guo, Dayong; Harris, Marie A.; Ray, Manas; Harris, Stephan E.; Mishina, Yuji

2009-01-01

Bone morphogenetic protein 2 (BMP2) was originally found by its osteoinductive ability, and recent genetic analyses have revealed that it plays critical roles during early embryogenesis, cardiogenesis, decidualization as well as skeletogenesis. During a course of evaluation of the conditional allele for Bmp2, we found that the presence of a neo cassette, a selection marker needed for gene targeting events in embryonic stem cells, in the 3’ untranslated region of exon 3 of Bmp2, reduced the expression levels of Bmp2 both in embryonic and maternal tissues. Some of the embryos that were genotyped as transheterozygous for the floxed allele with the neo cassette over the conventional null allele (fn/−) showed a lethal phenotype including defects in cephalic neural tube closure and ventral abdominal wall closure. Embryos exhibiting these abnormalities were increased when genotypes of the pregnant females were different; when expression levels of Bmp2 in maternal tissues were lower, a larger proportion of fn/− embryos exhibit these abnormalities. These results suggest that the expression levels of Bmp2 together in both in embryonic and maternal tissues influence the normal neural tube closure and body wall closure with different thresholds. PMID:18769073

TORC2 signaling antagonizes SKN-1 to induce C. elegans mesendodermal embryonic development

PubMed Central

Ruf, Vanessa; Holzem, Christina; Peyman, Tobias; Walz, Gerd; Blackwell, T. Keith; Neumann-Haefelin, Elke

2013-01-01

The evolutionarily conserved target of rapamycin (TOR) kinase controls fundamental metabolic processes to support cell and tissue growth. TOR functions within the context of two distinct complexes, TORC1 and TORC2. TORC2, with its specific component Rictor, has been recently implicated in aging and regulation of growth and metabolism. Here, we identify rict-1/Rictor as a regulator of embryonic development in C. elegans. The transcription factor skn-1 establishes development of the mesendoderm in embryos, and is required for cellular homeostasis and longevity in adults. Loss of maternal skn-1 function leads to misspecification of the mesendodermal precursor and failure to form intestine and pharynx. We found that genetic inactivation of rict-1 suppressed skn-1-associated lethality by restoring mesendodermal specification in skn-1 deficient embryos. Inactivation of other TORC2 but not TORC1 components also partially rescued skn-1 embryonic lethality. The SGK-1 kinase mediated these functions downstream of rict-1/TORC2, as a sgk-1 gain-of-function mutant suppressed the rict-1 mutant phenotype. These data indicate that TORC2 and SGK-1 antagonize SKN-1 during embryonic development. PMID:23973804

High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice

PubMed Central

Slevin, John C; Byers, Lois; Gertsenstein, Marina; Qu, Dawei; Mu, Junwu; Sunn, Nana; Kingdom, John CP; Rossant, Janet; Adamson, S Lee

2006-01-01

Background In utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5–E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure. Results In 40 MHz ultrasound images at E6.5, the ectoplacental cone region and proamniotic cavity could be visualized. The ectoplacental cone region was successfully targeted with 13.8 nL of a fluorescent bead suspension with few or no beads off-target in 51% of concepti microinjected at E6.5 (28/55 injected). Seventy eight percent of the embryos survived 2 to 12 days post injection (93/119), 73% (41/56) survived to term of which 68% (38/56) survived and appeared normal one week after birth. At E7.5, the amniotic and exocoelomic cavities, and ectoplacental cone region were discernable. Our success at targeting with few or no beads off-target was 90% (36/40) for the ectoplacental cone region and 81% (35/43) for the exocoelomic cavity but tended to be less, 68% (34/50), for the smaller amniotic cavity. At E11.5, beads microinjected at E7.5 into the ectoplacental cone region were found in the placental spongiotrophoblast layer, those injected into the exocoelomic cavity were found on the surface or within the placental labyrinth, and those injected into the amniotic cavity were found on the surface or within the embryo. Following microinjection at E7.5, survival one week after birth was 60% (26/43) when the amniotic cavity was the target and 66% (19/29) when the target was the ectoplacental cone region. The survival rate was

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

PubMed

Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

2016-07-07

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

Developmental staging of male murine embryonic gonad by SAGE analysis

PubMed Central

Lee, Tin-Lap; Li, Yunmin; Alba, Diana; Vong, Queenie P.; Wu, Shao-Ming; Baxendale, Vanessa; Rennert, Owen M.; Lau, Yun-Fai Chris; Chan, Wai-Yee

2012-01-01

Despite the identification of key genes such as Sry integral to embryonic gonadal development, the genomic classification and identification of chromosomal activation of this process is still poorly understood. To better understand the genetic regulation of gonadal development, we performed Serial Analysis of Gene Expression (SAGE) to profile the genes and novel transcripts, and an average of 152,000 tags from male embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5 and E17.5 were analyzed. A total of 275,583 non-singleton tags that do not map to any annotated sequence were identified in the six gonad libraries, and 47,255 tags were mapped to 24,975 annotated sequences, among which 987 sequences were uncharacterized. Utilizing an unsupervised pattern identification technique, we established molecular staging of male gonadal development. Rather than providing a static descriptive analysis, we developed algorithms to cluster the SAGE data and assign SAGE tags to a corresponding chromosomal position; these data are displayed in chromosome graphic format. A prominent increase in global genomic activity from E10.5 to E17.5 was observed. Important chromosomal regions related to the developmental processes were identified and validated based on established mouse models with developmental disorders. These regions may represent markers for early diagnosis for disorders of male gonad development as well as potential treatment targets. PMID:19376482

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish

NASA Astrophysics Data System (ADS)

Lee, Kerry J.; Browning, Lauren M.; Nallathamby, Prakash D.; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

2013-11-01

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 +/- 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c << 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target

Placenta Defects and Embryonic Lethality Resulting from Disruption of Mouse Hydroxysteroid (17-β) Dehydrogenase 2 Gene

PubMed Central

Rantakari, Pia; Strauss, Leena; Kiviranta, Riku; Lagerbohm, Heidi; Paviala, Jenni; Holopainen, Irma; Vainio, Seppo; Pakarinen, Pirjo; Poutanen, Matti

2008-01-01

Hydroxysteroid (17-β) dehydrogenase 2 (HSD17B2) is a member of aldo-keto reductase superfamily, known to catalyze the inactivation of 17β-hydroxysteroids to less active 17-keto forms and catalyze the conversion of 20α-hydroxyprogesterone to progesterone in vitro. To examine the role of HSD17B2 in vivo, we generated mice deficient in Hsd17b2 [HSD17B2 knockout (KO)] by a targeted gene disruption in embryonic stem cells. From the homozygous mice carrying the disrupted Hsd17b2, 70% showed embryonic lethality appearing at the age of embryonic d 11.5 onward. The embryonic lethality was associated with reduced placental size measured at embryonic d 17.5. The HSD17B2KO mice placentas presented with structural abnormalities in all three major layers: the decidua, spongiotrophoblast, and labyrinth. Most notable was the disruption of the spongiotrophoblast and labyrinthine layers, together with liquid-filled cysts in the junctional region and the basal layer. Treatments with an antiestrogen or progesterone did not rescue the embryonic lethality or the placenta defect in the homozygous mice. In hybrid background used, 24% of HSD17B2KO mice survived through the fetal period but were born growth retarded and displayed a phenotype in the brain with enlargement of ventricles, abnormal laminar organization, and increased cellular density in the cortex. Furthermore, the HSD17B2KO mice had unilateral renal degeneration, the affected kidney frequently appearing as a fluid-filled sac. Our results provide evidence for a role for HSD17B2 enzyme in the cellular organization of the mouse placenta. PMID:18048640

Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

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

Peng, Yin; Zhao, Shaomin; School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069

2013-11-29

Highlights: •SERTAD1 interacts with SMAD1. •Sertad1 is expressed in mouse embryonic hearts. •SERTAD1 is localized in both cytoplasm and nucleus of cardiomyocytes. •SERTAD1 enhances expression of BMP target cardiogenic genes as a SMAD1 co-activator. -- Abstract: Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, themore » activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.« less

Effects and possible mechanisms of simulated-microgravity on zebrafish embryonic cell

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Wang, Ruonan

2016-07-01

Cellular level studies are helpful for revealing the underlying mechanisms of microgravity effects on living organisms. Many cell types, ranging from bacteria to mammalian cells, are sensitive to the microgravity environment. In this study, zebrafish embryonic cells (ZF4) were exposed to simulated-microgravity (SMG) for different times to investigate the effects and possible mechanisms of microgravity on fibroblasts. A significant arrest in G2/M phase was detected in ZF4 cells after 24 or 48 hour of SMG exposure, respectively. The mRNA levels of G2/M phase regulators cyclinB1 and cdc2 were significantly decreased, while wee1 was significantly increased. Additionally, CEP135, a core centrosome protein throughout the cell cycle, seems to play a key role in modulating this effect. Quantitative analysis showed that cep135 expression was significantly increased, while CEP135 protein expression level was significantly decreased two times after SMG. Further investigation demonstrated the transfection of dre-miR-22a, a miRNA for targeting cep135, also induced G2/M arrest in ZF4 cells. These results suggest that SMG induced G2/M arrest in ZF4 cells may due to the regulation of dre-miR-22a and its target cep135. Key Words: Simulated-microgravity; zebrafish embryonic cell; G2/M arrest; molecular mechanism

Induction of murine embryonic stem cell differentiation by medicinal plant extracts

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

Reynertson, Kurt A.; Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065; Charlson, Mary E.

Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extractsmore » for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells.« less

Induction of murine embryonic stem cell differentiation by medicinal plant extracts.

PubMed

Reynertson, Kurt A; Charlson, Mary E; Gudas, Lorraine J

2011-01-01

Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

PubMed Central

Inoue, Kimiko; Ogura, Atsuo

2013-01-01

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT. PMID:24146866

Gene expression dynamics during embryonic development in rainbow trout

USDA-ARS?s Scientific Manuscript database

The supply of maternal RNAs in fertilized egg and activation of embryonic genome during maternal-zygotic transition (MZT) are important for normal embryonic development. In order to identify genes and gene products that are essential in the regulation of embryonic development in rainbow trout, RNA-S...

“Addition” and “Subtraction”: Selectivity Design for Type II Maternal Embryonic Leucine Zipper Kinase Inhibitors

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

Chen, Xin; Giraldes, John; Sprague, Elizabeth R.

2017-02-17

While adding the structural features that are more favored by on-target activity is the more common strategy in selectivity optimization, the opposite strategy of subtracting the structural features that contribute more to off-target activity can also be very effective. Reported here is our successful effort of improving the kinase selectivity of type II maternal embryonic leucine zipper kinase inhibitors by applying these two complementary approaches together, which clearly demonstrates the powerful synergy between them.

Heart failure-associated changes in RNA splicing of sarcomere genes.

PubMed

Kong, Sek Won; Hu, Yong Wu; Ho, Joshua W K; Ikeda, Sadakatsu; Polster, Sean; John, Ranjit; Hall, Jennifer L; Bisping, Egbert; Pieske, Burkert; dos Remedios, Cristobal G; Pu, William T

2010-04-01

Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared with control hearts. We used the Affymetrix Exon array to globally evaluate mRNA splicing in left ventricular myocardial RNA from controls (n=15) and patients with ischemic cardiomyopathy (n=15). We observed a broad and significant decrease in mRNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ischemic cardiomyopathy and control samples, suggesting distinct changes in mRNA splicing between groups. Reverse transcription-polymerase chain reaction validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of 4 key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C, gamma (FLNC), was significantly altered in ischemic cardiomyopathy and in dilated cardiomyopathy and aortic stenosis. In aortic stenosis samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with >98% accuracy. Our data indicate that mRNA splicing is broadly altered in human heart disease and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes.

Scaffolding for Three-Dimensional Embryonic Vasculogenesis

NASA Astrophysics Data System (ADS)

Kraehenbuehl, Thomas P.; Aday, Sezin; Ferreira, Lino S.

Biomaterial scaffolds have great potential to support efficient vascular differentiation of embryonic stem cells. Vascular cell fate-specific biochemical and biophysical cues have been identified and incorporated into three-dimensional (3D) biomaterials to efficiently direct embryonic vasculogenesis. The resulting vascular-like tissue can be used for regenerative medicine applications, further elucidation of biophysical and biochemical cues governing vasculogenesis, and drug discovery. In this chapter, we give an overview on the following: (1) developmental cues for directed differentiation of human embryonic stem cells (hESCs) into vascular cells, (2) 3D vascular differentiation in embryoid bodies (EBs), (3) preparation of 3D scaffolds for the vascular differentiation of hESCs, and (4) the most significant studies combining scaffolding and hESCs for development of vascular-like tissue.

Growth enhancement by embryonic fibroblasts upon cotransplantation of noncommitted pig embryonic tissues with fully committed organs.

PubMed

Cohen, Sivan; Tchorsh-Yutsis, Dalit; Aronovich, Anna; Tal, Orna; Eventov-Friedman, Smadar; Katchman, Helena; Klionsky, Yael; Shezen, Elias; Reisner, Yair

2010-05-27

We recently defined the optimal gestational time windows for the transplantation of several embryonic tissues. We showed that the liver and kidney obtained from E28 pig embryos can grow and differentiate normally after transplantation, whereas 1 week earlier in gestation, these tissues develop into teratoma-like structures or fibrotic mass. In this study, we investigated whether cotransplantation of E28 with E21 tissue could control its tumorogenic potential, or alternatively whether the stem cells derived from the earlier tissue contribute to the growth of the more committed one. Pig embryonic precursors from E21 and E28 gestational age were transplanted alone or together, into nonobese diabetic/severe combined immunodeficiency mice, and their growth and differentiation was evaluated by immunohistology. In situ analysis, based on sex disparity between the E21 and E28 tissues, was used to identify the tissue source. In some experiments, mouse embryonic fibroblasts (MEF) were cotransplanted with E28 liver, and their effect was evaluated. E28 tissues could not abrogate the propensity of the cells within the undifferentiated tissue to form teratoma-like structures. However, E21 kidney or liver tissue markedly enhanced the growth and function of E28 kidney, liver, and heart grafts. Moreover, similar growth enhancement was observed on coimplantation of E28 liver tissue with MEF or on infusion of MEF culture medium, indicating that this enhancement is likely mediated through soluble factors secreted by the fibroblasts. Our results suggest a novel approach for the enhancement of growth and differentiation of transplanted embryonic tissues by the use of soluble factors secreted by embryonic fibroblasts.

Murine mesenchymal and embryonic stem cells express a similar Hox gene profile.

PubMed

Phinney, Donald G; Gray, Andrew J; Hill, Katy; Pandey, Amitabh

2005-12-30

Using degenerate oligonucleotide primers targeting the homeobox domain, we amplified by PCR and sequenced 723 clones from five murine cell populations and lines derived from embryonic mesoderm and adult bone marrow. Transcripts from all four vertebrate Hox clusters were expressed by the different populations. Hierarchical clustering of the data revealed that mesenchymal stem cells (MSCs) and the embryonic stem (ES) cell line D3 shared a similar Hox expression profile. These populations exclusively expressed Hoxb2, Hoxb5, Hoxb7, and Hoxc4, transcripts regulating self-renewal and differentiation of other stem cells. Additionally, Hoxa7 transcript quantified by real-time PCR strongly correlated (r2=0.89) with the number of Hoxa7 clones identified by sequencing, validating that data from the PCR screen reflects differences in Hox mRNA abundance between populations. This is the first study to catalogue Hox transcripts in murine MSCs and by comparative analyses identify specific Hox genes that may contribute to their stem cell character.

Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice.

PubMed

Nakagawa, Kimie; Sawada, Natsumi; Hirota, Yoshihisa; Uchino, Yuri; Suhara, Yoshitomo; Hasegawa, Tomoka; Amizuka, Norio; Okamoto, Tadashi; Tsugawa, Naoko; Kamao, Maya; Funahashi, Nobuaki; Okano, Toshio

2014-01-01

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

The ‘Ventral Organs’ of Pycnogonida (Arthropoda) Are Neurogenic Niches of Late Embryonic and Post-Embryonic Nervous System Development

PubMed Central

Brenneis, Georg; Scholtz, Gerhard

2014-01-01

Early neurogenesis in arthropods has been in the focus of numerous studies, its cellular basis, spatio-temporal dynamics and underlying genetic network being by now comparably well characterized for representatives of chelicerates, myriapods, hexapods and crustaceans. By contrast, neurogenesis during late embryonic and/or post-embryonic development has received less attention, especially in myriapods and chelicerates. Here, we apply (i) immunolabeling, (ii) histology and (iii) scanning electron microscopy to study post-embryonic ventral nerve cord development in Pseudopallene sp., a representative of the sea spiders (Pycnogonida), the presumable sister group of the remaining chelicerates. During early post-embryonic development, large neural stem cells give rise to additional ganglion cell material in segmentally paired invaginations in the ventral ectoderm. These ectodermal cell regions – traditionally designated as ‘ventral organs’ – detach from the surface into the interior and persist as apical cell clusters on the ventral ganglion side. Each cluster is a post-embryonic neurogenic niche that features a tiny central cavity and initially still houses larger neural stem cells. The cluster stays connected to the underlying ganglionic somata cortex via an anterior and a posterior cell stream. Cell proliferation remains restricted to the cluster and streams, and migration of newly produced cells along the streams seems to account for increasing ganglion cell numbers in the cortex. The pycnogonid cluster-stream-systems show striking similarities to the life-long neurogenic system of decapod crustaceans, and due to their close vicinity to glomerulus-like neuropils, we consider their possible involvement in post-embryonic (perhaps even adult) replenishment of olfactory neurons – as in decapods. An instance of a potentially similar post-embryonic/adult neurogenic system in the arthropod outgroup Onychophora is discussed. Additionally, we document two transient

[Low expression of activin A in mouse and human embryonic teratocarcinoma cells].

PubMed

Gordeeva, O F

2014-01-01

TGFP3 family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFbeta family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGF@[beta] family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty 1, TGFbeta1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFbeta family (ACTIVINA, NODAL, LEFTY 1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells: Thus, in mouse and human nullipotent teratocarcinoma cells, theexpression of ActivinA is significantly reduced com- pared ivith embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.

Maternal Lifestyle Impairs Embryonic Growth: The Rotterdam Periconception Cohort.

PubMed

Van Dijk, Matthijs R; Borggreven, Nicole V; Willemsen, Sten P; Koning, Anton H J; Steegers-Theunissen, Régine P M; Koster, Maria P H

2018-06-01

Previously, embryonic growth has been assumed to be uniform, but in recent years, it has become more clear that genetic and environmental factors may influence the intrauterine environment and therefore embryonic growth trajectories as well as pregnancy course and outcome. The objective of this study was to investigate associations between modifiable maternal nutrition and lifestyle factors during the periconception period and embryonic growth. We established a prospective cohort including 342 women less than 13 weeks pregnant. At enrollment, women filled out a questionnaire regarding demographic and medical data and a validated food frequency questionnaire. Participants received multiple 3-dimensional ultrasound examinations up until the 12th week of pregnancy, and crown-rump length (CRL) and embryonic volume (EV) were measured offline using V-Scope Virtual Reality software (version 1.0.0) in a Barco I-Space. Associations between maternal periconception vegetable and fruit intake, folic acid supplement use, smoking, and alcohol consumption and embryonic growth measurements were assessed by linear mixed models adjusted for potential confounders. No or postconception initiation of folic acid supplement use was significantly associated with a 0.76 mm (-7.8%) and 1.63 mm (-3.7%) smaller CRL and a 0.01 cm 3 (-19.5%) and 0.86 cm 3 (-12.2%) smaller EV at 7 +0 and 11 +0 weeks of gestation, respectively. Smoking, alcohol consumption, and inadequate fruit and vegetable intake showed weaker associations with embryonic growth parameters. These results emphasize the influence of periconceptional maternal folic acid supplement use on embryonic growth. Results regarding maternal nutrition and lifestyle factors also suggest an association with embryonic growth, but this has to be confirmed in a larger study.

Simulated-microgravity induced G2/M arrest in zebrafish embryonic cell is regulated by dre-miR-22a and its target cep135

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Wang, Ruonan

2016-07-01

Microgravity has been recognized as a major environmental factor that can induce a number of adverse effects such as bone loss, skeletal muscle atrophy, cardiovascular problems and immune system dysregulation, etc. The underlying mechanisms are not absolutely identified yet. Our previous study demonstrated centrosomal protein of 135 kDa (CEP135) might be a microgravity sensitive molecule. In this study, the expression and regulation of CEP135 and its possible roles in cell cycle regulation under simulated microgravity (SMG) condition were investigated. SMG can induce significant increasing of cep135 in zebrafish embryos, detected by both in situ hybridization and RT-qPCR, while CEP135 protein level was significantly decreased, tested by western blot. The similar results were also obtained in zebrafish embryonic cells (ZF4) exposed to SMG. Accordingly, the expression level of dre-miR-22a, which might be the potential miRNA for targeting cep135, was significantly increased in SMG exposed ZF4 cells. By combining the results obtained from transfection and dual luciferase reporter assay, we firstly confirmed that dre-miR-22a regulated the expression of cep135 in ZF4 cells. Further investigation on cell cycle demonstrated SMG induced a significant arrest in G2/M phase. Transfection of dre-miR-22a also induced G2/M arrest in ZF4 cells. These results suggest that SMG induced G2/M arrest in ZF4 cells is via cep135, while dre-miR-22a plays a key role in modulating this effect. Key Words: Simulated-microgravity; cep135; dre-miR-22a; G2/M arrest; zebrafish embryonic cell

How the embryonic chick brain twists.

PubMed

Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A

2016-11-01

During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left-right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic morphology and mechanics analysis that the vitelline membrane (VM) exerts an external load on the brain that drives torsion. Our theoretical analysis showed that the force is of the order of 10 micronewtons. We also designed an experiment to use fluid surface tension to replace the mechanical role of the VM, and the estimated magnitude of the force owing to surface tension was shown to be consistent with the above theoretical analysis. We further discovered that the asymmetry of the looping heart determines the chirality of the twisted brain via physical mechanisms, demonstrating the mechanical transfer of left-right asymmetry between organs. Our experiments also implied that brain flexure is a necessary condition for torsion. Our work clarifies the mechanical origin of torsion and the development of left-right asymmetry in the early embryonic brain. © 2016 The Author(s).

Fine-tuning of chromatin composition and Polycomb recruitment by two Mi2 homologues during C. elegans early embryonic development.

PubMed

Käser-Pébernard, Stéphanie; Pfefferli, Catherine; Aschinger, Caroline; Wicky, Chantal

2016-01-01

The nucleosome remodeling and deacetylase complex promotes cell fate decisions throughout embryonic development. Its core enzymatic subunit, the SNF2-like ATPase and Helicase Mi2, is well conserved throughout the eukaryotic kingdom and can be found in multiple and highly homologous copies in all vertebrates and some invertebrates. However, the reasons for such duplications and their implications for embryonic development are unknown. Here we studied the two C. elegans Mi2 homologues, LET-418 and CHD-3, which displayed redundant activities during early embryonic development. At the transcriptional level, these two Mi2 homologues redundantly repressed the expression of a large gene population. We found that LET-418 physically accumulated at TSS-proximal regions on transcriptionally active genomic targets involved in growth and development. Moreover, LET-418 acted redundantly with CHD-3 to block H3K4me3 deposition at these genes. Our study also revealed that LET-418 was partially responsible for recruiting Polycomb to chromatin and for promoting H3K27me3 deposition. Surprisingly, CHD-3 displayed opposite activities on Polycomb, as it was capable of moderating its LET-418-dependent recruitment and restricted the amount of H3K27me3 on the studied target genes. Although closely homologous, LET-418 and CHD-3 showed both redundant and opposite functions in modulating the chromatin environment at developmental target genes. We identified the interplay between LET-418 and CHD-3 to finely tune the levels of histone marks at developmental target genes. More than just repressors, Mi2-containing complexes appear as subtle modulators of gene expression throughout development. The study of such molecular variations in vertebrate Mi2 counterparts might provide crucial insights to our understanding of the epigenetic control of early development.

Dual effects of fluoxetine on mouse early embryonic development.

PubMed

Kim, Chang-Woon; Choe, Changyong; Kim, Eun-Jin; Lee, Jae-Ik; Yoon, Sook-Young; Cho, Young-Woo; Han, Sunkyu; Tak, Hyun-Min; Han, Jaehee; Kang, Dawon

2012-11-15

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50μM) for different durations. When late 2-cells were incubated with 5μM fluoxetine for 6h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5μM) over 24h showed a reduction in blastocyst formation. The addition of fluoxetine (5μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K(+) channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ~30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Copyright © 2012 Elsevier Inc. All rights reserved.

Targeting autophagy to modulate cell survival: a comparative analysis in cancer, normal and embryonic cells.

PubMed

Divac Rankov, Aleksandra; Ljujić, Mila; Petrić, Marija; Radojković, Dragica; Pešić, Milica; Dinić, Jelena

2017-11-01

Autophagy is linked to multiple cancer-related signaling pathways, and represents a defense mechanism for cancer cells under therapeutic stress. The crosstalk between apoptosis and autophagy is essential for both tumorigenesis and embryonic development. We studied the influence of autophagy on cell survival in pro-apoptotic conditions induced by anticancer drugs in three model systems: human cancer cells (NCI-H460, COR-L23 and U87), human normal cells (HaCaT and MRC-5) and zebrafish embryos (Danio rerio). Autophagy induction with AZD2014 and tamoxifen antagonized the pro-apoptotic effect of chemotherapeutics doxorubicin and cisplatin in cell lines, while autophagy inhibition by wortmannin and chloroquine synergized the action of both anticancer agents. This effect was further verified by assessing cleaved caspase-3 and PARP-1 levels. Autophagy inhibitors significantly increased both apoptotic markers when applied in combination with doxorubicin while autophagy inducers had the opposite effect. In a similar manner, autophagy induction in zebrafish embryos prevented cisplatin-induced apoptosis in the tail region while autophagy inhibition increased cell death in the tail and retina of cisplatin-treated animals. Autophagy modulation with direct inhibitors of the PI3kinase/Akt/mTOR pathway (AZD2014 and wortmannin) triggered the cellular response to anticancer drugs more effectively in NCI-H460 and zebrafish embryonic models compared to HaCaT suggesting that these modulators are selective towards rapidly proliferating cells. Therefore, evaluating the autophagic properties of chemotherapeutics could help determine more accurately the fate of different cell types under treatment. Our study underlines the importance of testing autophagic activity of potential anticancer agents in a comparative approach to develop more rational anticancer therapeutic strategies.

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells.

PubMed

Shi, Cheng; Shen, Huan; Jiang, Wei; Song, Zhi-Hua; Wang, Cheng-Yan; Wei, Li-Hui

2011-04-01

Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability. Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propagate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells. We generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line. This newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo

Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

PubMed

Huang, Boxian; Ning, Song; Zhuang, Lili; Jiang, Chunyan; Cui, Yugui; Fan, Guoping; Qin, Lianju; Liu, Jiayin

2015-01-01

Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

Embryonic stem cells: testing the germ-cell theory.

PubMed

Hochedlinger, Konrad

2011-10-25

The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state. Copyright © 2011 Elsevier Ltd. All rights reserved.

Embryonic integument and "molts" in Manduca sexta (Insecta, Lepidoptera).

PubMed

Ziese, Stefanie; Dorn, August

2003-02-01

In Manduca sexta the germ band is formed 12 h post-oviposition (p.o.) (=10% development completed) and is located above the yolk at the egg surface. The cells show a polar organization. They are engaged in the uptake and degradation of yolk globules, pinched off from the yolk cells. This process can be observed in the integumental cells during the first growth phase of the embryo that lasts until "katatrepsis," an embryonic movement that takes place at 40% development completed. At 37% development completed, the ectoderm deposits a thin membrane at its apical surface, the first embryonic membrane, which detaches immediately before katatrepsis. The second period of embryonic growth--from katatrepsis to 84 h p.o. (70% development completed)--starts with the deposition of a second embryonic membrane that is somewhat thicker than the first one and shows a trilaminar, cuticulin-like structure. Whereas the apical cell surface is largely smooth during the deposition of the first embryonic membrane, it forms microvilli during deposition of the second one. At the same time, uptake of formed yolk material ceases and the epidermal cells now contain clusters of mitochondria below the apical surface. Rough endoplasmic reticulum (RER) increases in the perinuclear region. The second embryonic membrane detaches about 63 h p.o. At 69 h p.o., a new generation of microvilli forms and islands of a typical cuticulin layer indicate the onset of the deposition of the larval cuticle. The third growth phase is characterized by a steady increase in the embryo length, the deposition of the larval procuticle, and by cuticular tanning at about 100 h p.o. Beginning at that stage, electron-lucent vesicles aggregate below the epidermal surface and are apparently released below the larval cuticle. Manduca sexta is the first holometabolous insect in which the deposition of embryonic membranes and cuticles has been examined by electron microscopy. In correspondence with hemimetabolous insects, the

Autophagy in Human Embryonic Stem Cells

PubMed Central

Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J.; Wolvetang, Ernst; Prescott, Mark

2011-01-01

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC. PMID:22110659

The primary role of zebrafish nanog is in extra-embryonic tissue.

PubMed

Gagnon, James A; Obbad, Kamal; Schier, Alexander F

2018-01-09

The role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the proper formation of the extra-embryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. In an alternative scenario, Nanog has been proposed to directly regulate transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafish nanog mutants. Whereas zygotic nanog mutants survive to adulthood, maternal-zygotic (MZ nanog ) and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, YSL formation and epiboly are abnormal, embryonic tissue detaches from the yolk, and the expression of dozens of YSL and embryonic genes is reduced. Epiboly defects can be rescued by generating chimeric embryos of MZ nanog embryonic tissue with wild-type vegetal tissue that includes the YSL and yolk cell. Notably, cells lacking Nanog readily respond to Nodal signals and when transplanted into wild-type hosts proliferate and contribute to embryonic tissues and adult organs from all germ layers. These results indicate that zebrafish Nanog is necessary for proper YSL development but is not directly required for embryonic cell differentiation. © 2018. Published by The Company of Biologists Ltd.

Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

PubMed

Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

2017-10-01

Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Monosaccharide uptake by erythrocytes of the embryonic and adult chicken.

PubMed

Ingermann, R L; Stock, M K; Metcalfe, J; Bissonnette, J M

1985-01-01

Rates of monosaccharide uptake by adult and 10-18 day old embryonic chicken erythrocytes were quantitated. The rate of carrier-mediated, stereospecific transport decreased 28% from day 10 to day 14 of incubation and was unchanged thereafter. At no time, however, did the rate of carrier-mediated transport by embryonic erythrocytes differ significantly from that of the adult cells. The rate of transfer by simple diffusion was 3-5 fold faster in embryonic than in adult erythrocytes. Uptake by simple diffusion decreased slightly as the embryo developed. Chronic hyperoxic incubation (70% O2) had little influence on total monosaccharide uptake by embryonic erythrocytes.

Generation of embryos directly from embryonic stem cells by tetraploid embryo complementation reveals a role for GATA factors in organogenesis.

PubMed

Duncan, S A

2005-12-01

Gene targeting in ES (embryonic stem) cells has been used extensively to study the role of proteins during embryonic development. In the traditional procedure, this requires the generation of chimaeric mice by introducing ES cells into blastocysts and allowing them to develop to term. Once chimaeric mice are produced, they are bred into a recipient mouse strain to establish germline transmission of the allele of interest. Although this approach has been used very successfully, the breeding cycles involved are time consuming. In addition, genes that are essential for organogenesis often have roles in the formation of extra-embryonic tissues that are essential for early stages of post-implantation development. For example, mice lacking the GATA transcription factors, GATA4 or GATA6, arrest during gastrulation due to an essential role for these factors in differentiation of extra-embryonic endoderm. This lethality has frustrated the study of these factors during the development of organs such as the liver and heart. Extraembryonic defects can, however, be circumvented by generating clonal mouse embryos directly from ES cells by tetraploid complementation. Here, we describe the usefulness and efficacy of this approach using GATA factors as an example.

Transgenerational Epigenetic Programming of the Embryonic Testis Transcriptome

PubMed Central

Anway, Matthew D.; Rekow, Stephen S.; Skinner, Michael K.

2008-01-01

Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination appears to promote an epigenetic reprogramming of the male germ-line that is associated with transgenerational adult onset disease states. Transgenerational effects on the embryonic day 16 (E16) testis demonstrated reproducible changes in the testis transcriptome for multiple generations (F1-F3). The expression of 196 genes were found to be influenced, with the majority of gene expression being decreased or silenced. Dramatic changes in the gene expression of methyltransferases during gonadal sex determination were observed in the F1 and F2 vinclozolin generation (E16) embryonic testis, but the majority returned to control generation levels by the F3 generation. The most dramatic effects were on the germ-line associated Dnmt3A and Dnmt3L isoforms. Observations demonstrate that an embryonic exposure to vinclozolin appears to promote an epigenetic reprogramming of the male germ-line that correlates with transgenerational alterations in the testis transcriptome in subsequent generations. PMID:18042343

Targeted disruption of a novel gene contiguous to both glucocerebrodisidase (GC) and thrombospondin 3 (TSP3), results in an embryonic lethal phenotype in the mouse

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

Bornstein, P.; Shingu, T.; LaMarca, M.E.

1994-09-01

We have identified a new murine gene, termed gene X, that spans the 6 kb interval separating GC from TSP3. Mutations in GC result in Gaucher disease, the most common lysosomal storage disorder. Gene X and GC are transcribed convergently; their major polyadenylation sites are separated by only 431 bp. On the other hand, gene X and TSP3 are transcribed divergently and share a bidirectional promoter. The cDNA for gene X encodes a 317 amino acid protein, without either a signal sequence or N-linked glycosylation. Gene X is expressed ubiquitously in tissues of the young adult mouse, but no closemore » homologues have been found in the DNA or protein data bases. A targeted point mutation was introduced into the GC gene (Asn to Ser in exon 9) by homologous recombination in embryonic stem cells to establish a mouse model for a mild form of Gaucher disease. In the process, a PGK-neomycin gene cassette was inserted in the 3{prime} flanking region of GC as a selectable marker, in a sequence that was subsequently identified as exon 8 of gene X. Mice homozygous for the combined mutation die early in gestation. Since the amino acid mutation in humans is associated with milder type 1 Gaucher disease, we conclude that gene X is essential for embryonic development in mice. The locations of human and murine GC, gene X and TSP3 are similar, but the human genome includes a duplication that has produced GC and gene X pseudogenes. We are currently studying the possible functional interactions of GC, gene X and TSP3 in both mice and humans.« less

Cardiac troponin T is necessary for normal development in the embryonic chick heart.

PubMed

England, Jennifer; Pang, Kar Lai; Parnall, Matthew; Haig, Maria Isabel; Loughna, Siobhan

2016-09-01

The heart is the first functioning organ to develop during embryogenesis. The formation of the heart is a tightly regulated and complex process, and alterations to its development can result in congenital heart defects. Mutations in sarcomeric proteins, such as alpha myosin heavy chain and cardiac alpha actin, have now been associated with congenital heart defects in humans, often with atrial septal defects. However, cardiac troponin T (cTNT encoded by gene TNNT2) has not. Using gene-specific antisense oligonucleotides, we have investigated the role of cTNT in chick cardiogenesis. TNNT2 is expressed throughout heart development and in the postnatal heart. TNNT2-morpholino treatment resulted in abnormal atrial septal growth and a reduction in the number of trabeculae in the developing primitive ventricular chamber. External analysis revealed the development of diverticula from the ventricular myocardial wall which showed no evidence of fibrosis and still retained a myocardial phenotype. Sarcomeric assembly appeared normal in these treated hearts. In humans, congenital ventricular diverticulum is a rare condition, which has not yet been genetically associated. However, abnormal haemodynamics is known to cause structural defects in the heart. Further, structural defects, including atrial septal defects and congenital diverticula, have previously been associated with conduction anomalies. Therefore, to provide mechanistic insights into the effect that cTNT knockdown has on the developing heart, quantitative PCR was performed to determine the expression of the shear stress responsive gene NOS3 and the conduction gene TBX3. Both genes were differentially expressed compared to controls. Therefore, a reduction in cTNT in the developing heart results in abnormal atrial septal formation and aberrant ventricular morphogenesis. We hypothesize that alterations to the haemodynamics, indicated by differential NOS3 expression, causes these abnormalities in growth in cTNT knockdown

Imprinted expression in cystic embryoid bodies shows an embryonic and not an extra-embryonic pattern

PubMed Central

Kulinski, Tomasz M.; Casari, M. Rita T.; Guenzl, Philipp M.; Wenzel, Daniel; Andergassen, Daniel; Hladik, Anastasiya; Datlinger, Paul; Farlik, Matthias; Theussl, H. -Christian; Penninger, Josef M.; Knapp, Sylvia; Bock, Christoph; Barlow, Denise P.; Hudson, Quanah J.

2015-01-01

A large subset of mammalian imprinted genes show extra-embryonic lineage (EXEL) specific imprinted expression that is restricted to placental trophectoderm lineages and to visceral yolk sac endoderm (ysE). Isolated ysE provides a homogenous in vivo model of a mid-gestation extra-embryonic tissue to examine the mechanism of EXEL-specific imprinted gene silencing, but an in vitro model of ysE to facilitate more rapid and cost-effective experiments is not available. Reports indicate that ES cells differentiated into cystic embryoid bodies (EBs) contain ysE, so here we investigate if cystic EBs model ysE imprinted expression. The imprinted expression pattern of cystic EBs is shown to resemble fetal liver and not ysE. To investigate the reason for this we characterized the methylome and transcriptome of cystic EBs in comparison to fetal liver and ysE, by whole genome bisulphite sequencing and RNA-seq. Cystic EBs show a fetal liver pattern of global hypermethylation and low expression of repeats, while ysE shows global hypomethylation and high expression of IAPEz retroviral repeats, as reported for placenta. Transcriptome analysis confirmed that cystic EBs are more similar to fetal liver than ysE and express markers of early embryonic endoderm. Genome-wide analysis shows that ysE shares epigenetic and repeat expression features with placenta. Contrary to previous reports, we show that cystic EBs do not contain ysE, but are more similar to the embryonic endoderm of fetal liver. This explains why cystic EBs reproduce the imprinted expression seen in the embryo but not that seen in the ysE. PMID:25912690

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish.

PubMed

Lee, Kerry J; Browning, Lauren M; Nallathamby, Prakash D; Osgood, Christopher J; Xu, Xiao-Hong Nancy

2013-12-07

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 ± 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c < 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target

First trimester size charts of embryonic brain structures.

PubMed

Gijtenbeek, M; Bogers, H; Groenenberg, I A L; Exalto, N; Willemsen, S P; Steegers, E A P; Eilers, P H C; Steegers-Theunissen, R P M

2014-02-01

Can reliable size charts of human embryonic brain structures be created from three-dimensional ultrasound (3D-US) visualizations? Reliable size charts of human embryonic brain structures can be created from high-quality images. Previous studies on the visualization of both the cavities and the walls of the brain compartments were performed using 2D-US, 3D-US or invasive intrauterine sonography. However, the walls of the diencephalon, mesencephalon and telencephalon have not been measured non-invasively before. Last-decade improvements in transvaginal ultrasound techniques allow a better visualization and offer the tools to measure these human embryonic brain structures with precision. This study is embedded in a prospective periconceptional cohort study. A total of 141 pregnancies were included before the sixth week of gestation and were monitored until delivery to assess complications and adverse outcomes. For the analysis of embryonic growth, 596 3D-US scans encompassing the entire embryo were obtained from 106 singleton non-malformed live birth pregnancies between 7(+0) and 12(+6) weeks' gestational age (GA). Using 4D View (3D software) the measured embryonic brain structures comprised thickness of the diencephalon, mesencephalon and telencephalon, and the total diameter of the diencephalon and mesencephalon. Of 596 3D scans, 161 (27%) high-quality scans of 79 pregnancies were eligible for analysis. The reliability of all embryonic brain structure measurements, based on the intra-class correlation coefficients (ICCs) (all above 0.98), was excellent. Bland-Altman plots showed moderate agreement for measurements of the telencephalon, but for all other measurements the agreement was good. Size charts were constructed according to crown-rump length (CRL). The percentage of high-quality scans suitable for analysis of these brain structures was low (27%).  The size charts of human embryonic brain structures can be used to study normal and abnormal development of

Diverging functions of Scr between embryonic and post-embryonic development in a hemimetabolous insect, Oncopeltus fasciatus.

PubMed

Chesebro, John; Hrycaj, Steven; Mahfooz, Najmus; Popadić, Aleksandar

2009-05-01

Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development.

Diverging functions of Scr between embryonic and post-embryonic development in a hemimetabolous insect, Oncopeltus fasciatus

PubMed Central

Chesebro, John; Hrycaj, Steven; Mahfooz, Najmus; Popadić, Aleksandar

2009-01-01

Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development. PMID:19382295

Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells

PubMed Central

Ma, Yanni; Yao, Nan; Liu, Guang; Dong, Lei; Liu, Yufang; Zhang, Meili; Wang, Fang; Wang, Bin; Wei, Xueju; Dong, He; Wang, Lanlan; Ji, Shaowei; Zhang, Junwu; Wang, Yangming; Huang, Yue; Yu, Jia

2015-01-01

MicroRNAs play important roles in controlling the embryonic stem cell (ESC) state. Although much is known about microRNAs maintaining ESC state, microRNAs that are responsible for promoting ESC differentiation are less reported. Here, by screening 40 microRNAs pre-selected by their expression patterns and predicted targets in Dgcr8-null ESCs, we identify 14 novel differentiation-associated microRNAs. Among them, miR-27a and miR-24, restrained by c-Myc in ESC, exert their roles of silencing self-renewal through directly targeting several important pluripotency-associated factors, such as Oct4, Foxo1 and Smads. CRISPR/Cas9-mediated knockout of all miR-27/24 in ESCs leads to serious deficiency in ESC differentiation in vitro and in vivo. Moreover, depleting of them in mouse embryonic fibroblasts can evidently promote somatic cell reprogramming. Altogether, our findings uncover the essential role of miR-27 and miR-24 in ESC differentiation and also demonstrate novel microRNAs responsible for ESC differentiation. PMID:25519956

Childhood Central Nervous System Embryonal Tumors (PDQ®)—Health Professional Version

Cancer.gov

Pediatric CNS embryonal tumors are a collection of heterogeneous lesions (medulloblastoma, and nonmedulloblastoma). Molecular genetic studies are used to classify embryonal tumors, stratify risk, and plan treatment. Get detailed information about tumor biology, diagnosis, prognosis, and treatment of untreated and recurrent CNS embryonal tumors in this summary for clinicians.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta.

PubMed

Misra, Ashish; Feng, Zhonghui; Zhang, Jiasheng; Lou, Zhi-Yin; Greif, Daniel M

2017-09-12

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Expression and identification of 10 sarcomeric MyHC isoforms in human skeletal muscles of different embryological origin. Diversity and similarity in mammalian species.

PubMed

Mascarello, Francesco; Toniolo, Luana; Cancellara, Pasqua; Reggiani, Carlo; Maccatrozzo, Lisa

2016-09-01

In the mammalian genome, among myosin heavy chain (MyHC) isoforms a family can be identified as sarcomeric based on their molecular structure which allows thick filament formation. In this study we aimed to assess the expression of the 10 sarcomeric isoforms in human skeletal muscles, adopting this species as a reference for comparison with all other mammalian species. To this aim, we set up the condition for quantitative Real Time PCR assay to detect and quantify MyHC mRNA expression in a wide variety of human muscles from somitic, presomitic and preotic origin. Specific patterns of expression of the following genes MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH8, MYH13, MYH14/7b and MYH15 were demonstrated in various muscle samples. On the same muscle samples which were analysed for mRNA expression, the corresponding MyHC proteins were studied with SDS PAGE and Western blot. The mRNA-protein comparison allowed the identification of 10 distinct proteins based on the electrophoretic migration rate. Three groups were formed based on the migration rate: fast migrating comprising beta/slow/1, alpha cardiac and fast 2B, slow migrating comprising fast 2X, fast 2A and two developmental isoforms (NEO and EMB), intermediate migrating comprising EO MyHC, slow B (product of MYH15), slow tonic (product of MYH14/7b). Of special interest was the demonstration of a protein band corresponding to 2B-MyHC in laryngeal muscles and the finding that all 10 isoforms are expressed in extraocular muscles. These latter muscles are the unique localization for extraocular, slow B (product of MYH15) and slow tonic (product of MYH14/7b). Copyright © 2016 Elsevier GmbH. All rights reserved.

Flt1/VEGFR1 heterozygosity causes transient embryonic edema.

PubMed

Otowa, Yasunori; Moriwaki, Kazumasa; Sano, Keigo; Shirakabe, Masanori; Yonemura, Shigenobu; Shibuya, Masabumi; Rossant, Janet; Suda, Toshio; Kakeji, Yoshihiro; Hirashima, Masanori

2016-06-02

Vascular endothelial growth factor-A is a major player in vascular development and a potent vascular permeability factor under physiological and pathological conditions by binding to a decoy receptor Flt1 and its primary receptor Flk1. In this study, we show that Flt1 heterozygous (Flt1(+/-)) mouse embryos grow up to adult without life-threatening abnormalities but exhibit a transient embryonic edema around the nuchal and back regions, which is reminiscent of increased nuchal translucency in human fetuses. Vascular permeability is enhanced and an intricate infolding of the plasma membrane and huge vesicle-like structures are seen in Flt1(+/-) capillary endothelial cells. Flk1 tyrosine phosphorylation is elevated in Flt1(+/-) embryos, but Flk1 heterozygosity does not suppress embryonic edema caused by Flt1 heterozygosity. When Flt1 mutants are crossed with Aspp1(-/-) mice which exhibit a transient embryonic edema with delayed formation and dysfunction of lymphatic vessels, only 5.7% of Flt1(+/-); Aspp1(-/-) mice survive, compared to expected ratio (25%). Our results demonstrate that Flt1 heterozygosity causes a transient embryonic edema and can be a risk factor for embryonic lethality in combination with other mutations causing non-lethal vascular phenotype.

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

Generation of a Knockout Mouse Embryonic Stem Cell Line Using a Paired CRISPR/Cas9 Genome Engineering Tool.

PubMed

Wettstein, Rahel; Bodak, Maxime; Ciaudo, Constance

2016-01-01

CRISPR/Cas9, originally discovered as a bacterial immune system, has recently been engineered into the latest tool to successfully introduce site-specific mutations in a variety of different organisms. Composed only of the Cas9 protein as well as one engineered guide RNA for its functionality, this system is much less complex in its setup and easier to handle than other guided nucleases such as Zinc-finger nucleases or TALENs.Here, we describe the simultaneous transfection of two paired CRISPR sgRNAs-Cas9 plasmids, in mouse embryonic stem cells (mESCs), resulting in the knockout of the selected target gene. Together with a four primer-evaluation system, it poses an efficient way to generate new independent knockout mouse embryonic stem cell lines.

Critical components of the pluripotency network are targets for the p300/CBP interacting protein (p/CIP) in embryonic stem cells.

PubMed

Chitilian, J M; Thillainadesan, G; Manias, J L; Chang, W Y; Walker, E; Isovic, M; Stanford, W L; Torchia, J

2014-01-01

p/CIP, also known as steroid receptor coactivator 3 (SRC-3)/Nuclear Receptor Coactivator 3 (NCoA3), is a transcriptional coactivator that binds liganded nuclear hormone receptors, as well as other transcription factors, and facilitates transcription through direct recruitment of accessory factors. We have found that p/CIP is highly expressed in undifferentiated mouse embryonic stem cells (mESCs) and is downregulated during differentiation. siRNA-mediated knockdown of p/CIP decreased transcript levels of Nanog, but not Oct4 or Sox2. Microarray expression analysis showed that Klf4, Tbx3, and Dax-1 are significantly downregulated in mESCs when p/CIP is knocked down. Subsequent chromatin immunoprecipitation (ChIP) analysis demonstrated that Tbx3, Klf4, and Dax-1 are direct transcriptional targets of p/CIP. Using the piggyBac transposition system, a mouse ESC line that expresses Flag-p/CIP in a doxycycline-dependent manner was generated. p/CIP overexpression increased the level of target genes and promoted the formation of undifferentiated colonies. Collectively, these results indicate that p/CIP contributes to the maintenance of ESC pluripotency through direct regulation of essential pluripotency genes. To better understand the mechanism by which p/CIP functions in ESC pluripotency, we integrated our ChIP and transcriptome data with published protein-protein interaction and promoter occupancy data to draft a p/CIP gene regulatory network. The p/CIP gene regulatory network identifies various feed-forward modules including one in which p/CIP activates members of the extended pluripotency network, demonstrating that p/CIP is a component of this extended network. © AlphaMed Press.

Derivation of Multipotent Mesenchymal Precursors from Human Embryonic Stem Cells

PubMed Central

Barberi, Tiziano; Willis, Lucy M; Socci, Nicholas D; Studer, Lorenz

2005-01-01

Background Human embryonic stem cells provide access to the earliest stages of human development and may serve as a source of specialized cells for regenerative medicine. Thus, it becomes crucial to develop protocols for the directed differentiation of embryonic stem cells into tissue-restricted precursors. Methods and Findings Here, we present culture conditions for the derivation of unlimited numbers of pure mesenchymal precursors from human embryonic stem cells and demonstrate multilineage differentiation into fat, cartilage, bone, and skeletal muscle cells. Conclusion Our findings will help to elucidate the mechanism of mesoderm specification during embryonic stem cell differentiation and provide a platform to efficiently generate specialized human mesenchymal cell types for future clinical applications. PMID:15971941

PI3K/Akt-dependent functions of TFII-I transcription factors in mouse embryonic stem cells.

PubMed

Chimge, Nyam-Osor; Makeyev, Aleksandr V; Waigel, Sabine J; Enkhmandakh, Badam; Bayarsaihan, Dashzeveg

2012-04-01

Activation of PI3K/Akt signaling is sufficient to maintain the pluripotency of mouse embryonic stem cells (mESC) and results in down-regulation of Gtf2i and Gtf2ird1 encoding TFII-I family transcription factors. To investigate how these genes might be involved in the process of embryonic stem cell differentiation, we performed expression microarray profiling of mESC upon inhibition of PI3K by LY294002. This analysis revealed significant alterations in expression of genes for specific subsets of chromatin-modifying enzymes. Surprisingly, genome-wide promoter ChIP-chip mapping indicated that the majority of differently expressed genes could be direct targets of TFII-I regulation. The data support the hypothesis that upregulation of TFII-I factors leads to activation of a specific group of developmental genes during mESC differentiation. © 2011 Wiley Periodicals, Inc.

Dual effects of fluoxetine on mouse early embryonic development

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

Kim, Chang-Woon; Department of Obstetrics and Gynecology, Samsung Changwon Hospital, Sungkyunkwan University, Changwon 630-723; Choe, Changyong

2012-11-15

Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetinemore » (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K{sup +} channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from Ca

CNS embryonal tumours: WHO 2016 and beyond.

PubMed

Pickles, J C; Hawkins, C; Pietsch, T; Jacques, T S

2018-02-01

Embryonal tumours of the central nervous system (CNS) present a significant clinical challenge. Many of these neoplasms affect young children, have a very high mortality and therapeutic strategies are often aggressive with poor long-term outcomes. There is a great need to accurately diagnose embryonal tumours, predict their outcome and adapt therapy to the individual patient's risk. For the first time in 2016, the WHO classification took into account molecular characteristics for the diagnosis of CNS tumours. This integration of histological features with genetic information has significantly changed the diagnostic work-up and reporting of tumours of the CNS. However, this remains challenging in embryonal tumours due to their previously unaccounted tumour heterogeneity. We describe the recent revisions made to the 4th edition of the WHO classification of CNS tumours and review the main changes, while highlighting some of the more common diagnostic testing strategies. © 2017 British Neuropathological Society.

Embryonic origin of the gnathostome vertebral skeleton

PubMed Central

Gillis, J. Andrew

2017-01-01

The vertebral column is a key component of the jawed vertebrate (gnathostome) body plan, but the primitive embryonic origin of this skeleton remains unclear. In tetrapods, all vertebral components (neural arches, haemal arches and centra) derive from paraxial mesoderm (somites). However, in teleost fishes, vertebrae have a dual embryonic origin, with arches derived from somites, but centra formed, in part, by secretion of bone matrix from the notochord. Here, we test the embryonic origin of the vertebral skeleton in a cartilaginous fish (the skate, Leucoraja erinacea) which serves as an outgroup to tetrapods and teleosts. We demonstrate, by cell lineage tracing, that both arches and centra are somite-derived. We find no evidence of cellular or matrix contribution from the notochord to the skate vertebral skeleton. These findings indicate that the earliest gnathostome vertebral skeleton was exclusively of somitic origin, with a notochord contribution arising secondarily in teleosts. PMID:29167367

The embryonic mir-35 family of microRNAs promotes multiple aspects of fecundity in Caenorhabditis elegans.

PubMed

McJunkin, Katherine; Ambros, Victor

2014-07-21

MicroRNAs guide many aspects of development in all metazoan species. Frequently, microRNAs are expressed during a specific developmental stage to perform a temporally defined function. The C. elegans mir-35-42 microRNAs are expressed abundantly in oocytes and early embryos and are essential for embryonic development. Here, we show that these embryonic microRNAs surprisingly also function to control the number of progeny produced by adult hermaphrodites. Using a temperature-sensitive mir-35-42 family mutant (a deletion of the mir-35-41 cluster), we demonstrate three distinct defects in hermaphrodite fecundity. At permissive temperatures, a mild sperm defect partially reduces hermaphrodite fecundity. At restrictive temperatures, somatic gonad dysfunction combined with a severe sperm defect sharply reduces fecundity. Multiple lines of evidence, including a late embryonic temperature-sensitive period, support a role for mir-35-41 early during development to promote subsequent sperm production in later larval stages. We further show that the predicted mir-35 family target sup-26 (suppressor-26) acts downstream of mir-35-41 in this process, suggesting that sup-26 de-repression in mir-35-41 deletion mutants may contribute to temperature-sensitive loss of fecundity. In addition, these microRNAs play a role in male fertility, promoting proper morphogenesis of male-specific mating structures. Overall, our results demonstrate that robust activity of the mir-35-42 family microRNAs not only is essential for embryonic development across a range of temperatures but also enables the worm to subsequently develop full reproductive capacity. Copyright © 2014 McJunkin and Ambros.

Embryonic Heart Progenitors and Cardiogenesis

PubMed Central

Brade, Thomas; Pane, Luna S.; Moretti, Alessandra; Chien, Kenneth R.; Laugwitz, Karl-Ludwig

2013-01-01

The mammalian heart is a highly specialized organ, comprised of many different cell types arising from distinct embryonic progenitor populations during cardiogenesis. Three precursor populations have been identified to contribute to different myocytic and nonmyocytic cell lineages of the heart: cardiogenic mesoderm cells (CMC), the proepicardium (PE), and cardiac neural crest cells (CNCCs). This review will focus on molecular cues necessary for proper induction, expansion, and lineage-specific differentiation of these progenitor populations during cardiac development in vivo. Moreover, we will briefly discuss how the knowledge gained on embryonic heart progenitor biology can be used to develop novel therapeutic strategies for the management of congenital heart disease as well as for improvement of cardiac function in ischemic heart disease. PMID:24086063

Tension (re)builds: Biophysical mechanisms of embryonic wound repair.

PubMed

Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo

2017-04-01

Embryonic tissues display an outstanding ability to rapidly repair wounds. Epithelia, in particular, serve as protective layers that line internal organs and form the skin. Thus, maintenance of epithelial integrity is of utmost importance for animal survival, particularly at embryonic stages, when an immune system has not yet fully developed. Rapid embryonic repair of epithelial tissues is conserved across species, and involves the collective migration of the cells around the wound. The migratory cell behaviours associated with wound repair require the generation and transmission of mechanical forces, not only for the cells to move, but also to coordinate their movements. Here, we review the forces involved in embryonic wound repair. We discuss how different force-generating structures are assembled at the molecular level, and the mechanisms that maintain the balance between force-generating structures as wounds close. Finally, we describe the mechanisms that cells use to coordinate the generation of mechanical forces around the wound. Collective cell movements and their misregulation have been associated with defective tissue repair, developmental abnormalities and cancer metastasis. Thus, we propose that understanding the role of mechanical forces during embryonic wound closure will be crucial to develop therapeutic interventions that promote or prevent collective cell movements under pathological conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Three-dimensional microCT imaging of murine embryonic development from immediate post-implantation to organogenesis: application for phenotyping analysis of early embryonic lethality in mutant animals.

PubMed

Ermakova, Olga; Orsini, Tiziana; Gambadoro, Alessia; Chiani, Francesco; Tocchini-Valentini, Glauco P

2018-04-01

In this work, we applied three-dimensional microCT imaging to study murine embryogenesis in the range from immediate post-implantation period (embryonic day 5.5) to mid-gestation (embryonic day 12.5) with the resolution up to 1.4 µm/voxel. Also, we introduce an imaging procedure for non-invasive volumetric estimation of an entire litter of embryos within the maternal uterine structures. This method allows for an accurate, detailed and systematic morphometric analysis of both embryonic and extra-embryonic components during embryogenesis. Three-dimensional imaging of unperturbed embryos was performed to visualize the egg cylinder, primitive streak, gastrulation and early organogenesis stages of murine development in the C57Bl6/N mouse reference strain. Further, we applied our microCT imaging protocol to determine the earliest point when embryonic development is arrested in a mouse line with knockout for tRNA splicing endonuclease subunit Tsen54 gene. Our analysis determined that the embryonic development in Tsen54 null embryos does not proceed beyond implantation. We demonstrated that application of microCT imaging to entire litter of non-perturbed embryos greatly facilitate studies to unravel gene function during early embryogenesis and to determine the precise point at which embryonic development is arrested in mutant animals. The described method is inexpensive, does not require lengthy embryos dissection and can be applicable for detailed analysis of mutant mice at laboratory scale as well as for high-throughput projects.

Gravity and embryonic development

NASA Technical Reports Server (NTRS)

Young, R. S.

1976-01-01

The relationship between the developing embryo (both plant and animal) and a gravitational field has long been contemplated. The difficulty in designing critical experiments on the surface of the earth because of its background of 1 g, has been an obstacle to a resolution of the problem. Biological responses to gravity (particularly in plants) are obvious in many cases; however, the influence of gravity as an environmental input to the developing embryo is not as obvious and has proven to be extremely difficult to define. In spite of this, over the years numerous attempts have been made using a variety of embryonic materials to come to grips with the role of gravity in development. Three research tools are available: the centrifuge, the clinostat, and the orbiting spacecraft. Experimental results are now available from all three sources. Some tenuous conclusions are drawn, and an attempt at a unifying theory of gravitational influence on embryonic development is made.

Isolation and culture of rabbit embryonic stem cells.

PubMed

Honda, Arata

2013-01-01

Mammalian stem cells are invaluable research resources for the study of cell and embryonic development as well as practical tools for use in the production of genetically engineered animals and further therapeutics. It is important that we further our knowledge and understanding of a variety of stem cells from several different animal species before trials in humans commence. Here we describe methods for establishing rabbit embryonic stem (rES) cell lines with indefinite proliferation potential. rES cells attain maximum proliferation potential when cultured at a feeder cell density of one-sixth of that of full confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Fibroblast growth factor (FGF)2 can maintain the undifferentiated status of rES cells; however leukemia inhibitory factor (LIF) is dispensable. Under optimized conditions, rES cells could be passaged by trypsinization 50 times. This culture system enabled efficient gene transduction and clonal expansion from single cells. rES cells grew as flat monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo, respectively. Characterization of ES cells from different species is important for establishing common features of pluripotency. We have demonstrated the similarity of ES cells between rabbit and humans. These cell lines could be applied directly using gene-targeting techniques, or in combination with induced pluripotent stem cells. Thus, rES cells are a suitable model for studying human transplantation therapy and disease treatments.

Metabolic circadian rhythms in embryonic turtles.

PubMed

Loudon, Fiona Kay; Spencer, Ricky-John; Strassmeyer, Alana; Harland, Karen

2013-07-01

Oviparous species are model organisms for investigating embryonic development of endogenous physiological circadian rhythms without the influence of maternal biorhythms. Recent studies have demonstrated that heart rates and metabolic rates of embryonic turtles are not constant or always maximal and can be altered in response to the presence of embryos at a more advanced stage of development within the nest. A first step in understanding the physiological mechanisms underpinning these responses in embryonic ectothermic organisms is to develop metabolic profiles (e.g., heart rate) at different temperatures throughout incubation. Heart beat and rhythmic patterns or changes in development may represent important signals or cues within a nest and may be vital to coordinate synchronous hatching well in advance of the final stages of incubation. We developed baseline embryonic heart-rate profiles of embryos of the short-necked Murray River turtle (Emydura macquarii) to determine the stage of embryogenesis that metabolic circadian rhythms become established, if at all. Eggs were incubated at constant temperatures (26°C and 30°C) and heart rates were monitored at 6-h intervals over 24 h every 7-11 days until hatching. Circadian heart rate rhythms were detected at the mid-gestation period and were maintained until hatching. Heart rates throughout the day varied by up to 20% over 24 h and were not related to time of day. This study demonstrated that endogenous metabolic circadian rhythms in developing embryos in turtle eggs establish earlier in embryogenesis than those documented in other vertebrate taxa during embryogenesis. Early establishment of circadian rhythms in heart rates may be critical for communication among embryos and synchrony in hatching and emergence from the nest.

Ets-1 is a target of MAPK signaling in the embryonic anterior pituitary gland during glucocorticoid initiation of pituitary growth hormone expression

USDA-ARS?s Scientific Manuscript database

Glucocorticoids play a critical role in functional differentiation of somatotrophs, the growth hormone (GH)-producing cells within the anterior pituitary gland. In chicken embryonic day 11 (e11) pituitary cells, premature induction of growth hormone (GH) resulting from corticosterone (CORT) treatmen...

Embryonic kidney function in a chronic renal failure model in rodents.

PubMed

Fujimoto, Eisuke; Yamanaka, Shuichiro; Kurihara, Sho; Tajiri, Susumu; Izuhara, Luna; Katsuoka, Yuichi; Yokote, Shinya; Matsumoto, Kei; Kobayashi, Eiji; Okano, Hirotaka James; Chikaraishi, Tatsuya; Yokoo, Takashi

2017-08-01

Rapid advancements have been made in alternative treatments for renal diseases. Our goal for renal regeneration is to establish a kidney graft derived from human embryonic tissues. In this study, we investigated the effects of host renal failure on the structure and activity of transplanted embryonic kidney and bladder, and found that diuretics effectively induced urine production in the transplanted kidney. Uremic conditions were reproduced using a 5/6 renal infarction rat model. An embryonic kidney plus bladder (embryonic day 15) was isolated from a pregnant Lewis rat and transplanted into the para-aortic area of a 5/6 renal-infarcted Lewis rat. Following growth, the embryonic bladder was successfully anastomosed to the host ureter. We assessed graft function in terms of survival rates and found no differences between normal (n = 5) and renal failure (n = 8) groups (median survival: 70.5 vs 74.5 h; p = 0.331) in terms of survival, indicating that the grafts prolonged rat survival, even under renal failure conditions. Furosemide (n = 9) significantly increased urine volume compared with saline-treated controls (n = 7; p < 0.05), confirming that the grafts were functional. We also demonstrated the possibilities of an in vivo imaging system for determining the viability of transplanted embryonic kidney with bladder. The results of this study demonstrate that transplanted embryonic kidney and bladder can grow and function effectively, even under uremic conditions.

The ethics of patenting human embryonic stem cells.

PubMed

Chapman, Audrey R

2009-09-01

Just as human embryonic stem cell research has generated controversy about the uses of human embryos for research and therapeutic applications, human embryonic stem cell patents raise fundamental ethical issues. The United States Patent and Trademark Office has granted foundational patents, including a composition of matter (or product) patent to the Wisconsin Alumni Research Foundation (WARF), the University of Wisconsin-Madison's intellectual property office. In contrast, the European Patent Office rejected the same WARF patent application for ethical reasons. This article assesses the appropriateness of these patents placing the discussion in the context of the deontological and consequentialist ethical issues related to human embryonic stem cell patenting. It advocates for a patent system that explicitly takes ethical factors into account and explores options for new types of intellectual property arrangements consistent with ethical concerns.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

PubMed

Cong, Shan; Cao, Guifang; Liu, Dongjun

2014-12-01

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Role of adiponectin in delayed embryonic development of the short-nosed fruit bat, Cynopterus sphinx.

PubMed

Anuradha; Krishna, Amitabh

2014-12-01

The aim of this study was to evaluate the role of adiponectin in the delayed embryonic development of Cynopterus sphinx. Adiponectin receptor (ADIPOR1) abundance was first observed to be lower during the delayed versus non-delayed periods of utero-embryonic unit development. The effects of adiponectin treatment on embryonic development were then evaluated during the period of delayed development. Exogenous treatment increased the in vivo rate of embryonic development, as indicated by an increase in weight, ADIPOR1 levels in the utero-embryonic unit, and histological changes in embryonic development. Treatment with adiponectin during embryonic diapause showed a significant increase in circulating progesterone and estradiol concentrations, and in production of their receptors in the utero-embryonic unit. The adiponectin-induced increase in estradiol synthesis was correlated with increased cell survival (BCL2 protein levels) and cell proliferation (PCNA protein levels) in the utero-embryonic unit, suggesting an indirect effect of adiponectin via estradiol synthesis by the ovary. An in vitro study further confirmed the in vivo findings that adiponectin treatment increases PCNA levels together with increased uptake of glucose by increasing the abundance of glucose transporter 8 (GLUT8) in the utero-embryonic unit. The in vitro study also revealed that adiponectin, together with estradiol but not alone, significantly increased ADIPOR1 protein levels. Thus, adiponectin works in concert with estradiol to increase glucose transport to the utero-embryonic unit and promote cell proliferation, which together accelerate embryonic development. © 2014 Wiley Periodicals, Inc.

PTBP1 Is Required for Embryonic Development before Gastrulation

PubMed Central

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A. Francis; Solimena, Michele

2011-01-01

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures. PMID:21423341

PTBP1 is required for embryonic development before gastrulation.

PubMed

Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A Francis; Solimena, Michele

2011-02-17

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

Embryonic Stem Cells: Isolation, Characterization and Culture

NASA Astrophysics Data System (ADS)

Amit, Michal; Itskovitz-Eldor, Joseph

Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers

PubMed Central

2013-01-01

Introduction Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents. Methods We defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers. Results Small subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell

Embryonic development of the cricket Gryllus bimaculatus.

PubMed

Donoughe, Seth; Extavour, Cassandra G

2016-03-01

Extensive research into Drosophila melanogaster embryogenesis has improved our understanding of insect developmental mechanisms. However, Drosophila development is thought to be highly divergent from that of the ancestral insect and arthropod in many respects. We therefore need alternative models for arthopod development that are likely to be more representative of basally-branching clades. The cricket Gryllus bimaculatus is such a model, and currently has the most sophisticated functional genetic toolkit of any hemimetabolous insect. The existing cricket embryonic staging system is fragmentary, and it is based on morphological landmarks that are not easily visible on a live, undissected egg. To address this problem, here we present a complementary set of "egg stages" that serve as a guide for identifying the developmental progress of a cricket embryo from fertilization to hatching, based solely on the external appearance of the egg. These stages were characterized using a combination of brightfield timelapse microscopy, timed brightfield micrographs, confocal microscopy, and measurements of egg dimensions. These egg stages are particularly useful in experiments that involve egg injection (including RNA interference, targeted genome modification, and transgenesis), as injection can alter the speed of development, even in control treatments. We also use 3D reconstructions of fixed embryo preparations to provide a comprehensive description of the morphogenesis and anatomy of the cricket embryo during embryonic rudiment assembly, germ band formation, elongation, segmentation, and appendage formation. Finally, we aggregate and schematize a variety of published developmental gene expression patterns. This work will facilitate further studies on G. bimaculatus development, and serve as a useful point of reference for other studies of wild type and experimentally manipulated insect development in fields from evo-devo to disease vector and pest management. Copyright �

A toolbox to explore the mechanics of living embryonic tissues

PubMed Central

Campàs, Otger

2016-01-01

The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role of mechanics in embryonic development. Several methodologies are starting to enable quantitative studies of mechanics in vivo and in situ, opening new avenues to explore how mechanics contributes to shaping embryonic tissues and how it affects cell behavior within developing embryos. Here we review the present methodologies to study the role of mechanics in living embryonic tissues, considering their strengths and drawbacks as well as the conditions in which they are most suitable. PMID:27061360

Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice

PubMed Central

Kayser-Herold, Oliver; Stojanovic, Boban; Nedic, Djordje; Irving, Thomas C.; Geeves, Michael A.

2016-01-01

The effect of molecule tethering in three-dimensional (3-D) space on bimolecular binding kinetics is rarely addressed and only occasionally incorporated into models of cell motility. The simplest system that can quantitatively determine this effect is the 3-D sarcomere lattice of the striated muscle, where tethered myosin in thick filaments can only bind to a relatively small number of available sites on the actin filament, positioned within a limited range of thermal movement of the myosin head. Here we implement spatially explicit actomyosin interactions into the multiscale Monte Carlo platform MUSICO, specifically defining how geometrical constraints on tethered myosins can modulate state transition rates in the actomyosin cycle. The simulations provide the distribution of myosin bound to sites on actin, ensure conservation of the number of interacting myosins and actin monomers, and most importantly, the departure in behavior of tethered myosin molecules from unconstrained myosin interactions with actin. In addition, MUSICO determines the number of cross-bridges in each actomyosin cycle state, the force and number of attached cross-bridges per myosin filament, the range of cross-bridge forces and accounts for energy consumption. At the macroscopic scale, MUSICO simulations show large differences in predicted force-velocity curves and in the response during early force recovery phase after a step change in length comparing to the two simplest mass action kinetic models. The origin of these differences is rooted in the different fluxes of myosin binding and corresponding instantaneous cross-bridge distributions and quantitatively reflects a major flaw of the mathematical description in all mass action kinetic models. Consequently, this new approach shows that accurate recapitulation of experimental data requires significantly different binding rates, number of actomyosin states, and cross-bridge elasticity than typically used in mass action kinetic models to

Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice

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

Mijailovich, Srboljub M.; Kayser-Herold, Oliver; Stojanovic, Boban

2016-11-18

The effect of molecule tethering in three-dimensional (3-D) space on bimolecular binding kinetics is rarely addressed and only occasionally incorporated into models of cell motility. The simplest system that can quantitatively determine this effect is the 3-D sarcomere lattice of the striated muscle, where tethered myosin in thick filaments can only bind to a relatively small number of available sites on the actin filament, positioned within a limited range of thermal movement of the myosin head. Here we implement spatially explicit actomyosin interactions into the multiscale Monte Carlo platform MUSICO, specifically defining how geometrical constraints on tethered myosins can modulatemore » state transition rates in the actomyosin cycle. The simulations provide the distribution of myosin bound to sites on actin, ensure conservation of the number of interacting myosins and actin monomers, and most importantly, the departure in behavior of tethered myosin molecules from unconstrained myosin interactions with actin. In addition, MUSICO determines the number of cross-bridges in each actomyosin cycle state, the force and number of attached cross-bridges per myosin filament, the range of cross-bridge forces and accounts for energy consumption. At the macroscopic scale, MUSICO simulations show large differences in predicted force-velocity curves and in the response during early force recovery phase after a step change in length comparing to the two simplest mass action kinetic models. The origin of these differences is rooted in the different fluxes of myosin binding and corresponding instantaneous cross-bridge distributions and quantitatively reflects a major flaw of the mathematical description in all mass action kinetic models. Consequently, this new approach shows that accurate recapitulation of experimental data requires significantly different binding rates, number of actomyosin states, and cross-bridge elasticity than typically used in mass action kinetic models

Melatonin regulates delayed embryonic development in the short-nosed fruit bat, Cynopterus sphinx.

PubMed

Banerjee, Arnab; Meenakumari, K J; Udin, S; Krishna, A

2009-12-01

The aim of the present study was to evaluate the seasonal variation in serum melatonin levels and their relationship to the changes in the serum progesterone level, ovarian steroidogenesis, and embryonic development during two successive pregnancies of Cynopterus sphinx. Circulating melatonin concentrations showed two peaks; one coincided with the period of low progesterone synthesis and delayed embryonic development, whereas the second peak coincided with regressing corpus luteum. This finding suggests that increased serum melatonin level during November-December may be responsible for delayed embryonic development by suppressing progesterone synthesis. The study showed increased melatonin receptors (MTNR1A and MTNR1B) in the corpus luteum and in the utero-embryonic unit during the period of delayed embryonic development. The in vitro study showed that a high dose of melatonin suppressed progesterone synthesis, whereas a lower dose of melatonin increased progesterone synthesis by the ovary. The effects of melatonin on ovarian steroidogenesis are mediated through changes in the expression of peripheral-type benzodiazepine receptor, P450 side chain cleavage enzyme, and LH receptor proteins. This study further showed a suppressive impact of melatonin on the progesterone receptor (PGR) in the utero-embryonic unit; this effect might contribute to delayed embryonic development in C. sphinx. The results of the present study thus suggest that a high circulating melatonin level has a dual contribution in retarding embryonic development in C. sphinx by impairing progesterone synthesis as well as by inhibiting progesterone action by reducing expression of PGR in the utero-embryonic unit.

Muscle function during jumping in frogs. I. Sarcomere length change, EMG pattern, and jumping performance.

PubMed

Lutz, G J; Rome, L C

1996-08-01

We determined the influence of temperature on muscle function during jumping to better understand how the frog muscular system is designed to generate a high level of mechanical power. Maximal jumping performance and the in vivo operating conditions of the semimembranosus muscle (SM), a hip extensor, were measured and related to the mechanical properties of the isolated SM in the accompanying paper [Muscle function during jumping in frogs. II. Mechanical properties of muscle: implication for system design. Am. J. Physiol. 271 (Cell Physiol. 40): C571-C578, 1996]. Reducing temperature from 25 to 15 degrees C caused a 1.75-fold decline in peak mechanical power generation and a proportional decline in aerial jump distance. The hip and knee joint excursions were nearly the same at both temperatures. Accordingly, sarcomeres shortened over the same range (2.4 to 1.9 microns) at both temperatures, corresponding to myofilament overlap at least 90% of maximal. At the low temperature, however, movements were made more slowly. Angular velocities were 1.2- to 1.4-fold lower, and ground contact time was increased by 1.33-fold at 15 degrees C. Average shortening velocity of the SM was only 1.2-fold lower at 15 degrees C than at 25 degrees C. The low Q10 of velocity is in agreement with that predicted for muscles shortening against an inertial load.

High-throughput identification of small molecules that affect human embryonic vascular development

PubMed Central

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

2017-01-01

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

High-throughput identification of small molecules that affect human embryonic vascular development.

PubMed

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R; Honório, Inês; de Vries, Margreet R; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H A; Pereira, Carlos F; Mercader, Nadia; Fernandes, Hugo; Ferreira, Lino

2017-04-11

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Gene targeting and subsequent site-specific transgenesis at the β-actin (ACTB) locus in common marmoset embryonic stem cells.

PubMed

Shiozawa, Seiji; Kawai, Kenji; Okada, Yohei; Tomioka, Ikuo; Maeda, Takuji; Kanda, Akifumi; Shinohara, Haruka; Suemizu, Hiroshi; James Okano, Hirotaka; Sotomaru, Yusuke; Sasaki, Erika; Okano, Hideyuki

2011-09-01

Nonhuman primate embryonic stem (ES) cells have vast promise for preclinical studies. Genetic modification in nonhuman primate ES cells is an essential technique for maximizing the potential of these cells. The common marmoset (Callithrix jacchus), a nonhuman primate, is expected to be a useful transgenic model for preclinical studies. However, genetic modification in common marmoset ES (cmES) cells has not yet been adequately developed. To establish efficient and stable genetic modifications in cmES cells, we inserted the enhanced green fluorescent protein (EGFP) gene with heterotypic lox sites into the β-actin (ACTB) locus of the cmES cells using gene targeting. The resulting knock-in ES cells expressed EGFP ubiquitously under the control of the endogenous ACTB promoter. Using inserted heterotypic lox sites, we demonstrated Cre recombinase-mediated cassette exchange (RMCE) and successfully established a monomeric red fluorescent protein (mRFP) knock-in cmES cell line. Further, a herpes simplex virus-thymidine kinase (HSV-tk) knock-in cmES cell line was established using RMCE. The growth of tumor cells originating from the cell line was significantly suppressed by the administration of ganciclovir. Therefore, the HSV-tk/ganciclovir system is promising as a safeguard for stem cell therapy. The stable and ubiquitous expression of EGFP before RMCE enables cell fate to be tracked when the cells are transplanted into an animal. Moreover, the creation of a transgene acceptor locus for site-specific transgenesis will be a powerful tool, similar to the ROSA26 locus in mice.

Familial hypertrophic cardiomyopathy: functional variance among individual cardiomyocytes as a trigger of FHC-phenotype development

PubMed Central

Brenner, Bernhard; Seebohm, Benjamin; Tripathi, Snigdha; Montag, Judith; Kraft, Theresia

2014-01-01

Familial hypertrophic cardiomyopathy (FHC) is the most frequent inherited cardiac disease. It has been related to numerous mutations in many sarcomeric and even some non-sarcomeric proteins. So far, however, no common mechanism has been identified by which the many different mutations in different sarcomeric and non-sarcomeric proteins trigger development of the FHC phenotype. Here we show for different MYH7 mutations variance in force pCa-relations from normal to highly abnormal as a feature common to all mutations we studied, while direct functional effects of the different FHC-mutations, e.g., on force generation, ATPase or calcium sensitivity of the contractile system, can be quite different. The functional variation among individual M. soleus fibers of FHC-patients is accompanied by large variation in mutant vs. wildtype β-MyHC-mRNA. Preliminary results show a similar variation in mutant vs. wildtype β-MyHC-mRNA among individual cardiomyocytes. We discuss our previously proposed concept as to how different mutations in the β-MyHC and possibly other sarcomeric and non-sarcomeric proteins may initiate an FHC-phenotype by functional variation among individual cardiomyocytes that results in structural distortions within the myocardium, leading to cellular and myofibrillar disarray. In addition, distortions can activate stretch-sensitive signaling in cardiomyocytes and non-myocyte cells which is known to induce cardiac remodeling with interstitial fibrosis and hypertrophy. Such a mechanism will have major implications for therapeutic strategies to prevent FHC-development, e.g., by reducing functional imbalances among individual cardiomyocytes or by inhibition of their triggering of signaling paths initiating remodeling. Targeting increased or decreased contractile function would require selective targeting of mutant or wildtype protein to reduce functional imbalances. PMID:25346696

Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom's syndrome gene.

PubMed

Chester, N; Kuo, F; Kozak, C; O'Hara, C D; Leder, P

1998-11-01

Bloom's syndrome is a human autosomal genetic disorder characterized at the cellular level by genome instability and increased sister chomatid exchanges (SCEs). Clinical features of the disease include proportional dwarfism and a predisposition to develop a wide variety of malignancies. The human BLM gene has been cloned recently and encodes a DNA helicase. Mouse embryos homozygous for a targeted mutation in the murine Bloom's syndrome gene (Blm) are developmentally delayed and die by embryonic day 13.5. The fact that the interrupted gene is the homolog of the human BLM gene was confirmed by its homologous sequence, its chromosomal location, and by demonstrating high numbers of SCEs in cultured murine Blm-/- fibroblasts. The proportional dwarfism seen in the human is consistent with the small size and developmental delay (12-24 hr) seen during mid-gestation in murine Blm-/- embryos. Interestingly, the growth retardation in mutant embryos can be accounted for by a wave of increased apoptosis in the epiblast restricted to early post-implantation embryogenesis. Mutant embryos do not survive past day 13.5, and at this time exhibit severe anemia. Red blood cells and their precursors from Blm-/- embryos are heterogeneous in appearance and have increased numbers of macrocytes and micronuclei. Both the apoptotic wave and the appearance of micronuclei in red blood cells are likely cellular consequences of damaged DNA caused by effects on replicating or segregating chromosomes.

Embryonated chicken eggs: An experimental model for Pythium insidiosum infection.

PubMed

Verdi, Camila M; Jesus, Francielli P K; Kommers, Glaucia; Ledur, Pauline C; Azevedo, Maria I; Loreto, Erico S; Tondolo, Juliana S M; Andrade, Eduardo N C; Schlemmer, Karine B; Alves, Sydney H; Santurio, Janio M

2018-02-01

Pythiosis is a severe disease caused by Pythium insidiosum. Currently, the research on the treatment of pythiosis uses rabbits as an experimental infection model. To reduce the use of animals in scientific experimentation, alternative models are increasingly necessary options. The objective of this study was to establish a new experimental infection model for pythiosis using embryonated chicken eggs. First, we tested the inoculation of 4 zoospore concentrations into the egg allantoic cavity at 3 embryonic days. We observed that increased zoospore concentration causes a decrease in survival time, and at a later embryonic day (the 14th) of infection, embryos showed delayed mortality. To confirm the reproducibility of the model, we chose the 14th embryonic day for the inoculation of 50 zoospores/egg, and the experiment was repeated twice. Mortality began with 30% embryos 48 hours after inoculation, and 95% embryos died within 72 hours. There was no mortality in the uninfected control group. The infection was confirmed by culture, PCR and histopathology. Immunohistochemistry confirmed the presence of hyphae in blood vessels in the umbilical cords in 95% of embryos and only 1 liver (5%). Our results suggest that embryonated eggs can be a very useful alternative infection model to study pythiosis. © 2017 Blackwell Verlag GmbH.

A toolbox to explore the mechanics of living embryonic tissues.

PubMed

Campàs, Otger

2016-07-01

The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role of mechanics in embryonic development. Several methodologies are starting to enable quantitative studies of mechanics in vivo and in situ, opening new avenues to explore how mechanics contributes to shaping embryonic tissues and how it affects cell behavior within developing embryos. Here we review the present methodologies to study the role of mechanics in living embryonic tissues, considering their strengths and drawbacks as well as the conditions in which they are most suitable. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chromatin in embryonic stem cell neuronal differentiation.

PubMed

Meshorer, E

2007-03-01

Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.

Smoc2 modulates embryonic myelopoiesis during zebrafish development.

PubMed

Mommaerts, Hendrik; Esguerra, Camila V; Hartmann, Ursula; Luyten, Frank P; Tylzanowski, Przemko

2014-11-01

SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development. Copyright © 2014 Wiley Periodicals, Inc.

The death-inducer obliterator 1 (Dido1) gene regulates embryonic stem cell self-renewal.

PubMed

Liu, Yinyin; Kim, Hyeung; Liang, Jiancong; Lu, Weisi; Ouyang, Bin; Liu, Dan; Songyang, Zhou

2014-02-21

The regulatory network of factors that center on master transcription factors such as Oct4, Nanog, and Sox2 help maintain embryonic stem (ES) cells and ensure their pluripotency. The target genes of these master transcription factors define the ES cell transcriptional landscape. In this study, we report our findings that Dido1, a target of canonical transcription factors such as Oct4, Sox2, and Nanog, plays an important role in regulating ES cell maintenance. We found that depletion of Dido1 in mouse ES cells led to differentiation, and ectopic expression of Dido1 inhibited differentiation induced by leukemia inhibitory factor withdrawal. We further demonstrated that whereas Nanog and Oct4 could occupy the Dido1 locus and promote its transcription, Dido1 could also target to the loci of pluripotency factors such as Nanog and Oct4 and positively regulate their expression. Through this feedback and feedforward loop, Dido1 is able to regulate self-renewal of mouse ES cells.

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions

PubMed Central

Gerson-Gurwitz, Adina; Wang, Shaohe; Sathe, Shashank; Green, Rebecca; Yeo, Gene W.; Oegema, Karen; Desai, Arshad

2016-01-01

SUMMARY Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition. PMID:27020753

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung

PubMed Central

Lange, Alexander W.; Sridharan, Anusha; Xu, Yan; Stripp, Barry R.; Perl, Anne-Karina; Whitsett, Jeffrey A.

2015-01-01

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. PMID:25480985

Embryonic senescence and laminopathies in a progeroid zebrafish model.

PubMed

Koshimizu, Eriko; Imamura, Shintaro; Qi, Jie; Toure, Jamal; Valdez, Delgado M; Carr, Christopher E; Hanai, Jun-ichi; Kishi, Shuji

2011-03-30

Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with

Maternal Diabetes Leads to Adaptation in Embryonic Amino Acid Metabolism during Early Pregnancy.

PubMed

Gürke, Jacqueline; Hirche, Frank; Thieme, René; Haucke, Elisa; Schindler, Maria; Stangl, Gabriele I; Fischer, Bernd; Navarrete Santos, Anne

2015-01-01

During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA) metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2), branched chain ketoacid dehydrogenase (Bckdha) and dehydrolipoyl dehydrogenase (Dld), were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR) was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling.

Maternal Diabetes Leads to Adaptation in Embryonic Amino Acid Metabolism during Early Pregnancy

PubMed Central

Gürke, Jacqueline; Hirche, Frank; Thieme, René; Haucke, Elisa; Schindler, Maria; Stangl, Gabriele I.; Fischer, Bernd; Navarrete Santos, Anne

2015-01-01

During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA) metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2), branched chain ketoacid dehydrogenase (Bckdha) and dehydrolipoyl dehydrogenase (Dld), were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR) was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling. PMID:26020623

In utero mouse embryonic imaging with OCT for ophthalmologic research

NASA Astrophysics Data System (ADS)

Syed, Saba H.; Larina, Irina V.; Dickinson, Mary E.; Larin, Kirill V.

2011-03-01

Live imaging of an eye during embryonic development in mammalian model is important for understanding dynamic aspects of normal and abnormal eye morphogenesis. In this study, we used Swept Source Optical Coherence Tomography (SS-OCT) for live structural imaging of mouse embryonic eye through the uterine wall. The eye structure was reconstructed in mouse embryos at 13.5 to 17.5 days post coitus (dpc). Despite the limited imaging depth of OCT in turbid tissues, we were able to visualize the whole eye globe at these stages. These results suggest that live in utero OCT imaging is a useful tool to study embryonic eye development in the mouse model.

Spatial distribution of endogenous retinoids in the murine embryonic mandible.

PubMed

Kronmiller, J E; Beeman, C S

1994-12-01

Retinoids play an important part in pattern formation during embryonic development. Exogenous retinoids alter the pattern of skeletal, neural and odontogenic tissues. Endogenous retinoids have been demonstrated previously in the murine embryonic mandible, reaching a concentration peak during the initiation of odontogenesis. It was now found that endogenous retinoids are present in a concentration gradient in the embryonic mouse mandible at the time of the initiation of the dental lamina. All-trans-retinoic acid was more concentrated in the incisor region and retinol in the molar region. These results, and the fact that exogenous retinoids produce supernumerary incisors and missing molars, suggest that all-trans-retinoic acid may instruct incisor morphology.

G-quadruplexes as novel cis-elements controlling transcription during embryonic development

PubMed Central

David, Aldana P.; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B.

2016-01-01

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology. PMID:26773060

Overexpression of Trophoblast Stem Cell-Enriched MicroRNAs Promotes Trophoblast Fate in Embryonic Stem Cells.

PubMed

Nosi, Ursula; Lanner, Fredrik; Huang, Tsu; Cox, Brian

2017-05-09

The first cell fate choice of the preimplantation embryo generates the extraembryonic trophoblast and embryonic epiblast lineages. Embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) can be utilized to investigate molecular mechanisms of this first cell fate decision. It has been established that ESCs can be induced to acquire trophoblast lineage characteristics upon manipulation of lineage-determining transcription factors. Here, we have interrogated the potential of microRNAs (miRNAs) to drive trans-differentiation of ESCs into the trophoblast lineage. Analysis of gene expression data identified a network of TSC-enriched miRNAs that were predicted to target mRNAs enriched in ESCs. Ectopic expression of these miRNAs in ESCs resulted in a stable trophoblast phenotype, supported by gene expression changes and in vivo contribution potential. This process is highly miRNA-specific and dependent on Hdac2 inhibition. Our experimental evidence suggests that these miRNAs promote a mural trophectoderm (TE)-like cell fate with physiological properties that differentiate them from the polar TE. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The embryonic development of the cnidarian Hydractinia echinata.

PubMed

Kraus, Yulia; Flici, Hakima; Hensel, Katrin; Plickert, Günter; Leitz, Thomas; Frank, Uri

2014-01-01

With the rapid increase of the quantity of molecular data, many animals joined the ranks of the so-called 'emerging models' of Evo-Devo. One of the necessary steps in converting an emerging model into an established one is gaining comprehensive knowledge of its normal embryonic development. The marine colonial hydrozoan Hydractinia echinata - an excellent model for research on stem cells, metamorphosis, and allorecognition - has been studied for decades. Yet knowledge of its embryonic development remains fragmentary and incomplete. Here we provide a detailed account of H. echinata embryonic development using in vivo observations, histology, immunohistochemistry, and electron microscopy. Furthermore, we propose a model describing the cellular basis of the morphogenetic movements occurring during development and also reveal a functional link between canonical Wnt signaling and regional differences in the morphology of the embryo. Hydractinia embryogenesis is an example of the diversity and plasticity of hydrozoan development where multiple routes lead to the same result - the formation of a normal planula larva. © 2014 Wiley Periodicals, Inc.

Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Hepatocytes

PubMed Central

Basma, Hesham; Soto-Gutiérrez, Alejandro; Yannam, Govardhana Rao; Liu, Liping; Ito, Ryotaro; Yamamoto, Toshiyuki; Ellis, Ewa; Carson, Steven D.; Sato, Shintaro; Chen, Yong; Muirhead, David; Navarro-Álvarez, Nalu; Wong, Ron; Roy-Chowdhury, Jayanta; Platt, Jeffrey L.; Mercer, David F.; Miller, John D.; Strom, Stephen C.; Kobayashi, Noaya; Fox, Ira J.

2009-01-01

Background & Aims The ability to obtain unlimited numbers of human hepatocytes would improve development of cell-based therapies for liver diseases, facilitate the study of liver biology and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, can potentially differentiate into any cell type and could therefore be developed as a source of human hepatocytes. Methods To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human Activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein receptor expression. Characterization was performed by real-time PCR, imunohistochemistry, immunoblot, functional assays and transplantation. Results Embryonic stem cell-derived hepatocytes expressed liver-specific genes but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes and demonstrated human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha-1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. Conclusion Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein receptor expression and could potentially be used in drug discovery research and developed as therapeutics. PMID:19026649

Human embryonic curvature studied with 3D ultrasound in ongoing pregnancies and miscarriages.

PubMed

Bogers, Hein; van Uitert, Evelyne M; van Ginkel, Sharon; van der Mooren, Elisabeth D H; Groenenberg, Irene A L; Eilers, Paul H C; Exalto, Niek; Steegers, Eric A P; Steegers-Theunissen, Régine P M

2018-05-01

Embryonic growth is often impaired in miscarriages. It is postulated that derangements in embryonic growth result in abnormalities of the embryonic curvature. This study aims to create first trimester reference charts of the human embryonic curvature and investigate differences between ongoing pregnancies and miscarriages. Weekly ultrasonographic scans from ongoing pregnancies and miscarriages were used from the Rotterdam periconceptional cohort and a cohort of recurrent miscarriages. In 202 ongoing pregnancies and 33 miscarriages, first trimester crown rump length and total arch length were measured to assess the embryonic curvature. The results show that the total arch length increases and shows more variation with advanced gestation. The crown rump length/total arch length ratio shows a strong increase from 8 +0 to 10 +0 weeks and flattening thereafter. No significant difference was observed between the curvature of embryos of ongoing pregnancies and miscarriages. The majority of miscarried embryos could not be measured. Therefore, this technique is too limited to recommend the measurement of the embryonic curvature in clinical practice. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

The roles of ERAS during cell lineage specification of mouse early embryonic development.

PubMed

Zhao, Zhen-Ao; Yu, Yang; Ma, Huai-Xiao; Wang, Xiao-Xiao; Lu, Xukun; Zhai, Yanhua; Zhang, Xiaoxin; Wang, Haibin; Li, Lei

2015-08-01

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development. © 2015 The Authors.

GLUT3 gene expression is critical for embryonic growth, brain development and survival.

PubMed

Carayannopoulos, Mary O; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U

2014-04-01

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. Copyright © 2014 Elsevier Inc. All rights reserved.

GLUT3 Gene Expression is Critical for Embryonic Growth, Brain Development and Survival

PubMed Central

Carayannopoulos, Mary O.; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U.

2015-01-01

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. PMID:24529979

Mechanical signaling coordinates the embryonic heartbeat.

PubMed

Chiou, Kevin K; Rocks, Jason W; Chen, Christina Yingxian; Cho, Sangkyun; Merkus, Koen E; Rajaratnam, Anjali; Robison, Patrick; Tewari, Manorama; Vogel, Kenneth; Majkut, Stephanie F; Prosser, Benjamin L; Discher, Dennis E; Liu, Andrea J

2016-08-09

In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts-consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats.

Mechanical signaling coordinates the embryonic heartbeat

PubMed Central

Chiou, Kevin K.; Rocks, Jason W.; Chen, Christina Yingxian; Cho, Sangkyun; Merkus, Koen E.; Rajaratnam, Anjali; Robison, Patrick; Tewari, Manorama; Vogel, Kenneth; Majkut, Stephanie F.; Prosser, Benjamin L.; Discher, Dennis E.; Liu, Andrea J.

2016-01-01

In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats. PMID:27457951

Delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

PubMed

Meenakumari, Karukayil J; Krishna, Amitabh

2005-01-01

The unusual feature of the breeding cycle of Cynopterus sphinx at Varanasi is the significant variation in gestation length of the two successive pregnancies of the year. The aim of this study was to investigate whether the prolongation of the first pregnancy in C. sphinx is due to delayed embryonic development. The first (winter) pregnancy commences in late October and lasts until late March and has a gestation period of about 150 days. The second (summer) pregnancy commences in April and lasts until the end of July or early August with a gestation period of about 125 days. Changes in the size and weight of uterine cornua during the two successive pregnancies suggest retarded embryonic growth during November and December. Histological analysis during the period of retarded embryonic development in November and December showed a slow gastrulation process. The process of amniogenesis was particularly slow. When the embryos attained the early primitive streak stage, their developmental rate suddenly increased considerably. During the summer pregnancy, on the other hand, the process of gastrulation was much faster and proceeded quickly. A comparison of the pattern of embryonic development for 4 consecutive years consistently showed retarded or delayed embryonic development during November and December. The time of parturition and post-partum oestrus showed only a limited variation from 1 year to another. This suggests that delayed embryonic development in C. sphinx may function to synchronize parturition among females. The period of delayed embryonic development in this species clearly coincides with the period of fat deposition. The significance of this correlation warrants further investigation.

Embryonic development during chronic acceleration

NASA Technical Reports Server (NTRS)

Smith, A. H.; Abbott, U. K.

1982-01-01

Experiments carried out on chicken eggs indicate that the embryo is affected during very early development, especially over the first four days, and during hatching. In the first four days, the brain develops as well as the anlage for all other organs. In addition, the heart commences to function and the extraembryonic membranes that compartmentalize the egg contents form. The latter require an appreciable extension and folding of tissue which may be disrupted by the mechanical load. Observations of embryonic abnormalities that occur during chronic acceleration suggest an inhibition of development of the axial skeleton, which is rarely seen otherwise, a general retardation of embryonic growth, and circulatory problems. The final stages of development (after 18 days) involve the uptake of fluids, the transition to aerial respiration, and the reorientation of the embryo into a normal hatching position. At 4 G mortality is very high during this period, with a majority of embryos failing to reorient into the normal hatching position.

Embryonic Stem Cell Patents and Human Dignity

PubMed Central

Resnik, David B.

2009-01-01

This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells. PMID:17922198

The influence of IVF/ICSI treatment on human embryonic growth trajectories.

PubMed

Eindhoven, S C; van Uitert, E M; Laven, J S E; Willemsen, S P; Koning, A H J; Eilers, P H C; Exalto, N; Steegers, E A P; Steegers-Theunissen, R P M

2014-12-01

Is in vitro fertilization treatment with or without intracytoplasmatic sperm injection (IVF/ICSI) associated with changes in first and second trimester embryonic and fetal growth trajectories and birthweight in singleton pregnancies? Embryonic and fetal growth trajectories and birthweight are not significantly different between pregnancies conceived with IVF/ICSI treatment and spontaneously conceived pregnancies with reliable pregnancy dating. IVF/ICSI treatment has been associated with increased risks of preterm birth, fetal growth restriction and low birthweight. Decreased first-trimester crown-rump length (CRL) in the general population has been inversely associated with the same adverse pregnancy outcomes. In a prospective periconception birth cohort study conducted in a tertiary centre, 146 singleton pregnancies with reliable pregnancy dating and nonmalformed live borns were investigated, comprised of 88 spontaneous and 58 IVF/ICSI pregnancies. Serial 3D ultrasound scans were performed from 6 to 12 weeks of gestation. As estimates of embryonic growth, CRL and embryonic volume (EV) were measured using the I-Space virtual reality system. General characteristics were obtained from self-administered questionnaires at enrolment. Fetal growth parameters at 20 weeks and birthweight were obtained from medical records. To assess associations between IVF/ICSI and embryonic growth trajectories, estimated fetal weight and birthweight, stepwise linear mixed model analyses and linear regression analyses were performed using square root transformed CRL and fourth root transformed EV. In 146 pregnancies, 934 ultrasound scans were performed of which 849 (90.9%) CRLs and 549 (58.8%) EVs could be measured. Embryonic growth trajectories were comparable between IVF/ICSI pregnancies and spontaneously conceived pregnancies (CRL: βIVF/ICSI = 0.10√mm; P = 0.10; EV: βIVF/ICSI = 0.03(4)√cm³; P = 0.13). Estimated fetal weight and birthweight were also comparable between both

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

PubMed Central

Fan, Yong; Li, Rong; Huang, Jin; Yu, Yang; Qiao, Jie

2013-01-01

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells. PMID:23255130

Case Study: Organotypic human in vitro models of embryonic morphogenetic fusion

EPA Science Inventory

Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell...

Do sarcomere length, collagen content, pH, intramuscular fat and desmin degradation explain variation in the tenderness of three ovine muscles?

PubMed

Starkey, Colin P; Geesink, Geert H; Collins, Damian; Hutton Oddy, V; Hopkins, David L

2016-03-01

The longissimus (n=118) (LL), semimembranosus (n=104) (SM) and biceps femoris (n=134) (BF) muscles were collected from lamb and sheep carcases and aged for 5days (LL and SM) and 14days (BF) to study the impact of muscle characteristics on tenderness as assessed by shear force (SF) and sensory evaluation. The impact of gender, animal age, collagen content, sarcomere length (SL), desmin degradation, ultimate pH and intramuscular fat (IMF) on tenderness was examined. The main factors which influenced SF of the LL were IMF, SL and desmin degradation, but for sensory tenderness, IMF, ultimate pH and gender were the main factors. The SF and sensory tenderness of the SM was best predicted by the degree of desmin degradation. For the BF soluble collagen and animal age both influenced SF. Different factors affect tenderness across muscles and not one prediction model applied across all muscles equally well. Copyright © 2015 Elsevier Ltd. All rights reserved.

Microrna expression signatures predict patient progression and disease outcome in pediatric embryonal central nervous system neoplasms.

PubMed

Braoudaki, Maria; Lambrou, George I; Giannikou, Krinio; Milionis, Vasilis; Stefanaki, Kalliopi; Birks, Diane K; Prodromou, Neophytos; Kolialexi, Aggeliki; Kattamis, Antonis; Spiliopoulou, Chara A; Tzortzatou-Stathopoulou, Fotini; Kanavakis, Emmanouel

2014-12-31

pediatric embryonal CNS neoplasms studied. Deeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.

Endothelin-1 signalling controls early embryonic heart rate in vitro and in vivo.

PubMed

Karppinen, S; Rapila, R; Mäkikallio, K; Hänninen, S L; Rysä, J; Vuolteenaho, O; Tavi, P

2014-02-01

Spontaneous activity of embryonic cardiomyocytes originates from sarcoplasmic reticulum (SR) Ca(2+) release during early cardiogenesis. However, the regulation of heart rate during embryonic development is still not clear. The aim of this study was to determine how endothelin-1 (ET-1) affects the heart rate of embryonic mice, as well as the pathway through which it exerts its effects. The effects of ET-1 and ET-1 receptor inhibition on cardiac contraction were studied using confocal Ca(2+) imaging of isolated mouse embryonic ventricular cardiomyocytes and ultrasonographic examination of embryonic cardiac contractions in utero. In addition, the amount of ET-1 peptide and ET receptor a (ETa) and b (ETb) mRNA levels were measured during different stages of development of the cardiac muscle. High ET-1 concentration and expression of both ETa and ETb receptors was observed in early cardiac tissue. ET-1 was found to increase the frequency of spontaneous Ca(2+) oscillations in E10.5 embryonic cardiomyocytes in vitro. Non-specific inhibition of ET receptors with tezosentan caused arrhythmia and bradycardia in isolated embryonic cardiomyocytes and in whole embryonic hearts both in vitro (E10.5) and in utero (E12.5). ET-1-mediated stimulation of early heart rate was found to occur via ETb receptors and subsequent inositol trisphosphate receptor activation and increased SR Ca(2+) leak. Endothelin-1 is required to maintain a sufficient heart rate, as well as to prevent arrhythmia during early development of the mouse heart. This is achieved through ETb receptor, which stimulates Ca(2+) leak through IP3 receptors. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Transplantation of embryonic stem cell-derived dopaminergic neurons in MPTP-treated monkeys.

PubMed

Takahashi, Jun; Takagi, Yasushi; Saiki, Hidemoto

2009-01-01

One of the target diseases of cell-replacement therapy is Parkinson's disease. Clinical experiences with fetal dopaminergic cell graft have shown that the therapy is effective, but limited and accompanied by side effects, such as dyskinesia. So, the therapy needs to be further improved and sophisticated. Embryonic stem (ES) cells are expected to be another donor cell for the treatment, because of its proliferative and differentiation capacities. For clinical application, experiments using non-human primates are important, because size, anatomy, and biological characteristics of the brain are different between rodents and primates. Here, we would like to discuss induction of dopaminergic neurons from monkey ES cells and cell transplantation into the brain of monkey Parkinson's disease model.

Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

EPA Science Inventory

The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

EXTRA-EMBRYONIC-SPECIFIC IMPRINTED EXPRESSION IS RESTRICTED TO DEFINED LINEAGES IN THE POST-IMPLANTATION EMBRYO

PubMed Central

Hudson, Quanah J.; Seidl, Christine I.M.; Kulinski, Tomasz M.; Huang, Ru; Warczok, Katarzyna E.; Bittner, Romana; Bartolomei, Marisa S.; Barlow, Denise P.

2011-01-01

A subset of imprinted genes in the mouse have been reported to show imprinted expression that is restricted to the placenta, a short-lived extra-embryonic organ. Notably these so-called 'placental-specific' imprinted genes are expressed from both parental alleles in embryo and adult tissues. The placenta is an embryonic-derived organ that is closely associated with maternal tissue and as a consequence, maternal contamination can be mistaken for maternal-specific imprinted expression. The complexity of the placenta, which arises from multiple embryonic lineages, poses additional problems in accurately assessing allele-specific repressive epigenetic modifications in genes that also show lineage-specific silencing in this organ. These problems require that extra evidence be obtained to support the imprinted status of genes whose imprinted expression is restricted to the placenta. We show here that the extra-embryonic visceral yolk sac (VYS), a nutritive membrane surrounding the developing embryo, shows a similar 'extra-embryonic-lineage-specific' pattern of imprinted expression. We present an improved enzymatic technique for separating the bilaminar VYS and show that this pattern of imprinted expression is restricted to the endoderm layer. Finally, we show that VYS 'extra-embryonic-lineage-specific' imprinted expression is regulated by DNA methylation in a similar manner as shown for genes showing multi-lineage imprinted expression in extra-embryonic, embryonic and adult tissues. These results show that the VYS is an improved model for studying the epigenetic mechanisms regulating extra-embryonic-lineage-specific imprinted expression. PMID:21354127

Ras-dva Is a Novel Pit-1- and Glucocorticoid-Regulated Gene in the Embryonic Anterior Pituitary Gland

PubMed Central

Ellestad, Laura E.

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5′-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5′-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland. PMID:23161868

Ras-dva is a novel Pit-1- and glucocorticoid-regulated gene in the embryonic anterior pituitary gland.

PubMed

Ellestad, Laura E; Porter, Tom E

2013-01-01

Glucocorticoids play a role in functional differentiation of pituitary somatotrophs and lactotrophs during embryogenesis. Ras-dva was identified as a gene regulated by anterior neural fold protein-1/homeobox expressed in embryonic stem cells-1, a transcription factor known to be critical in pituitary development, and has an expression profile in the chicken embryonic pituitary gland that is consistent with in vivo regulation by glucocorticoids. The objective of this study was to characterize expression and regulation of ras-dva mRNA in the developing chicken anterior pituitary. Pituitary ras-dva mRNA levels increased during embryogenesis to a maximum on embryonic day (e) 18 and then decreased and remained low or undetectable after hatch. Ras-dva expression was highly enriched in the pituitary gland on e18 relative to other tissues examined. Glucocorticoid treatment of pituitary cells from mid- and late-stage embryos rapidly increased ras-dva mRNA, suggesting it may be a direct transcriptional target of glucocorticoids. A reporter construct driven by 4 kb of the chicken ras-dva 5'-flanking region, containing six putative pituitary-specific transcription factor-1 (Pit-1) binding sites and two potential glucocorticoid receptor (GR) binding sites, was highly activated in embryonic pituitary cells and up-regulated by corticosterone. Mutagenesis of the most proximal Pit-1 site decreased promoter activity in chicken e11 pituitary cells, indicating regulation of ras-dva by Pit-1. However, mutating putative GR binding sites did not substantially reduce induction of ras-dva promoter activity by corticosterone, suggesting additional DNA elements within the 5'-flanking region are responsible for glucocorticoid regulation. We have identified ras-dva as a glucocorticoid-regulated gene that is likely expressed in cells of the Pit-1 lineage within the developing anterior pituitary gland.

Do embryonic polar bodies commit suicide?

PubMed

Fabian, Dušan; Čikoš, Štefan; Rehák, Pavol; Koppel, Juraj

2014-02-01

The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

The business of human embryonic stem cell research and an international analysis of relevant laws.

PubMed

De Trizio, Ella; Brennan, Christopher S

2004-01-01

Few sciences have held out such therapeutic promise and correspondingly stirred so much controversy in countries throughout the world as the developing science surrounding human embryonic stem cells. Since the first reported development of several lines of human embryonic stem cells in 1988, many governments around the world have attempted to address the thorny ethical issues raised by human embryonic stem cell research by the passage of laws. In some cases these laws have directly regulated governmental funding of the science; in other cases they have created a legal environment that has either encouraged or discouraged both governmental and private funding of the science. This article first differentiates human embryonic stem cells from other types of stem cells and frames the ethical controversy surrounding human embryonic stem cell research, then surveys laws governing human embryonic stem cell research in various scientifically advanced countries located throughout the Pacific Rim, Europe and North America and explains the impact these laws have had on governmental and private funding of human embryonic stem cell research.

Maternal Gdf3 is an obligatory cofactor in Nodal signaling for embryonic axis formation in zebrafish

PubMed Central

Bisgrove, Brent W; Su, Yi-Chu

2017-01-01

Zebrafish Gdf3 (Dvr1) is a member of the TGFβ superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3. Surprisingly, engineered homozygous mutants in zebrafish have no apparent phenotype. Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA, demonstrating that Gdf3 is required only early in development, beyond which mutants are viable and fertile. Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1. Signaling driven by TGFβ ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants, indicating that Gdf3 functions at the same pathway step as Nodal. Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when co-expressed with endogenous Nodal. Together, these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis. PMID:29140249

Redundant role of protein kinase C delta and epsilon during mouse embryonic development.

PubMed

Carracedo, Sergio; Sacher, Frank; Brandes, Gudrun; Braun, Ursula; Leitges, Michael

2014-01-01

Protein Kinase C delta and epsilon are mediators of important cellular events, such as cell proliferation, migration or apoptosis. The formation of blood vessels, i.e., vasculo- and angiogenesis, is a process where these isoforms have also been shown to participate. However, mice deficient in either Protein Kinase C delta or epsilon are viable and therefore their individual contribution to the formation of the vasculature appeared so far dispensable. In this study, we show that double null mutation of Protein Kinase C delta and epsilon causes embryonic lethality at approximately E9.5. At this stage, whole mount staining of the endothelial marker CD31 in double null embryos revealed defective blood vessel formation. Moreover, culture of double deficient mouse allantois showed impaired endothelial cell organization, and analyses of double deficient embryo sections showed dilated vessels, decreased endothelial-specific adherent junctions, and decreased contact of endothelial cells with mural cells. Protein kinase C delta and epsilon also appeared essential for vascular smooth muscle cell differentiation, since α-smooth muscle actin, a classical marker for vascular smooth muscle cells, was almost undetectable in double deficient embryonic aorta at E9.5. Subsequent qPCR analyses showed decreased VE-cadherin, Vegfr2, Cd31, Cdh2, Ets1, and Fli-1, among other angiogenesis related transcripts in double deficient embryos. Taken together, these data suggest for the first time an in vivo redundant role between members of the novel Protein Kinase C subfamily that allows for mutual compensation during mouse embryonic development, with vasculogenesis/angiogenesis as an obvious common function of these two Protein Kinase Cs. Protein Kinase C delta and epsilon might therefore be useful targets for inhibiting vasculo- and/or angiogenesis.

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

PubMed

Rubinstein, M; Japón, M A; Low, M J

1993-06-11

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

PubMed Central

Rubinstein, M; Japón, M A; Low, M J

1993-01-01

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes. Images PMID:8392702

Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

PubMed Central

Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

2014-01-01

Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

Intact calcium signaling in adrenergic-deficient embryonic mouse hearts.

PubMed

Peoples, Jessica N; Taylor, David G; Katchman, Alexander N; Ebert, Steven N

2018-01-22

Mouse embryos that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to disruption of the dopamine beta-hydroxylase (Dbh -/- ) gene inevitably perish from heart failure during mid-gestation. Since adrenergic stimulation is well-known to enhance calcium signaling in developing as well as adult myocardium, and impairments in calcium signaling are typically associated with heart failure, we hypothesized that adrenergic-deficient embryonic hearts would display deficiencies in cardiac calcium signaling relative to adrenergic-competent controls at a developmental stage immediately preceding the onset of heart failure, which first appears beginning or shortly after mouse embryonic day 10.5 (E10.5). To test this hypothesis, we used ratiometric fluorescent calcium imaging techniques to measure cytosolic calcium transients, [Ca 2+ ] i in isolated E10.5 mouse hearts. Our results show that spontaneous [Ca 2+ ] i oscillations were intact and robustly responded to a variety of stimuli including extracellular calcium (5 mM), caffeine (5 mM), and NE (100 nM) in a manner that was indistinguishable from controls. Further, we show similar patterns of distribution (via immunofluorescent histochemical staining) and activity (via patch-clamp recording techniques) for the major voltage-gated plasma membrane calcium channel responsible for the L-type calcium current, I Ca,L , in adrenergic-deficient and control embryonic cardiac cells. These results demonstrate that despite the absence of vital adrenergic hormones that consistently leads to embryonic lethality in vivo, intracellular and extracellular calcium signaling remain essentially intact and functional in embryonic mouse hearts through E10.5. These findings suggest that adrenergic stimulation is not required for the development of intracellular calcium oscillations or extracellular calcium signaling through I Ca,L and that aberrant calcium signaling does not likely contribute

Hippo/Yap signaling controls epithelial progenitor cell proliferation and differentiation in the embryonic and adult lung.

PubMed

Lange, Alexander W; Sridharan, Anusha; Xu, Yan; Stripp, Barry R; Perl, Anne-Karina; Whitsett, Jeffrey A

2015-02-01

The Hippo/Yap pathway is a well-conserved signaling cascade that regulates cell proliferation and differentiation to control organ size and stem/progenitor cell behavior. Following airway injury, Yap was dynamically regulated in regenerating airway epithelial cells. To determine the role of Hippo signaling in the lung, the mammalian Hippo kinases, Mst1 and Mst2, were deleted in epithelial cells of the embryonic and mature mouse lung. Mst1/2 deletion in the fetal lung enhanced proliferation and inhibited sacculation and epithelial cell differentiation. The transcriptional inhibition of cell proliferation and activation of differentiation during normal perinatal lung maturation were inversely regulated following embryonic Mst1/2 deletion. Ablation of Mst1/2 from bronchiolar epithelial cells in the adult lung caused airway hyperplasia and altered differentiation. Inhibitory Yap phosphorylation was decreased and Yap nuclear localization and transcriptional targets were increased after Mst1/2 deletion, consistent with canonical Hippo/Yap signaling. YAP potentiated cell proliferation and inhibited differentiation of human bronchial epithelial cells in vitro. Loss of Mst1/2 and expression of YAP regulated transcriptional targets controlling cell proliferation and differentiation, including Ajuba LIM protein. Ajuba was required for the effects of YAP on cell proliferation in vitro. Hippo/Yap signaling regulates Ajuba and controls proliferation and differentiation of lung epithelial progenitor cells. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Dihydroartemisinin promotes angiogenesis during the early embryonic development of zebrafish

PubMed Central

Ba, Qian; Duan, Juan; Tian, Jia-qiang; Wang, Zi-liang; Chen, Tao; Li, Xiao-guang; Chen, Pei-zhan; Wu, Song-jie; Xiang, Li; Li, Jing-quan; Chu, Rui-ai; Wang, Hui

2013-01-01

Aim: To investigate the embryotoxicity of dihydroartemisinin (DHA), the main active metabolite of artemisinin, in zebrafish, and explore the corresponding mechanisms. Methods: The embryos of wild type and TG (flk1:GFP) transgenic zebrafish were exposed to DHA. Developmental phenotypes of the embryos were observed. Development of blood vessels was directly observed in living embryos of TG (flk1:GFP) transgenic zebrafish under fluorescence microscope. The expression of angiogenesis marker genes vegfa, flk1, and flt1 in the embryos was detected using real-time PCR and RNA in situ hybridization assays. Results: Exposure to DHA (1–10 mg/L) dose-dependently caused abnormal zebrafish embryonic phenotypes in the early developmental stage. Furthermore, exposure to DHA (10 mg/L) resulted in more pronounced embryonic angiogenesis in TG (flk1:GFP) zebrafish line. Exposure to DHA (10 mg/L) significantly increased the mRNA expression of vegfa, flk1, and flt1 in the embryos. Knockdown of the flk1 protein partially blocked the effects of DHA on embryogenesis. Conclusion: DHA causes abnormal embryonic phenotypes and promotes angiogenesis in zebrafish early embryonic development, demonstrating the potential embryotoxicity of DHA. PMID:23708556

A new role for muscle segment homeobox genes in mammalian embryonic diapause

PubMed Central

Cha, Jeeyeon; Sun, Xiaofei; Bartos, Amanda; Fenelon, Jane; Lefèvre, Pavine; Daikoku, Takiko; Shaw, Geoff; Maxson, Robert; Murphy, Bruce D.; Renfree, Marilyn B.; Dey, Sudhansu K.

2013-01-01

Mammalian embryonic diapause is a phenomenon defined by the temporary arrest in blastocyst growth and metabolic activity within the uterus which synchronously becomes quiescent to blastocyst activation and implantation. This reproductive strategy temporally uncouples conception from parturition until environmental or maternal conditions are favourable for the survival of the mother and newborn. The underlying molecular mechanism by which the uterus and embryo temporarily achieve quiescence, maintain blastocyst survival and then resume blastocyst activation with subsequent implantation remains unknown. Here, we show that uterine expression of Msx1 or Msx2, members of an ancient, highly conserved homeobox gene family, persists in three unrelated mammalian species during diapause, followed by rapid downregulation with blastocyst activation and implantation. Mice with uterine inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation. Remarkably, the North American mink and Australian tammar wallaby share similar expression patterns of MSX1 or MSX2 as in mice—it persists during diapause and is rapidly downregulated upon blastocyst activation and implantation. Evidence from mouse studies suggests that the effects of Msx genes in diapause are mediated through Wnt5a, a known transcriptional target of uterine Msx. These studies provide strong evidence that the Msx gene family constitutes a common conserved molecular mediator in the uterus during embryonic diapause to improve female reproductive fitness. PMID:23615030

A new role for muscle segment homeobox genes in mammalian embryonic diapause.

PubMed

Cha, Jeeyeon; Sun, Xiaofei; Bartos, Amanda; Fenelon, Jane; Lefèvre, Pavine; Daikoku, Takiko; Shaw, Geoff; Maxson, Robert; Murphy, Bruce D; Renfree, Marilyn B; Dey, Sudhansu K

2013-04-24

Mammalian embryonic diapause is a phenomenon defined by the temporary arrest in blastocyst growth and metabolic activity within the uterus which synchronously becomes quiescent to blastocyst activation and implantation. This reproductive strategy temporally uncouples conception from parturition until environmental or maternal conditions are favourable for the survival of the mother and newborn. The underlying molecular mechanism by which the uterus and embryo temporarily achieve quiescence, maintain blastocyst survival and then resume blastocyst activation with subsequent implantation remains unknown. Here, we show that uterine expression of Msx1 or Msx2, members of an ancient, highly conserved homeobox gene family, persists in three unrelated mammalian species during diapause, followed by rapid downregulation with blastocyst activation and implantation. Mice with uterine inactivation of Msx1 and Msx2 fail to achieve diapause and reactivation. Remarkably, the North American mink and Australian tammar wallaby share similar expression patterns of MSX1 or MSX2 as in mice-it persists during diapause and is rapidly downregulated upon blastocyst activation and implantation. Evidence from mouse studies suggests that the effects of Msx genes in diapause are mediated through Wnt5a, a known transcriptional target of uterine Msx. These studies provide strong evidence that the Msx gene family constitutes a common conserved molecular mediator in the uterus during embryonic diapause to improve female reproductive fitness.

Functional Analysis of the Drosophila Embryonic Germ Cell Transcriptome by RNA Interference

PubMed Central

Bujna, Ágnes; Vilmos, Péter; Spirohn, Kerstin; Boutros, Michael; Erdélyi, Miklós

2014-01-01

In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general. PMID:24896584

Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells.

PubMed

Lu, Qin; Liu, Yinan; Wang, Yang; Wang, Weiping; Yang, Zhe; Li, Tao; Tian, Yuyao; Chen, Ping; Ma, Kangtao; Jia, Zhuqing; Zhou, Chunyan

2017-06-30

To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10 -4 M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata 4, α- Mhc , β- Mhc , and Tnnt 2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp 1, Brachyury T, Eomes, Isl 1 , Gata 4 , Nkx 2.5 , Tbx 5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/β-catenin and Notch signaling but promotes the expression of fibroblast growth factor ( Fgf 8), Fgf 10, and Nodal at early stage, and bone morphogenetic protein 2 ( Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 µM) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin. © 2017 The Author(s).

Growth trajectories of the human embryonic head and periconceptional maternal conditions.

PubMed

Koning, I V; Baken, L; Groenenberg, I A L; Husen, S C; Dudink, J; Willemsen, S P; Gijtenbeek, M; Koning, A H J; Reiss, I K M; Steegers, E A P; Steegers-Theunissen, R P M

2016-05-01

Can growth trajectories of the human embryonic head be created using 3D ultrasound (3D-US) and virtual reality (VR) technology, and be associated with second trimester fetal head size and periconceptional maternal conditions? Serial first trimester head circumference (HC) and head volume (HV) measurements were used to create reliable growth trajectories of the embryonic head, which were significantly associated with fetal head size and periconceptional maternal smoking, age and ITALIC! in vitro fertilization (IVF)/intra-cytoplasmic sperm injection (ICSI) treatment. Fetal growth is influenced by periconceptional maternal conditions. We selected 149 singleton pregnancies with a live born non-malformed fetus from the Rotterdam periconception cohort. Bi-parietal diameter and occipital frontal diameter to calculate HC, HV and crown-rump length (CRL) were measured weekly between 9 + 0 and 12 + 6 weeks gestational age (GA) using 3D-US and VR. Fetal HC was obtained from second trimester structural anomaly scans. Growth trajectories of the embryonic head were created with general additive models and linear mixed models were used to estimate associations with maternal periconceptional conditions as a function of GA and CRL, respectively. A total of 303 3D-US images of 149 pregnancies were eligible for embryonic head measurements (intra-class correlation coefficients >0.99). Associations were found between embryonic HC and fetal HC ( ITALIC! ρ = 0.617, ITALIC! P < 0.001) and between embryonic HV and fetal HC ( ITALIC! ρ = 0.660, ITALIC! P < 0.001) in ITALIC! Z-scores. Maternal periconceptional smoking was associated with decreased, and maternal age and IVF/ICSI treatment with increased growth trajectories of the embryonic head measured by HC and HV (All ITALIC! P < 0.05). The consequences of the small effect sizes for neurodevelopmental outcome need further investigation. As the study population consists largely of tertiary hospital patients, external validity should be

Real-time analysis of Drosophila post-embryonic haemocyte behaviour.

PubMed

Sampson, Christopher J; Williams, Michael J

2012-01-01

The larval stage of the model organism Drosophila is frequently used to study host-pathogen interactions. During embryogenesis the cellular arm of the immune response, consisting of macrophage-like cells known as plasmatocytes, is extremely motile and functions to phagocytise pathogens and apoptotic bodies, as well as produce extracellular matrix. The cellular branch of the larval (post-embryonic) innate immune system consists of three cell types--plasmatocytes, crystal cells and lamellocytes--which are involved in the phagocytosis, encapsulation and melanisation of invading pathogens. Post-embryonic haemocyte motility is poorly understood thus further characterisation is required, for the purpose of standardisation. In order to examine post-embryonic haemocyte cytoskeletal dynamics or migration, the most commonly used system is in vitro cell lines. The current study employs an ex vivo system (an adaptation of in vitro cell incubation using primary cells), in which primary larval or pre-pupal haemocytes are isolated for short term analysis, in order to discover various aspects of their behaviour during events requiring cytoskeleton dynamics. The ex vivo method allows for real-time analysis and manipulation of primary post-embryonic haemocytes. This technique was used to characterise, and potentially standardised, larval and pre-pupal haemocyte cytoskeleton dynamics, assayed on different extracellular matrices. Using this method it was determined that, while larval haemocytes are unable to migrate, haemocytes recovered from pre-pupae are capable of migration.

Targeted Lipidomic Analysis of Oxysterols in the Embryonic Central Nervous System

PubMed Central

Wang, Yuqin; Sousa, Kyle M.; Bodin, Karl; Theofilopoulos, Spyridon; Sacchetti, Paola; Hornshaw, Martin; Woffendin, Gary; Karu, Kersti; Sjövall, Jan; Arenas, Ernest; Griffiths, William J.

2009-01-01

Summary In this study two regions of embryonic (E11) mouse central nervous system (CNS) have been profiled for their unesterified sterol content. Using high-performance liquid chromatography (HPLC) – mass spectrometry (MS) and tandem mass spectrometry (MSn) low levels of oxysterols (estimated 2 – 165 ng/g wet weight) were identified in cortex (Ctx) and spinal cord (Sc). The identified oxysterols include 7α-, 7β-, 22R-, 24S-, 25- and 27-hydroxycholesterol; 24,25- and 24,27-dihydroxycholesterol; and 24S,25-epoxycholesterol. Of these, 24S-hydroxycholesterol is biosynthesised exclusively in brain. In comparison to adult mouse where the 24S-hydroxycholesterol level is about 40 μg/g in brain the level of 24S-hydroxycholesterol reported here (estimated 26 ng/g in Ctx and 13 ng/g in Sc) is extremely low. Interestingly, the level of 24S,25-epoxycholesterol in both CNS regions (estimated 165 ng/g in Ctx and 91 ng/g in Sc) is somewhat higher than the levels of the hydroxycholesterols. This oxysterol is formed in parallel to cholesterol via a shunt of the mevalonate pathway and its comparatively high abundance may be a reflection of a high rate of cholesterol synthesis at this stage of development. Levels of cholesterol (estimated 1.25 mg/g in Ctx and 1.15 mg/g in Sc) and its precursors were determined by gas chromatography – mass spectrometry (GC-MS). In both CNS regions cholesterol levels were found to be lower than those reported in the adult, but in relation to cholesterol the levels of cholesterol precursors were higher than found in adult indicating a high rate of cholesterol synthesis. In summary, our data provide evidence for the presence of endogenous oxysterols in two brain regions of the developing CNS. Moreover, while most of the enzymes involved in hydroxysterol synthesis are minimally active at E11, our results suggest that the mevalonate pathway is significantly active, opening up the possibility for a function of 24S,25-epoxycholesterol during brain

Human embryonic stem cell research: an intercultural perspective.

PubMed

Walters, LeRoy

2004-03-01

In 1998, researchers discovered that embryonic stem cells could be derived from early human embryos. This discovery has raised a series of ethical and public-policy questions that are now being confronted by multiple international organizations, nations, cultures, and religious traditions. This essay surveys policies for human embryonic stem cell research in four regions of the world, reports on the recent debate at the United Nations about one type of such research, and reviews the positions that various religious traditions have adopted regarding this novel type of research. In several instances the religious traditions seem to have influenced the public-policy debates.

Maintaining embryonic stem cell pluripotency with Wnt signaling.

PubMed

Sokol, Sergei Y

2011-10-01

Wnt signaling pathways control lineage specification in vertebrate embryos and regulate pluripotency in embryonic stem (ES) cells, but how the balance between progenitor self-renewal and differentiation is achieved during axis specification and tissue patterning remains highly controversial. The context- and stage-specific effects of the different Wnt pathways produce complex and sometimes opposite outcomes that help to generate embryonic cell diversity. Although the results of recent studies of the Wnt/β-catenin pathway in ES cells appear to be surprising and controversial, they converge on the same conserved mechanism that leads to the inactivation of TCF3-mediated repression.

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates.

PubMed

Sternberg, Hal; Kidd, Jennifer; Murai, James T; Jiang, Jianjie; Rinon, Ariel; Erickson, Isaac E; Funk, Walter D; Wang, Qian; Chapman, Karen B; Vangsness, C Thomas; West, Michael D

2013-03-01

The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

Human Embryonic Stem Cell Therapy in Crohn’s Disease: A Case Report

PubMed Central

Shroff, Geeta

2016-01-01

Patient: Male, 21 Final Diagnosis: Crohn’s disease Symptoms: Intolerance to specific foods • abdominal pain and diarrhea Medication: Human embryonic stem cell therapy Clinical Procedure: Human embryonic stem cell transplantation Specialty: Gastroenterology Objective: Unusual or unexpected effect of treatment Background: Crohn’s disease is a chronic inflammatory disease of the intestines, mainly the colon and ileum, related with ulcers and fistulae. It is estimated to affect 565 000 people in the United States. Currently available therapies, such as antibiotics, thiopurines, and anti-tumor necrosis factor-alpha agents, are only observed to reduce the complications associated with Crohn’s disease and to improve quality of life, but cannot cure the disease. Stem cell therapy appears to have certain advantages over conventional therapies. Our study aimed to evaluate the efficacy of human embryonic stem cell therapy in a patient with Crohn’s disease. Case Report: A 21-year-old male with chief complaints of intolerance to specific foods, abdominal pain, and diarrhea underwent human embryonic stem cell therapy for two months. After undergoing human embryonic stem cell therapy, the patient showed symptomatic relief. He had no complaints of back pain, abdominal pain, or diarrhea and had improved digestion. The patient had no signs and symptoms of skin infection, and had improved limb stamina, strength, and endurance. The condition of patient was stable after the therapy. Conclusions: Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn’s disease. PMID:26923312

G-quadruplexes as novel cis-elements controlling transcription during embryonic development.

PubMed

David, Aldana P; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B

2016-05-19

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

PubMed Central

Min, Irene M.; Waterfall, Joshua J.; Core, Leighton J.; Munroe, Robert J.; Schimenti, John; Lis, John T.

2011-01-01

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, “bivalent” ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5′ proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation. PMID:21460038

Nitric Oxide Synthase-3 Promotes Embryonic Development of Atrioventricular Valves

PubMed Central

Liu, Yin; Lu, Xiangru; Xiang, Fu-Li; Lu, Man; Feng, Qingping

2013-01-01

Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3−/− mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3−/− compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3−/− mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1+ cells in the AV cushion were decreased in NOS3−/− compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ), bone morphogenetic protein (BMP2) and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3−/− compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency. PMID:24204893

Genetic engineering of human embryonic stem cells with lentiviral vectors.

PubMed

Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

2005-08-01

Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

The birth of embryonic pluripotency

PubMed Central

Boroviak, Thorsten; Nichols, Jennifer

2014-01-01

Formation of a eutherian mammal requires concurrent establishment of embryonic and extraembryonic lineages. The functions of the trophectoderm and primitive endoderm are to enable implantation in the maternal uterus, axis specification and delivery of nutrients. The pluripotent epiblast represents the founding cell population of the embryo proper, which is protected from ectopic and premature differentiation until it is required to respond to inductive cues to form the fetus. While positional information plays a major role in specifying the trophoblast lineage, segregation of primitive endoderm from epiblast depends upon gradual acquisition of transcriptional identity, directed but not initiated by fibroblast growth factor (FGF) signalling. Following early cleavage divisions and formation of the blastocyst, cells of the inner cell mass lose totipotency. Developing epiblast cells transiently attain the state of naive pluripotency and competence to self-renew in vitro as embryonic stem cells and in vivo by means of diapause. This property is lost after implantation as the epiblast epithelializes and becomes primed in preparation for gastrulation and subsequent organogenesis. PMID:25349450

GLUCOCORTICOID RECEPTOR EXPRESSION DURING THE DEVELOPMENT OF THE EMBRYONIC MOUSE SECONDARY PALATE

EPA Science Inventory

Glucocorticoids are important regulators of embryonic growth and development. hese effects are mediated through glucocorticoid receptors (GR) which bind to glucocorticoid response elements upstream of regulated genes. his study examines the expression of GR and GR mRNA in embryon...

Phenotypically anchored transcriptome profiling of developmental exposure to the antimicrobial agent, triclosan, reveals hepatotoxicity in embryonic zebrafish

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

Haggard, Derik E.

Triclosan (TCS) is an antimicrobial agent commonly found in a variety of personal care products and cosmetics. TCS readily enters the environment through wastewater and is detected in human plasma, urine, and breast milk due to its widespread use. Studies have implicated TCS as a disruptor of thyroid and estrogen signaling; therefore, research examining the developmental effects of TCS is warranted. In this study, we used embryonic zebrafish to investigate the developmental toxicity and potential mechanism of action of TCS. Embryos were exposed to graded concentrations of TCS from 6 to 120 hours post-fertilization (hpf) and the concentration where 80%more » of the animals had mortality or morbidity at 120 hpf (EC{sub 80}) was calculated. Transcriptomic profiling was conducted on embryos exposed to the EC{sub 80} (7.37 μM). We identified a total of 922 significant differentially expressed transcripts (FDR adjusted P-value ≤ 0.05; fold change ≥ 2). Pathway and gene ontology enrichment analyses identified biological networks and transcriptional hubs involving normal liver functioning, suggesting TCS may be hepatotoxic in zebrafish. Tissue-specific gene enrichment analysis further supported the role of the liver as a target organ for TCS toxicity. We also examined the in vitro bioactivity profile of TCS reported by the ToxCast screening program. TCS had a diverse bioactivity profile and was a hit in 217 of the 385 assay endpoints we identified. We observed similarities in gene expression and hepatic steatosis assays; however, hit data for TCS were more concordant with the hypothesized CAR/PXR activity of TCS from rodent and human in vitro studies. - Highlights: • Triclosan is a common antimicrobial agent with widespread human exposure. • Exposure to the triclosan EC{sub 80} causes robust gene expression changes in zebrafish. • The liver may be a target organ of triclosan toxicity in embryonic zebrafish. • Triclosan disrupts normal liver functioning and

Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

PubMed Central

Kharraz, Yacine; Salmand, Pierre-Adrien; Camus, Anne; Auriol, Jacques; Gueydan, Cyril; Kruys, Véronique; Morello, Dominique

2010-01-01

Background TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation. Methodology/Principal Findings To examine potential TIAR tissue-specificity in various cellular contexts, either embryonic or adult, we constructed a TIAR transgenic allele (loxPGFPloxPTIAR) allowing the conditional expression of TIAR protein upon Cre recombinase activity. Here, we report the role of TIAR during mouse embryogenesis. We observed that early TIAR overexpression led to low transgene transmission associated with embryonic lethality starting at early post-implantation stages. Interestingly, while pre-implantation steps evolved correctly in utero, in vitro cultured embryos were very sensitive to culture medium. Control and transgenic embryos developed equally well in the G2 medium, whereas culture in M16 medium led to the phosphorylation of eIF2α that accumulated in cytoplasmic granules precluding transgenic blastocyst hatching. Our results thus reveal a differential TIAR-mediated embryonic response following artificial or natural growth environment. Conclusions/Significance This study reports the importance of the tightly balanced expression of the RNA-binding protein TIAR for normal embryonic development, thereby emphasizing the role of post-transcriptional regulations in early embryonic programming. PMID:20596534

Density gradient electrophoresis of cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Giranda, V.; Todd, P. W.

1985-01-01

Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

Jaw muscle development as evidence for embryonic repatterning in direct-developing frogs.

PubMed Central

Hanken, J; Klymkowsky, M W; Alley, K E; Jennings, D H

1997-01-01

The Puerto Rican direct-developing frog Eleutherodactylus coqui (Leptodactylidae) displays a novel mode of jaw muscle development for anuran amphibians. Unlike metamorphosing species, several larval-specific features never form in E. coqui; embryonic muscle primordia initially assume an abbreviated, mid-metamorphic configuration that is soon remodelled to form the adult morphology before hatching. Also lacking are both the distinct population of larval myofibres and the conspicuous, larval-to-adult myofibre turnover that are characteristic of muscle development in metamorphosing species. These modifications are part of a comprehensive alteration in embryonic cranial patterning that has accompanied life history evolution in this highly speciose lineage. Embryonic 'repatterning' in Eleutherodactylus may reflect underlying developmental mechanisms that mediate the integrated evolution of complex structures. Such mechanisms may also facilitate, in organisms with a primitively complex life cycle, the evolutionary dissociation of embryonic, larval, and adult features. PMID:9332017

Informing Stem Cell-Based Tendon Tissue Engineering Approaches with Embryonic Tendon Development.

PubMed

Okech, William; Kuo, Catherine K

Adult tendons fail to regenerate normal tissue after injury, and instead form dysfunctional scar tissue with abnormal mechanical properties. Surgical repair with grafts is the current standard to treat injuries, but faces significant limitations including pain and high rates of re-injury. To address this, we aim to regenerate new, normal tendons to replace dysfunctional tendons. A common approach to tendon tissue engineering is to design scaffolds and bioreactors based on adult tendon properties that can direct adult stem cell tenogenesis. Despite significant progress, advances have been limited due, in part, to a need for markers and potent induction cues. Our goal is to develop novel tendon tissue engineering approaches informed by embryonic tendon development. We are characterizing structure-property relationships of embryonic tendon to identify design parameters for three-dimensional scaffolds and bioreactor mechanical loading systems to direct adult stem cell tenogenesis. We will review studies in which we quantified changes in the mechanical and biochemical properties of tendon during embryonic development and elucidated specific mechanisms of functional property elaboration. We then examined the effects of these mechanical and biochemical factors on embryonic tendon cell behavior. Using custom-designed bioreactors, we also examined the effects of dynamic mechanical loading and growth factor treatment on embryonic tendon cells. Our findings have established cues to induce tenogenesis as well as metrics to evaluate differentiation. We finish by discussing how we have evaluated the tenogenic differentiation potential of adult stem cells by comparing their responses to that of embryonic tendon cells in these culture systems.

Intrauterine air impairs embryonic postimplantation development in mice.

PubMed

Liu, Ruonan; Li, Yimeng; Miao, Yanping; Wei, Yanhui; Guan, Mo; Zhou, Rongyan; Li, Xiangyun

2017-12-01

Although most embryologists load air bubbles into the catheter along with embryos during embryo transfer, the effects of these air bubbles on embryo transfer success rate are not clear. Air bubbles were nonsurgically injected into unilateral uterine horns of mice to demonstrate the negative effects of intrauterine air bubbles on embryonic development. Our data showed that when air bubbles are nonsurgically injected into unilateral uterine horns of pregnant 4days mice the litter size is significantly decreased. Four days after the introduction of air, abnormal decidua and dead conceptuses were detected in the uterine horns receiving the air bubbles. In addition, intrauterine air also significantly impaired murine embryo transfer success rates, and induced an increase in endometrial capillary permeability and decidualization in mice on day 4 of pseudopregnancy. These results strongly indicated that the air bubbles loaded into embryo transfer catheters to bracket the embryo-containing medium may have negative effect on embryonic implantation and development. Intrauterine air impaired murine embryonic postimplantation development, and this provided some clues for improving embryo transfer techniques in human. Copyright © 2017 Elsevier B.V. All rights reserved.

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

PubMed Central

Cohen-Tannoudji, Michel; Robine, Sylvie; Choulika, André; Pinto, Daniel; El Marjou, Fatima; Babinet, Charles; Louvard, Daniel; Jaisser, Frédéric

1998-01-01

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 × 10−6) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest. PMID:9488460

The Evolutionary Economics of Embryonic-Sac Fluids in Squamate Reptiles.

PubMed

Bonnet, Xavier; Naulleau, Guy; Shine, Richard

2017-03-01

The parchment-shelled eggs of squamate reptiles take up substantial water from the nest environment, enabling the conversion of yolk into neonatal tissue and buffering the embryo against the possibility of subsequent dry weather. During development, increasing amounts of water are stored in the embryonic sacs (i.e., membranes around the embryo: amnion, allantois, and chorion). The evolution of viviparity (prolonged uterine retention of developing embryos) means that embryonic-sac fluid storage now imposes a cost (increased maternal burdening), confers less benefit (because the mother buffers fetal water balance), and introduces a potential conflict among uterine siblings (for access to finite water supplies). Our data on nine species of squamate reptiles and published information on three species show that the embryonic-sac fluids comprise around 33% of neonatal mass in viviparous species versus 94% in full-term eggs of oviparous squamates. Data on parturition in 149 vipers (Vipera aspis, a viviparous species) show that larger offspring store more fluids in their fetal sacs and that an increase in litter size is associated with a decrease in fluid-sac mass per offspring. Overall, the evolutionary transition from oviparity to viviparity may have substantially altered selective forces on offspring packaging and created competition among offspring for access to water reserves during embryonic development.

Ethics and policy in embryonic stem cell research.

PubMed

Robertson, John A

1999-06-01

Embryonic stem cells, which have the potential to save many lives, must be recovered from aborted fetuses or live embyros. Although tissue from aborted fetuses can be used without moral complicity in the underlying abortion, obtaining stem cells from embryos necessarily kills them, thus raising difficult questions about the use of embryonic human material to save others. This article draws on previous controversies over embryo research and distinctions between intrinsic and symbolic moral status to analyze these issues. It argues that stem cell research with spare embryos produced during infertility treatment, or even embryos created specifically for research or therapeutic purposes, is ethically acceptable and should receive federal funding.

Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

PubMed

Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

2016-10-01

The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface. Copyright © 2016 Elsevier Inc. All rights reserved.

Cis-regulatory underpinnings of human GLI3 expression in embryonic craniofacial structures and internal organs.

PubMed

Abbasi, Amir A; Minhas, Rashid; Schmidt, Ansgar; Koch, Sabine; Grzeschik, Karl-Heinz

2013-10-01

The zinc finger transcription factor Gli3 is an important mediator of Sonic hedgehog (Shh) signaling. During early embryonic development Gli3 participates in patterning and growth of the central nervous system, face, skeleton, limb, tooth and gut. Precise regulation of the temporal and spatial expression of Gli3 is crucial for the proper specification of these structures in mammals and other vertebrates. Previously we reported a set of human intronic cis-regulators controlling almost the entire known repertoire of endogenous Gli3 expression in mouse neural tube and limbs. However, the genetic underpinning of GLI3 expression in other embryonic domains such as craniofacial structures and internal organs remain elusive. Here we demonstrate in a transgenic mice assay the potential of a subset of human/fish conserved non-coding sequences (CNEs) residing within GLI3 intronic intervals to induce reporter gene expression at known regions of endogenous Gli3 transcription in embryonic domains other than central nervous system (CNS) and limbs. Highly specific reporter expression was observed in craniofacial structures, eye, gut, and genitourinary system. Moreover, the comparison of expression patterns directed by these intronic cis-acting regulatory elements in mouse and zebrafish embryos suggests that in accordance with sequence conservation, the target site specificity of a subset of these elements remains preserved among these two lineages. Taken together with our recent investigations, it is proposed here that during vertebrate evolution the Gli3 expression control acquired multiple, independently acting, intronic enhancers for spatiotemporal patterning of CNS, limbs, craniofacial structures and internal organs. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

PubMed

Cebola, Inês; Rodríguez-Seguí, Santiago A; Cho, Candy H-H; Bessa, José; Rovira, Meritxell; Luengo, Mario; Chhatriwala, Mariya; Berry, Andrew; Ponsa-Cobas, Joan; Maestro, Miguel Angel; Jennings, Rachel E; Pasquali, Lorenzo; Morán, Ignasi; Castro, Natalia; Hanley, Neil A; Gomez-Skarmeta, Jose Luis; Vallier, Ludovic; Ferrer, Jorge

2015-05-01

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas.

Genetic Tools for Self-Organizing Culture of Mouse Embryonic Stem Cells via Small Regulatory RNA-Mediated Technologies, CRISPR/Cas9, and Inducible RNAi.

PubMed

Takata, Nozomu; Sakakura, Eriko; Sakuma, Tetsushi; Yamamoto, Takashi

2017-01-01

Approaches to investigate gene functions in experimental biology are becoming more diverse and reliable. Furthermore, several kinds of tissues and organs that possess their original identities can be generated in petri dishes from stem cells including embryonic, adult and induced pluripotent stem cells. Researchers now have several choices of experimental methods and their combinations to analyze gene functions in various biological systems. Here, as an example we describe one of the better protocols, which combines three-dimensional embryonic stem cell culture with small regulatory RNA-mediated technologies, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), and inducible RNA interference (RNAi). This protocol allows investigation of genes of interest to better understand gene functions in target tissues (or organs) during in vitro development.

Arrested embryonic development: a review of strategies to delay hatching in egg-laying reptiles

PubMed Central

Rafferty, Anthony R.; Reina, Richard D.

2012-01-01

Arrested embryonic development involves the downregulation or cessation of active cell division and metabolic activity, and the capability of an animal to arrest embryonic development results in temporal plasticity of the duration of embryonic period. Arrested embryonic development is an important reproductive strategy for egg-laying animals that provide no parental care after oviposition. In this review, we discuss each type of embryonic developmental arrest used by oviparous reptiles. Environmental pressures that might have directed the evolution of arrest are addressed and we present previously undiscussed environmentally dependent physiological processes that may occur in the egg to bring about arrest. Areas for future research are proposed to clarify how ecology affects the phenotype of developing embryos. We hypothesize that oviparous reptilian mothers are capable of providing their embryos with a level of phenotypic adaptation to local environmental conditions by incorporating maternal factors into the internal environment of the egg that result in different levels of developmental sensitivity to environmental conditions after they are laid. PMID:22438503

Embryonic vaccines against cancer: an early history.

PubMed

Brewer, Bradley G; Mitchell, Robert A; Harandi, Amir; Eaton, John W

2009-06-01

Almost 100 years have passed since the seminal observations of Schöne showing that vaccination of animals with fetal tissue would prevent the growth of transplantable tumors. Many subsequent reports have affirmed the general idea that immunologic rejection of transplantable tumors, as well as prevention of carcinogenesis, may be affected by vaccination with embryonic/fetal material. Following a decade of intense research on this phenomenon during approximately 1964-1974, interest appears to have waned. This earlier experimental work may be particularly pertinent in view of the rising interest in so-called cancer stem cells. We believe that further work - perhaps involving the use of embryonic stem cells as immunogens - is warranted and that the results reviewed herein support the concept that vaccination against the appearance of cancers of all kinds is a real possibility.

In utero imaging of mouse embryonic development with optical coherence tomography

NASA Astrophysics Data System (ADS)

Syed, Saba H.; Dickinson, Mary E.; Larin, Kirill V.; Larina, Irina V.

2011-03-01

Studying progression of congenital diseases in animal models can greatly benefit from live embryonic imaging Mouse have long served as a model of mammalian embryonic developmental processes, however, due to intra-uterine nature of mammalian development live imaging is challenging. In this report we present results on live mouse embryonic imaging in utero with Optical Coherence Tomography. Embryos from 12.5 through 17.5 days post-coitus (dpc) were studied through the uterine wall. In longitudinal studies, same embryos were imaged at developmental stages 13.5, 15.5 and 17.5 dpc. This study suggests that OCT can serve as a powerful tool for live mouse embryo imaging. Potentially this technique can contribute to our understanding developmental abnormalities associated with mutations, toxic drugs.

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting.

PubMed

Kim, So-Jung; Habib, Omer; Kim, Jin-Soo; Han, Hyo-Won; Koo, Soo Kyung; Kim, Jung-Hyun

2017-03-01

Kelch-like ECH-associated protein 1 (keap1) is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a). Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC). The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Stage-dependent and locus-specific role of histone demethylase Jumonji D3 (JMJD3) in the embryonic stages of lung development.

PubMed

Li, Qingtian; Wang, Helen Y; Chepelev, Iouri; Zhu, Qingyuan; Wei, Gang; Zhao, Keji; Wang, Rong-Fu

2014-07-01

Histone demethylases have emerged as important players in developmental processes. Jumonji domain containing-3 (Jmjd3) has been identified as a key histone demethylase that plays a critical role in the regulation of gene expression; however, the in vivo function of Jmjd3 in embryonic development remains largely unknown. To this end, we generated Jmjd3 global and conditional knockout mice. Global deletion of Jmjd3 induces perinatal lethality associated with defective lung development. Tissue and stage-specific deletion revealed that Jmjd3 is dispensable in the later stage of embryonic lung development. Jmjd3 ablation downregulates the expression of genes critical for lung development and function, including AQP-5 and SP-B. Jmjd3-mediated alterations in gene expression are associated with locus-specific changes in the methylation status of H3K27 and H3K4. Furthermore, Jmjd3 is recruited to the SP-B promoter through interactions with the transcription factor Nkx2.1 and the epigenetic protein Brg1. Taken together, these findings demonstrate that Jmjd3 plays a stage-dependent and locus-specific role in the mouse lung development. Our study provides molecular insights into the mechanisms by which Jmjd3 regulates target gene expression in the embryonic stages of lung development.

hnRNP K Coordinates Transcriptional Silencing by SETDB1 in Embryonic Stem Cells

PubMed Central

Thompson, Peter J.; Dulberg, Vered; Moon, Kyung-Mee; Foster, Leonard J.; Chen, Carol; Karimi, Mohammad M.; Lorincz, Matthew C.

2015-01-01

Retrotransposition of endogenous retroviruses (ERVs) poses a substantial threat to genome stability. Transcriptional silencing of a subset of these parasitic elements in early mouse embryonic and germ cell development is dependent upon the lysine methyltransferase SETDB1, which deposits H3K9 trimethylation (H3K9me3) and the co-repressor KAP1, which binds SETDB1 when SUMOylated. Here we identified the transcription co-factor hnRNP K as a novel binding partner of the SETDB1/KAP1 complex in mouse embryonic stem cells (mESCs) and show that hnRNP K is required for ERV silencing. RNAi-mediated knockdown of hnRNP K led to depletion of H3K9me3 at ERVs, concomitant with de-repression of proviral reporter constructs and specific ERV subfamilies, as well as a cohort of germline-specific genes directly targeted by SETDB1. While hnRNP K recruitment to ERVs is dependent upon KAP1, SETDB1 binding at these elements requires hnRNP K. Furthermore, an intact SUMO conjugation pathway is necessary for SETDB1 recruitment to proviral chromatin and depletion of hnRNP K resulted in reduced SUMOylation at ERVs. Taken together, these findings reveal a novel regulatory hierarchy governing SETDB1 recruitment and in turn, transcriptional silencing in mESCs. PMID:25611934

Hedgehog Is a Positive Regulator of FGF Signalling during Embryonic Tracheal Cell Migration

PubMed Central

Butí, Elisenda; Mesquita, Duarte; Araújo, Sofia J.

2014-01-01

Cell migration is a widespread and complex process that is crucial for morphogenesis and for the underlying invasion and metastasis of human cancers. During migration, cells are steered toward target sites by guidance molecules that induce cell direction and movement through complex intracellular mechanisms. The spatio-temporal regulation of the expression of these guidance molecules is of extreme importance for both normal morphogenesis and human disease. One way to achieve this precise regulation is by combinatorial inputs of different transcription factors. Here we used Drosophila melanogaster mutants with migration defects in the ganglionic branches of the tracheal system to further clarify guidance regulation during cell migration. By studying the cellular consequences of overactivated Hh signalling, using ptc mutants, we found that Hh positively regulates Bnl/FGF levels during embryonic stages. Our results show that Hh modulates cell migration non-autonomously in the tissues surrounding the action of its activity. We further demonstrate that the Hh signalling pathway regulates bnl expression via Stripe (Sr), a zinc-finger transcription factor with homology to the Early Growth Response (EGR) family of vertebrate transcription factors. We propose that Hh modulates embryonic cell migration by participating in the spatio-temporal regulation of bnl expression in a permissive mode. By doing so, we provide a molecular link between the activation of Hh signalling and increased chemotactic responses during cell migration. PMID:24651658

Hedgehog is a positive regulator of FGF signalling during embryonic tracheal cell migration.

PubMed

Butí, Elisenda; Mesquita, Duarte; Araújo, Sofia J

2014-01-01

Cell migration is a widespread and complex process that is crucial for morphogenesis and for the underlying invasion and metastasis of human cancers. During migration, cells are steered toward target sites by guidance molecules that induce cell direction and movement through complex intracellular mechanisms. The spatio-temporal regulation of the expression of these guidance molecules is of extreme importance for both normal morphogenesis and human disease. One way to achieve this precise regulation is by combinatorial inputs of different transcription factors. Here we used Drosophila melanogaster mutants with migration defects in the ganglionic branches of the tracheal system to further clarify guidance regulation during cell migration. By studying the cellular consequences of overactivated Hh signalling, using ptc mutants, we found that Hh positively regulates Bnl/FGF levels during embryonic stages. Our results show that Hh modulates cell migration non-autonomously in the tissues surrounding the action of its activity. We further demonstrate that the Hh signalling pathway regulates bnl expression via Stripe (Sr), a zinc-finger transcription factor with homology to the Early Growth Response (EGR) family of vertebrate transcription factors. We propose that Hh modulates embryonic cell migration by participating in the spatio-temporal regulation of bnl expression in a permissive mode. By doing so, we provide a molecular link between the activation of Hh signalling and increased chemotactic responses during cell migration.

Patterns of muscular strain in the embryonic heart wall.

PubMed

Damon, Brooke J; Rémond, Mathieu C; Bigelow, Michael R; Trusk, Thomas C; Xie, Wenjie; Perucchio, Renato; Sedmera, David; Denslow, Stewart; Thompson, Robert P

2009-06-01

The hypothesis that inner layers of contracting muscular tubes undergo greater strain than concentric outer layers was tested by numerical modeling and by confocal microscopy of strain within the wall of the early chick heart. We modeled the looped heart as a thin muscular shell surrounding an inner layer of sponge-like trabeculae by two methods: calculation within a two-dimensional three-variable lumped model and simulated expansion of a three-dimensional, four-layer mesh of finite elements. Analysis of both models, and correlative microscopy of chamber dimensions, sarcomere spacing, and membrane leaks, indicate a gradient of strain decreasing across the wall from highest strain along inner layers. Prediction of wall thickening during expansion was confirmed by ultrasonography of beating hearts. Degree of stretch determined by radial position may thus contribute to observed patterns of regional myocardial conditioning and slowed proliferation, as well as to the morphogenesis of ventricular trabeculae and conduction fascicles. Developmental Dynamics 238:1535-1546, 2009. (c) 2009 Wiley-Liss, Inc.

Case Study: Organotypic human in vitro models of embryonic ...

EPA Pesticide Factsheets

Morphogenetic fusion of tissues is a common event in embryonic development and disruption of fusion is associated with birth defects of the eye, heart, neural tube, phallus, palate, and other organ systems. Embryonic tissue fusion requires precise regulation of cell-cell and cell-matrix interactions that drive proliferation, differentiation, and morphogenesis. Chemical low-dose exposures can disrupt morphogenesis across space and time by interfering with key embryonic fusion events. The Morphogenetic Fusion Task uses computer and in vitro models to elucidate consequences of developmental exposures. The Morphogenetic Fusion Task integrates multiple approaches to model responses to chemicals that leaad to birth defects, including integrative mining on ToxCast DB, ToxRefDB, and chemical structures, advanced computer agent-based models, and human cell-based cultures that model disruption of cellular and molecular behaviors including mechanisms predicted from integrative data mining and agent-based models. The purpose of the poster is to indicate progress on the CSS 17.02 Virtual Tissue Models Morphogenesis Task 1 products for the Board of Scientific Counselors meeting on Nov 16-17.

Human Embryonic Stem Cell Therapy in Crohn's Disease: A Case Report.

PubMed

Shroff, Geeta

2016-02-29

Crohn's disease is a chronic inflammatory disease of the intestines, mainly the colon and ileum, related with ulcers and fistulae. It is estimated to affect 565,000 people in the United States. Currently available therapies, such as antibiotics, thiopurines, and anti-tumor necrosis factor-alpha agents, are only observed to reduce the complications associated with Crohn's disease and to improve quality of life, but cannot cure the disease. Stem cell therapy appears to have certain advantages over conventional therapies. Our study aimed to evaluate the efficacy of human embryonic stem cell therapy in a patient with Crohn's disease. A 21-year-old male with chief complaints of intolerance to specific foods, abdominal pain, and diarrhea underwent human embryonic stem cell therapy for two months. After undergoing human embryonic stem cell therapy, the patient showed symptomatic relief. He had no complaints of back pain, abdominal pain, or diarrhea and had improved digestion. The patient had no signs and symptoms of skin infection, and had improved limb stamina, strength, and endurance. The condition of patient was stable after the therapy. Human embryonic stem cell therapy might serve as a new optimistic treatment approach for Crohn's disease.

Erythropoiesis from Human Embryonic Stem Cells Through Erythropoietin-Independent AKT Signaling

PubMed Central

Kim, William S.; Zhu, Yuhua; Deng, Qiming; Chin, Chee Jia; He, Chong Bin; Grieco, Amanda J.; Dravid, Gautam G.; Parekh, Chintan; Hollis, Roger P.; Lane, Timothy F.; Bouhassira, Eric E.; Kohn, Donald B.; Crooks, Gay M.

2014-01-01

Unlimited self renewal capacity and differentiation potential make human pluripotent stem cells (PSC) a promising source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods to induce erythropoiesis from PSC suffer from low yields of RBCs, most of which are immature and contain embryonic and fetal rather than adult hemoglobins. We have previously shown that homo-dimerization of the intracellular component of MPL (ic-MPL) induces erythropoiesis from human cord blood progenitors. The goal of the present study was to investigate the potential of ic-MPL dimerization to induce erythropoiesis from human embryonic stem cells (hESC) and to identify the signaling pathways activated by this strategy. We present here evidence that ic-MPL dimerization induces erythropoietin (EPO)-independent erythroid differentiation from hESC by inducing the generation of erythroid progenitors and by promoting more efficient erythroid maturation with increased RBC enucleation as well as increased gamma:epsilon globin ratio and production of beta-globin protein. ic-MPL dimerization is significantly more potent than EPO in inducing erythropoiesis and its effect is additive to EPO. Signaling studies show that dimerization of ic-MPL, unlike stimulation of the wild type MPL receptor, activates AKT in the absence of JAK2/STAT5 signaling. AKT activation upregulates the GATA-1 and FOXO3 transcriptional pathways with resulting inhibition of apoptosis, modulation of cell cycle and enhanced maturation of erythroid cells. These findings open up potential new targets for the generation of therapeutically relevant RBC products from hPSC. PMID:24677652

An Efficient System for Gene Perturbation in Embryonic Hippocampal Progenitors Using Ex Vivo Electroporation Followed by In Vitro Dissociated Cell Culture

PubMed Central

Muralidharan, Bhavana

2018-01-01

We established an efficient cell culture assay that permits combinatorial genetic perturbations in hippocampal progenitors to examine cell-autonomous mechanisms of fate specification. The procedure begins with ex vivo electroporation of isolated, intact embryonic brains, in a manner similar to in utero electroporation but with greatly improved access and targeting. The electroporated region is then dissected and transiently maintained in organotypic explant culture, followed by dissociation and plating of cells on coverslips for in vitro culture. This assay recapitulates data obtained in vivo with respect to the neuron-glia cell fate switch and can be effectively used to test intrinsic or extrinsic factors that regulate this process. The advantages of this ex vivo procedure over in utero electroporation include the fact that distinct combinations of perturbative reagents can be introduced in different embryos from a single litter, and issues related to embryonic lethality of transgenic animals can be circumvented. PMID:29760561

An Efficient System for Gene Perturbation in Embryonic Hippocampal Progenitors Using Ex Vivo Electroporation Followed by In Vitro Dissociated Cell Culture.

PubMed

Muralidharan, Bhavana; D'Souza, Leora; Tole, Shubha

2018-01-01

We established an efficient cell culture assay that permits combinatorial genetic perturbations in hippocampal progenitors to examine cell-autonomous mechanisms of fate specification. The procedure begins with ex vivo electroporation of isolated, intact embryonic brains, in a manner similar to in utero electroporation but with greatly improved access and targeting. The electroporated region is then dissected and transiently maintained in organotypic explant culture, followed by dissociation and plating of cells on coverslips for in vitro culture. This assay recapitulates data obtained in vivo with respect to the neuron-glia cell fate switch and can be effectively used to test intrinsic or extrinsic factors that regulate this process. The advantages of this ex vivo procedure over in utero electroporation include the fact that distinct combinations of perturbative reagents can be introduced in different embryos from a single litter, and issues related to embryonic lethality of transgenic animals can be circumvented.

Neurogenesis in the embryonic and adult brain: same regulators, different roles

PubMed Central

Urbán, Noelia; Guillemot, François

2014-01-01

Neurogenesis persists in adult mammals in specific brain areas, known as neurogenic niches. Adult neurogenesis is highly dynamic and is modulated by multiple physiological stimuli and pathological states. There is a strong interest in understanding how this process is regulated, particularly since active neuronal production has been demonstrated in both the hippocampus and the subventricular zone (SVZ) of adult humans. The molecular mechanisms that control neurogenesis have been extensively studied during embryonic development. Therefore, we have a broad knowledge of the intrinsic factors and extracellular signaling pathways driving proliferation and differentiation of embryonic neural precursors. Many of these factors also play important roles during adult neurogenesis, but essential differences exist in the biological responses of neural precursors in the embryonic and adult contexts. Because adult neural stem cells (NSCs) are normally found in a quiescent state, regulatory pathways can affect adult neurogenesis in ways that have no clear counterpart during embryogenesis. BMP signaling, for instance, regulates NSC behavior both during embryonic and adult neurogenesis. However, this pathway maintains stem cell proliferation in the embryo, while it promotes quiescence to prevent stem cell exhaustion in the adult brain. In this review, we will compare and contrast the functions of transcription factors (TFs) and other regulatory molecules in the embryonic brain and in adult neurogenic regions of the adult brain in the mouse, with a special focus on the hippocampal niche and on the regulation of the balance between quiescence and activation of adult NSCs in this region. PMID:25505873

Embryonic chirality and the evolution of spiralian left–right asymmetries

PubMed Central

2016-01-01

The group Spiralia includes species with one of the most significant cases of left–right asymmetries in animals: the coiling of the shell of gastropod molluscs (snails). In this animal group, an early event of embryonic chirality controlled by cytoskeleton dynamics and the subsequent differential activation of the genes nodal and Pitx determine the left–right axis of snails, and thus the direction of coiling of the shell. Despite progressive advances in our understanding of left–right axis specification in molluscs, little is known about left–right development in other spiralian taxa. Here, we identify and characterize the expression of nodal and Pitx orthologues in three different spiralian animals—the brachiopod Novocrania anomala, the annelid Owenia fusiformis and the nemertean Lineus ruber—and demonstrate embryonic chirality in the biradial-cleaving spiralian embryo of the bryozoan Membranipora membranacea. We show asymmetric expression of nodal and Pitx in the brachiopod and annelid, respectively, and symmetric expression of Pitx in the nemertean. Our findings indicate that early embryonic chirality is widespread and independent of the cleavage programme in the Spiralia. Additionally, our study illuminates the evolution of nodal and Pitx signalling by demonstrating embryonic asymmetric expression in lineages without obvious adult left–right asymmetries. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’. PMID:27821523

TOPICAL REVIEW: Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials

NASA Astrophysics Data System (ADS)

Amranul Haque, Md; Nagaoka, Masato; Hexig, Bayar; Akaike, Toshihiro

2010-02-01

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

Maintaining oncologic integrity with minimally invasive resection of pediatric embryonal tumors.

PubMed

Phelps, Hannah M; Ayers, Gregory D; Ndolo, Josephine M; Dietrich, Hannah L; Watson, Katherine D; Hilmes, Melissa A; Lovvorn, Harold N

2018-05-08

Embryonal tumors arise typically in infants and young children and are often massive at presentation. Operative resection is a cornerstone in the multimodal treatment of embryonal tumors but potentially disrupts therapeutic timelines. When used appropriately, minimally invasive surgery can minimize treatment delays. The oncologic integrity and safety attainable with minimally invasive resection of embryonal tumors, however, remains controversial. Query of the Vanderbilt Cancer Registry identified all children treated for intracavitary, embryonal tumors during a 15-year period. Tumors were assessed radiographically to measure volume (mL) and image-defined risk factors (neuroblastic tumors only) at time of diagnosis, and at preresection and postresection. Patient and tumor characteristics, perioperative details, and oncologic outcomes were compared between minimally invasive surgery and open resection of tumors of comparable size. A total of 202 patients were treated for 206 intracavitary embryonal tumors, of which 178 were resected either open (n = 152, 85%) or with minimally invasive surgery (n = 26, 15%). The 5-year, relapse-free, and overall survival were not significantly different after minimally invasive surgery or open resection of tumors having a volume less than 100 mL, corresponding to the largest resected with minimally invasive surgery (P = .249 and P = .124, respectively). No difference in margin status or lymph node sampling between the 2 operative approaches was detected (p = .333 and p = .070, respectively). Advantages associated with minimally invasive surgery were decreased blood loss (P < .001), decreased operating time (P = .002), and shorter hospital stay (P < .001). Characteristically, minimally invasive surgery was used for smaller volume and earlier stage neuroblastic tumors without image-defined risk factors. When selected appropriately, minimally invasive resection of pediatric embryonal tumors, particularly

Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

PubMed

Sleeboom-Faulkner, Margaret

2010-01-01

This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

Normal embryonic and germ cell development in mice lacking alpha 1,3-fucosyltransferase IX (Fut9) which show disappearance of stage-specific embryonic antigen 1.

PubMed

Kudo, Takashi; Kaneko, Mika; Iwasaki, Hiroko; Togayachi, Akira; Nishihara, Shoko; Abe, Kuniya; Narimatsu, Hisashi

2004-05-01

Stage-specific embryonic antigen 1 (SSEA-1), an antigenic epitope defined as a Lewis x carbohydrate structure, is expressed during the 8-cell to blastocyst stages in mouse embryos and in primordial germ cells, undifferentiated embryonic stem cells, and embryonic carcinoma cells. For many years, SSEA-1 has been implicated in the development of mouse embryos as a functional carbohydrate epitope in cell-to-cell interaction during morula compaction. In a previous study, alpha 1,3-fucosyltransferase IX (Fut9) exhibited very strong activity for the synthesis of Lewis x compared to other alpha 1,3-fucosyltransferases in an in vitro substrate specificity assay. Fut4 and Fut9 transcripts were expressed in mouse embryos. The Fut9 transcript was detected in embryonic-day-13.5 gonads containing primordial germ cells, but the Fut4 transcript was not. In order to identify the role of SSEA-1 and determine the key enzyme for SSEA-1 synthesis in vivo, we have generated Fut9-deficient (Fut9(-/-)) mice. Fut9(-/-) mice develop normally, with no gross phenotypic abnormalities, and are fertile. Immunohistochemical analysis revealed an absence of SSEA-1 expression in early embryos and primordial germ cells of Fut9(-/-) mice. Therefore, we conclude that expression of the SSEA-1 epitope in the developing mouse embryo is not essential for embryogenesis in vivo.

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

The miR-290-295 cluster as multi-faceted players in mouse embryonic stem cells.

PubMed

Yuan, Kai; Ai, Wen-Bing; Wan, Lin-Yan; Tan, Xiao; Wu, Jiang-Feng

2017-01-01

Increasing evidence indicates that embryonic stem cell specific microRNAs (miRNAs) play an essential role in the early development of embryo. Among them, the miR-290-295 cluster is the most highly expressed in the mouse embryonic stem cells and involved in various biological processes. In this paper, we reviewed the research progress of the function of the miR-290-295 cluster in embryonic stem cells. The miR-290-295 cluster is involved in regulating embryonic stem cell pluripotency maintenance, self-renewal, and reprogramming somatic cells to an embryonic stem cell-like state. Moreover, the miR-290-295 cluster has a latent pro-survival function in embryonic stem cells and involved in tumourigenesis and senescence with a great significance. Elucidating the interaction between the miR-290-295 cluster and other modes of gene regulation will provide us new ideas on the biology of pluripotent stem cells. In the near future, the broad prospects of the miRNA cluster will be shown in the stem cell field, such as altering cell identities with high efficiency through the transient introduction of tissue-specific miRNA cluster.

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy.

PubMed

Mosqueira, Diogo; Mannhardt, Ingra; Bhagwan, Jamie R; Lis-Slimak, Katarzyna; Katili, Puspita; Scott, Elizabeth; Hassan, Mustafa; Prondzynski, Maksymilian; Harmer, Stephen C; Tinker, Andrew; Smith, James G W; Carrier, Lucie; Williams, Philip M; Gaffney, Daniel; Eschenhagen, Thomas; Hansen, Arne; Denning, Chris

2018-05-08

Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-β-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-βMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient βMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.

Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update

PubMed Central

Miele, Lucio; Harris, Pamela Jo; Jeong, Woondong; Bando, Hideaki; Kahn, Michael; Yang, Sherry X.

2015-01-01

During the past decade, cancer stem cells (CSCs) have been increasingly identified in many malignancies. Although the origin and plasticity of these cells remain controversial, tumour heterogeneity and the presence of small populations of cells with stem-like characteristics is established in most malignancies. CSCs display many features of embryonic or tissue stem cells, and typically demonstrate persistent activation of one or more highly conserved signal transduction pathways involved in development and tissue homeostasis, including the Notch, Hedgehog (HH), and Wnt pathways. CSCs generally have slow growth rates and are resistant to chemotherapy and/or radiotherapy. Thus, new treatment strategies targeting these pathways to control stem-cell replication, survival and differentiation are under development. Herein, we provide an update on the latest advances in the clinical development of such approaches, and discuss strategies for overcoming CSC-associated primary or acquired resistance to cancer treatment. Given the crosstalk between the different embryonic developmental signalling pathways, as well as other pathways, designing clinical trials that target CSCs with rational combinations of agents to inhibit possible compensatory escape mechanisms could be of particular importance. We also share our views on the future directions for targeting CSCs to advance the clinical development of these classes of agents. PMID:25850553

Embryonic toxico-pathological effects of meglumine antimoniate using a chick embryo model.

PubMed

Khosravi, Ahmad; Sharifi, Iraj; Tavakkoli, Hadi; Derakhshanfar, Amin; Keyhani, Ali Reza; Salari, Zohreh; Mosallanejad, Seyedeh Saedeh; Bamorovat, Mehdi

2018-01-01

Leishmaniasis is one of the diverse and neglected tropical diseases. Embryo-toxicity of drugs has always been a major concern. Chick embryo is a preclinical model relevant in the assessment of adverse effects of drugs. The current study aimed to assess embryonic histopathological disorders and amniotic fluid biochemical changes following meglumine antimoniate treatment. The alteration of vascular branching pattern in the chick's extra-embryonic membrane and exploration of molecular cues to early embryonic vasculogenesis and angiogenesis were also quantified. Embryonated chicken eggs were treated with 75 or 150 mg/kg of meglumine antimoniate. Embryo malformations, growth retardation and haemorrhages on the external body surfaces were accompanied by histopathological lesions in the brain, kidney, liver and heart in a dose-dependent manner. Significant rise occurred in the biochemical indices of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase and amylase in the amniotic fluid. Quantification of the extra-embryonic membrane vasculature showed that the anti-angiogenic and anti-vasculogenic effects of the drug were revealed by a significant decrease in fractal dimension value and mean capillary area. The relative expression levels of vascular endothelial growth factor A and vascular endothelial growth factor receptor 2 mRNA also significantly reduced. Concerns of a probable teratogenicity of meglumine antimoniate were established by data presented in this study. It is concluded that tissue lesions, amniotic fluid disturbance, altered early extra-embryonic vascular development and gene expression as well as the consecutive cascade of events, might eventually lead to developmental defects in embryo following meglumine antimoniate treatment. Therefore, the use of meglumine antimoniate during pregnancy should be considered as potentially embryo-toxic. Hence, physicians should be aware of such teratogenic effects and limit the use of this drug

Asynchronous Replication and Autosome-Pair Non-Equivalence in Human Embryonic Stem Cells

PubMed Central

Dutta, Devkanya; Ensminger, Alexander W.; Zucker, Jacob P.; Chess, Andrew

2009-01-01

A number of mammalian genes exhibit the unusual properties of random monoallelic expression and random asynchronous replication. Such exceptional genes include genes subject to X inactivation and autosomal genes including odorant receptors, immunoglobulins, interleukins, pheromone receptors, and p120 catenin. In differentiated cells, random asynchronous replication of interspersed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair non-equivalence. Here we have investigated the replication pattern of the random asynchronously replicating genes in undifferentiated human embryonic stem cells, using fluorescence in situ hybridization based assay. We show that allele-specific replication of X-linked genes and random monoallelic autosomal genes occur in human embryonic stem cells. The direction of replication is coordinated at the whole chromosome level and can cross the centromere, indicating the existence of autosome-pair non-equivalence in human embryonic stem cells. These results suggest that epigenetic mechanism(s) that randomly distinguish between two parental alleles are emerging in the cells of the inner cell mass, the source of human embryonic stem cells. PMID:19325893

Targeting Tumor Oct4 to Deplete Prostate Tumor and Metastasis Initiating Cells

DTIC Science & Technology

2016-10-01

Award Number: W81XWH-13-1-0461 TITLE: Targeting Tumor Oct4 to Deplete Prostate Tumor- and Metastasis-Initiating Cells PRINCIPAL INVESTIGATOR: Daotai...29 2016 4. TITLE AND SUBTILE Targeting Tumor Oct4 to Deplete Prostate Tumor- and Metastasis-Initiating Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER...the c-MYC oncogene. POU5F1B is a pseudogene of embryonic Oct4 (POU5F1). A recent study found that tumor Oct4 found in prostate cancer cells is due

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

PubMed

Pandolfini, Luca; Luzi, Ettore; Bressan, Dario; Ucciferri, Nadia; Bertacchi, Michele; Brandi, Rossella; Rocchiccioli, Silvia; D'Onofrio, Mara; Cremisi, Federico

2016-05-06

Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

Embryonic Cerebrospinal Fluid Increases Neurogenic Activity in the Brain Ventricular-Subventricular Zone of Adult Mice.

PubMed

Alonso, Maria I; Lamus, Francisco; Carnicero, Estela; Moro, Jose A; de la Mano, Anibal; Fernández, Jose M F; Desmond, Mary E; Gato, Angel

2017-01-01

Neurogenesis is a very intensive process during early embryonic brain development, becoming dramatically restricted in the adult brain in terms of extension and intensity. We have previously demonstrated the key role of embryonic cerebrospinal fluid (CSF) in developing brain neurogenic activity. We also showed that cultured adult brain neural stem cells (NSCs) remain competent when responding to the neurogenic influence of embryonic CSF. However, adult CSF loses its neurogenic inductive properties. Here, by means of an organotypic culture of adult mouse brain sections, we show that local administration of embryonic CSF in the subventricular zone (SVZ) niche is able to trigger a neurogenic program in NSCs. This leads to a significant increase in the number of non-differentiated NSCs, and also in the number of new neurons which show normal migration, differentiation and maturation. These new data reveal that embryonic CSF activates adult brain NSCs, supporting the previous idea that it contains key instructive components which could be useful in adult brain neuroregenerative strategies.

Embryonic Cerebrospinal Fluid Increases Neurogenic Activity in the Brain Ventricular-Subventricular Zone of Adult Mice

PubMed Central

Alonso, Maria I.; Lamus, Francisco; Carnicero, Estela; Moro, Jose A.; de la Mano, Anibal; Fernández, Jose M. F.; Desmond, Mary E.; Gato, Angel

2017-01-01

Neurogenesis is a very intensive process during early embryonic brain development, becoming dramatically restricted in the adult brain in terms of extension and intensity. We have previously demonstrated the key role of embryonic cerebrospinal fluid (CSF) in developing brain neurogenic activity. We also showed that cultured adult brain neural stem cells (NSCs) remain competent when responding to the neurogenic influence of embryonic CSF. However, adult CSF loses its neurogenic inductive properties. Here, by means of an organotypic culture of adult mouse brain sections, we show that local administration of embryonic CSF in the subventricular zone (SVZ) niche is able to trigger a neurogenic program in NSCs. This leads to a significant increase in the number of non-differentiated NSCs, and also in the number of new neurons which show normal migration, differentiation and maturation. These new data reveal that embryonic CSF activates adult brain NSCs, supporting the previous idea that it contains key instructive components which could be useful in adult brain neuroregenerative strategies. PMID:29311854

Microfluidic-based patterning of embryonic stem cells for in vitro development studies.

PubMed

Suri, Shalu; Singh, Ankur; Nguyen, Anh H; Bratt-Leal, Andres M; McDevitt, Todd C; Lu, Hang

2013-12-07

In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments.

Microfluidic-based patterning of embryonic stem cells for in vitro development studies

PubMed Central

Suri, Shalu; Singh, Ankur; Nguyen, Anh H.; Bratt-Leal, Andres M.; McDevitt, Todd C.

2013-01-01

In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments. PMID:24113509

Derivation and characterization of gut-like structures from embryonic stem cells.

PubMed

Yamada, Takatsugu; Nakajima, Yoshiyuki

2006-01-01

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages of all three embryonic germ layers in vitro. The hanging drop culture of ES cell suspension in the absence of leukemia inhibitory factor induces aggregation and differentiation of the cells into simple or cystic embryoid bodies (EBs). After 6 d of hanging drop culture, the resulting EBs are plated onto plastic dishes for the outgrowth culture. At d 21 after outgrowth culture, cell populations of EBs can give rise to three-dimensional gut-like structures that exhibit spontaneous contraction and highly coordinated peristalsis. The gut-like structures have large lumens surrounded by three layers: epithelium, lamina propria, and muscularis. Ganglia are scattered along the periphery, and interstitial cells of Cajal are distributed among the smooth muscle cells. The fundamental process of formation of the in vitro organized gut-like structures is similar to embryonic gastrointestinal development in vivo. The EBs at the 6-d egg-cylinder stage may have the potential to regulate developmental programs associated with cell lineage commitment and provide an appropriate microenvironment to differentiate ES cells into enteric derivatives of all three embryonic germ layers and reproduce the gut organization process in vitro.

Mechanical signaling coordinates the embryonic heart

NASA Astrophysics Data System (ADS)

Chiou, Kevin; Rocks, Jason; Prosser, Benjamin; Discher, Dennis; Liu, Andrea

The heart is an active material which relies on robust signaling mechanisms between cells in order to produce well-timed, coordinated beats. Heart tissue is composed primarily of active heart muscle cells (cardiomyocytes) embedded in a passive extracellular matrix. During a heartbeat, cardiomyocyte contractions are coordinated across the heart to form a wavefront that propagates through the tissue to pump blood. In the adult heart, this contractile wave is coordinated via intercellular electrical signaling.Here we present theoretical and experimental evidence for mechanical coordination of embryonic heartbeats. We model cardiomyocytes as mechanically excitable Eshelby inclusions embedded in an overdamped elastic-fluid biphasic medium. For physiological parameters, this model replicates recent experimental measurements of the contractile wavefront which are not captured by electrical signaling models. We additionally challenge our model by pharmacologically blocking gap junctions, inhibiting electrical signaling between myocytes. We find that while adult hearts stop beating almost immediately after gap junctions are blocked, embryonic hearts continue beating even at significantly higher concentrations, providing strong support for a mechanical signaling mechanism.

Rotational imaging optical coherence tomography for full-body mouse embryonic imaging

PubMed Central

Wu, Chen; Sudheendran, Narendran; Singh, Manmohan; Larina, Irina V.; Dickinson, Mary E.; Larin, Kirill V.

2016-01-01

Abstract. Optical coherence tomography (OCT) has been widely used to study mammalian embryonic development with the advantages of high spatial and temporal resolutions and without the need for any contrast enhancement probes. However, the limited imaging depth of traditional OCT might prohibit visualization of the full embryonic body. To overcome this limitation, we have developed a new methodology to enhance the imaging range of OCT in embryonic day (E) 9.5 and 10.5 mouse embryos using rotational imaging. Rotational imaging OCT (RI-OCT) enables full-body imaging of mouse embryos by performing multiangle imaging. A series of postprocessing procedures was performed on each cross-section image, resulting in the final composited image. The results demonstrate that RI-OCT is able to improve the visualization of internal mouse embryo structures as compared to conventional OCT. PMID:26848543

Transcriptional Profiling of Ectoderm Specification to Keratinocyte Fate in Human Embryonic Stem Cells

PubMed Central

Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

2015-01-01

In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ–secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions. PMID:25849374

[Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

PubMed

Gordeeva, O F; Nikonova, T M; Lifantseva, N V

2009-01-01

The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

Involvement of 4E-BP phosphorylation in embryonic development of the silkworm, Bombyx mori.

PubMed

Gu, Shi-Hong; Young, Shun-Chieh; Tsai, Wen-Hsien; Lin, Ju-Ling; Lin, Pei-Ling

2011-07-01

Phosphorylation of the translational repressor 4E-binding protein (4E-BP) plays a critical role in regulating the overall translation levels in cells. In the present study, we investigated 4E-BP phosphorylation of Bombyx mori eggs by an immunoblot analysis of a conserved phospho-specific antibody to 4E-BP and demonstrated its role during embryonic development. When HCl treatment was applied to diapause-destined eggs at 20 h after oviposition, a dramatic increase in the phosphorylation of 4E-BP occurred 5 min after treatment with HCl, and high phosphorylation levels were maintained throughout embryonic stage in HCl-treated eggs compared to those in diapause (control) eggs. When HCl treatment was applied to diapause eggs on day 10 after oviposition, no dramatic activation in 4E-BP phosphorylation occurred, indicating stage-specific effects of HCl treatment. In both non-diapause eggs and eggs whose diapause had been terminated by chilling of diapausing eggs at 5°C for 70 days and then were transferred to 25°C, high phosphorylation levels of 4E-BP were also detected. Moreover, 4E-BP phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of rapamycin, a specific inhibitor of mammalian target of rapamycin (TOR) signaling, and LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, but not the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, dose-dependently inhibited 4E-BP phosphorylation in dechorionated eggs, indicating that PI3K/TOR signaling is an upstream signaling event involved in 4E-BP phosphorylation. Examination of 4E-BP gene expression levels showed no differences between treatments with HCl and water in the first hour after treatment, indicating that changes in phosphorylation of 4E-BP upon HCl treatment are mainly regulated at the post-transcriptional level. In addition, MAPK pathways and glycogen synthase kinase (GSK)-3β phosphorylation were

Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods

PubMed Central

González, Sheyla; Ibáñez, Elena

2010-01-01

Purpose The aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process. Methods Three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages. Results A total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%. Conclusions Using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process. PMID:20862536

Epigenetic modulation by TFII-I during embryonic stem cell differentiation.

PubMed

Bayarsaihan, Dashzeveg; Makeyev, Aleksandr V; Enkhmandakh, Badam

2012-10-01

TFII-I transcription factors play an essential role during early vertebrate embryogenesis. Genome-wide mapping studies by ChIP-seq and ChIP-chip revealed that TFII-I primes multiple genomic loci in mouse embryonic stem cells and embryonic tissues. Moreover, many TFII-I-bound regions co-localize with H3K4me3/K27me3 bivalent chromatin within the promoters of lineage-specific genes. This minireview provides a summary of current knowledge regarding the function of TFII-I in epigenetic control of stem cell differentiation. Copyright © 2012 Wiley Periodicals, Inc.

MRG15 Regulates Embryonic Development and Cell Proliferation

PubMed Central

Tominaga, Kaoru; Kirtane, Bhakti; Jackson, James G.; Ikeno, Yuji; Ikeda, Takayoshi; Hawks, Christina; Smith, James R.; Matzuk, Martin M.; Pereira-Smith, Olivia M.

2005-01-01

MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15−/− embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15−/− mouse embryonic fibroblasts. The hearts of the Mrg15−/− embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15−/− embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation. PMID:15798182

[Artificial propagation and embryonic development of Neolissochilus benasi].

PubMed

Pan, Xiao-Fu; Liu, Qian; Wang, Xiao-Ai; Yang, Jun-Xing; Chen, Xiao-Yong; Li, Zai-Yun; Li, Lie

2013-12-01

From 2009 to 2011, luteinizing hormone releasing hormone analogue (LHRH-A2) mixed with domperidon (DOM) was successfully applied during the artificial propagation of Neolissochilus benasi. Totally, 60 females and 100 males were injected with the hormone mixture, resulting in 47 (78.3%) females and 92 (92.0%) males being successfully spawned. A total of 1,986-5 854 eggs were spawned per female with an egg diameter varying between 2.2-2.8 mm, and an average nucleus deviation rate of 73.2%. Sperm density, vitality and life span were 16.32±2.89×10(9)/mL, 60.6±3.2% and 70.2±5.3 s, respectively. On the whole, the embryonic development of N. benasi was similar to that of zebra fish-albeit relatively slower-lasting approximately 120 hours. The development itself can be divided into six discrete stages: zygote, cleavage, blastula, gastrula, segmentation and hatching. Results showed that the average hatching rate was 32.4%, with 86.5% of larvae surviving 45 days after hatching. During embryonic development, deformities commonly occurred on the mouth, chest, ocular region, especially in the spinal column. To try to attempt improving future breeding efforts, we provided a survey of the embryonic developmental difficulties of N. benasi using LHRH-A2 followed by several potential solutions, including providing suitable breeding conditions and minimizing capture stresses.

The relationship of parthenogenesis in virgin Chinese Painted quail (Coturnix chinensis) hens with embryonic mortality and hatchability following mating.

PubMed

Parker, H M; Kiess, A S; Robertson, M L; Wells, J B; McDaniel, C D

2012-06-01

Unfertilized chicken, turkey, and quail eggs are capable of developing embryos by parthenogenesis. However, it is unknown if the physiological mechanisms regulating parthenogenesis in virgin hens may actually work against fertilization, embryonic development, and hatchability of eggs from these same hens following mating. Additionally, because most parthenogenic development closely resembles early embryonic mortality in fertilized eggs during the first 2 to 3 d of incubation, it is possible that many unhatched eggs classified as containing early embryonic mortality may actually be unfertilized eggs that contain parthenogens. Therefore, the objective of this study was to examine the relationship of parthenogenesis before mating with embryonic development and hatchability characteristics after mating. Based upon their ability to produce unfertilized eggs that contain parthenogens, 372 virgin Chinese Painted quail hens were divided into 7 groups, according to their incidence of parthenogenesis: 0, 10, 20, 30, 40, 50, and greater than 50% parthenogenesis. Males were then placed with these hens so that fertility, embryonic mortality, and hatchability could be evaluated for each hen. Hatchability of eggs set, hatchability of fertile eggs, and late embryonic mortality declined dramatically as the incidence of parthenogenesis increased. On the other hand, early embryonic mortality increased as parthenogenesis increased. Fertility was not different across the 7 parthenogenesis hen groups, perhaps because unfertilized eggs that exhibited parthenogenesis resembled and were therefore classified as early embryonic mortality. In conclusion, virgin quail hens that exhibit parthenogenesis appear to have impaired embryonic development and hatchability following mating. Additional sperm-egg interaction and embryonic research is needed to determine if a large portion of the early embryonic mortality experienced by mated hens that exhibit parthenogenesis as virgin hens is in fact

Intraspecific Variation in and Environment-Dependent Resource Allocation to Embryonic Development Time in Common Terns.

PubMed

Vedder, Oscar; Kürten, Nathalie; Bouwhuis, Sandra

Embryonic development time is thought to impact life histories through trade-offs against life-history traits later in life, yet the inference is based on interspecific comparative analyses only. It is largely unclear whether intraspecific variation in embryonic development time that is not caused by environmental differences occurs, which would be required to detect life-history trade-offs. Here we performed a classical common-garden experiment by incubating fresh eggs of free-living common terns (Sterna hirundo) in a controlled incubation environment at two different temperatures. Hatching success was high but was slightly lower at the lower temperature. While correcting for effects of year, incubation temperature, and laying order, we found significant variation in the incubation time embryos required until hatching and in their heart rate. Embryonic heart rate was significantly positively correlated within clutches, and a similar tendency was found for incubation time, suggesting that intrinsic differences in embryonic development rate between offspring of different parents exist. Incubation time and embryonic heart rate were strongly correlated: embryos with faster heart rates required shorter incubation time. However, after correction for heart rate, embryos still required more time for development at the lower incubation temperature. This suggests that processes other than development require a greater share of resources in a suboptimal environment and that relative resource allocation to development is, therefore, environment dependent. We conclude that there is opportunity to detect intraspecific life-history trade-offs with embryonic development time and that the resolution of trade-offs may differ between embryonic environments.

Degenerative changes and cell death in long-living homo- and heterotopic transplants from embryonic germ layers of rat neocortex.

PubMed

Petrova, E S; Otellin, V A

2003-09-01

Morphological study of allotransplants of rat embryonic neocortex 14-18 months after transplantation into the neocortex, lateral cerebral ventricle, and sciatic nerve of adult animals revealed death of nerve and glial cells in the delayed postoperation period independently on the site of transplantation. After heterotopic transplantation the count of degenerated neurons was 2 times higher that after homotopic transplantation. In heterotopic transplants a considerable number of grafted neurons underwent reversible and irreversible degenerative changes accompanied by their premature aging. Neuronal death is probably determined by insufficiency of trophic influence from afferent structures and target tissues. We hypothesized that antiapoptotic preparations can be used for prevention of transplanted cell death. It was also found that degeneration of neurons was associated with impaired vascularization of transplants and pronounced immune reaction of the recipient in late posttransplantation period. Transplantation of embryonic brain structures can serve as a model system in studies concerning involutive and pathological processes in the central nervous system and in the search for factors improving survival of neurons.

Melphalan, Carboplatin, Mannitol, and Sodium Thiosulfate in Treating Patients With Recurrent or Progressive CNS Embryonal or Germ Cell Tumors

ClinicalTrials.gov

2018-05-02

Adult Central Nervous System Germ Cell Tumor; Adult Embryonal Tumor With Multilayered Rosettes, C19MC-Altered; Adult Medulloblastoma; Adult Pineoblastoma; Adult Supratentorial Embryonal Tumor, Not Otherwise Specified; Atypical Teratoid/Rhabdoid Tumor; Childhood Atypical Teratoid/Rhabdoid Tumor; Childhood Central Nervous System Germ Cell Tumor; Childhood Embryonal Tumor With Multilayered Rosettes, C19MC-Altered; Medulloepithelioma; Ototoxicity; Recurrent Adult Brain Neoplasm; Recurrent Childhood Central Nervous System Embryonal Neoplasm; Recurrent Childhood Malignant Germ Cell Tumor; Recurrent Childhood Medulloblastoma; Recurrent Childhood Pineoblastoma; Recurrent Childhood Supratentorial Embryonal Tumor, Not Otherwise Specified

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development.

PubMed

Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie; Barozzi, Iros; Osterwalder, Marco; Akiyama, Jennifer A; Lincoln, Joy; Lopez-Rios, Javier; Visel, Axel; Zuniga, Aimée; Zeller, Rolf

2017-05-23

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development

DOE PAGES

Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie; ...

2017-05-23

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost frommore » Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.« less

HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development

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

Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie

The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost frommore » Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development.« less

Non-Neuronal Release of Gamma-Aminobutyric Acid by Embryonic Pluripotent Stem Cells

PubMed Central

Teng, Lin; Tang, Ya-Bin; Sun, Fan; An, Shi-Min; Zhang, Chun; Yang, Xin-Jie; Lv, Hao-Yu; Lu, Qin; Cui, Yong-Yao; Hu, Jin-Jia

2013-01-01

γ-Aminobutyric acid (GABA), the principle inhibitory transmitter in the mature central nervous system, is also involved in activities outside the nervous system. Recent studies have shown that functional GABA receptors are expressed in embryonic stem (ES) cells and these receptors control ES cell proliferation. However, it is not clear whether ES cells have their own GABAergic transmission output machinery that can fulfill GABA release or whether the cells merely process the GABA receptors by receiving and responding to the diffused GABA released elsewhere. To get further insight into this unresolved problem, we detected the repertoire of components for GABA synthesis, storage, reaction, and termination in ES and embryonal carcinoma stem cells by biological assays, and then directly quantified released GABA in the intercellular milieu from these pluripotent stem (PS) cells by an analytical chemical assay based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). We found that embryonic PS cells processed a GABAergic circuit machinery and spontaneously released GABA, which suggests the potential that embryonic PS cells could autonomously establish a GABA niche via release of the transmitter. PMID:23799822

Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

PubMed

Moralli, Daniela; Monaco, Zoia L

2015-02-01

De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

Msx-2 expression and glucocorticoid-induced overexpression in embryonic mouse submandibular glands.

PubMed

Jaskoll, T; Luo, W; Snead, M L

1998-01-01

It is well known that the process of branching morphogenesis requires epithelial-mesenchymal interactions. One outstanding model for the study of tissue interactions during branching morphogenesis is the embryonic mouse submandibular gland (SMG). Although it has been clearly demonstrated that the branching pattern is dependent on interactions between the epithelium and the surrounding mesenchyme, little is known about the molecular mechanism underlying the branching process. One group of transcription factors that likely participates in the control of epithelial-mesenchymal inductive interactions are the Msx-class of homeodomain-containing proteins. In this paper, we focus on Msx-2 because its developmental expression is correlated with inductive interactions, suggesting that Msx-2 may play a functional role during cell-cell interactions. We demonstrate the expression of Msx-2 mRNA and protein to be primarily in the branching epithelia with progressive embryonic (E13 to E15) SMG development and, to a lesser extent, in the mesenchyme. We also show that Msx-2 is expressed by embryonic SMG primordia cultured under defined conditions. In addition, to begin to delineate a functional role for Msx-2, we employed an experimental strategy by using exogenous glucocorticoid (CORT) treatment of embryonic SMGs in vitro and in vivo to significantly enhance branching morphogenesis and evaluate the effect of CORT treatment on embryonic SMG Msx-2 expression. A marked increase in Msx-2 transcripts and protein is detected with in vitro and in vivo CORT treatment. Our studies indicate that one mechanism of CORT regulation of salivary gland morphogenesis is likely through the modulation of Msx-2 gene expression.

AMPKα2 Suppresses Murine Embryonic Fibroblast Transformation and Tumorigenesis

PubMed Central

Phoenix, Kathryn N.; Devarakonda, Charan V.; Fox, Melissa M.; Stevens, Laura E.

2012-01-01

AMP-activated kinase (AMPK) is a key metabolic sensor and stress signaling kinase. AMPK activity is known to suppress anabolic processes such as protein and lipid biosynthesis and promote energy-producing pathways including fatty acid oxidation, resulting in increased cellular energy. In addition, AMPK localizes to centrosomes during cell division, plays a role in cellular polarization, and directly targets p53, affecting apoptosis. Two distinct catalytic AMPKα isoforms exist: α1 and α2. Multiple reports indicate that both common and distinct functions exist for each of the 2 α isoforms. AMPK activation has been shown to repress tumor growth, and it has been suggested that AMPK may function as a metabolic tumor suppressor. To evaluate the potential role of each of the AMPKα isoforms in modulating cellular transformation, susceptibility to Ras-induced transformation was evaluated in normal murine embryonic fibroblasts (MEFs) obtained from genetically deleted AMPKα1- or AMPKα2-null mice. This study demonstrated that while AMPKα1 is the dominant AMPK isoform expressed in MEFs, only the AMPKα2-null MEFs displayed increased susceptibility to H-RasV12 transformation in vitro and tumorigenesis in vivo. Conversely, AMPKα1-null MEFs, which demonstrated compensation with increased expression of AMPKα2, displayed minimal transformation susceptibility, decreased cell survival, decreased cell proliferation, and increased apoptosis. Finally, this study demonstrates that AMPKα2 was selectively responsible for targeting p53, thus contributing to the suppression of transformation and tumorigenic mechanisms. PMID:22893790

Multimodality optical imaging of embryonic heart microstructure

PubMed Central

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H.; Boudoux, Caroline; Vakoc, Benjamin J.; Bouma, Brett E.; Tearney, Guillermo J.

2009-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 µm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass. PMID:18163837

Multimodality optical imaging of embryonic heart microstructure.

PubMed

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H; Boudoux, Caroline; Vakoc, Benjamin J; Bouma, Brett E; Tearney, Guillermo J

2007-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

Utilization of ketone bodies by chick brain and spinal cord during embryonic and postnatal development.

PubMed

Linares, A; Caamaño, G J; Diaz, R; Gonzalez, F J; Garcia-Peregrin, E

1993-10-01

Lipid synthesis from acetoacetate and 3-hydroxybutyrate was studied in chick embryo from 15 to 21 days and in chick neonate from 1 to 21 days. Embryonic spinal cord showed higher ability than brain to incorporate acetoacetate into total lipids, although a sharp decrease was found at hatching. 3-Hydroxybutyrate incorporation into total lipids was also higher in spinal cord than in brain, especially during the embryonic period. Phospholipids were the main lipids formed in both tissues from both precursors. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. The developmental patterns of amino acid synthesis from acetoacetate and 3-hydroxybutyrate were similar in both tissues: a clear increase after hatching was followed by a decrease at day 4 of neonatal life. Acetoacetate was a better substrate for amino acid synthesis than 3-hydroxybutyrate during the embryonic development in both tissues. Oxidation of both precursors to CO2 strongly decreased between 15 and 21 days of embryonic development both in brain and spinal cord.

Derivation, propagation and differentiation of human embryonic stem cells.

PubMed

Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard

2004-04-01

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug

Protein targeting in the analysis of learning and memory: a potential alternative to gene targeting.

PubMed

Gerlai, R; Williams, S P; Cairns, B; Van Bruggen, N; Moran, P; Shih, A; Caras, I; Sauer, H; Phillips, H S; Winslow, J W

1998-11-01

Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location

The bHLH transcription factor, hairy, refines the terminal cell fate in the Drosophila embryonic trachea.

PubMed

Zhan, Yaoyao; Maung, Saw W; Shao, Bing; Myat, Monn Monn

2010-11-30

The pair-rule gene, hairy, encodes a basic helix-loop-helix transcription factor and is required for patterning of the early Drosophila embryo and for morphogenesis of the embryonic salivary gland. Although hairy was shown to be expressed in the tracheal primordia and in surrounding mesoderm, whether hairy plays a role in tracheal development is not known. Here, we report that hairy is required for refining the terminal cell fate in the embryonic trachea and that hairy's tracheal function is distinct from its earlier role in embryonic patterning. In hairy mutant embryos where the repressive activity of hairy is lost due to lack of its co-repressor binding site, extra terminal cells are specified in the dorsal branches. We show that hairy functions in the muscle to refine the terminal cell fate to a single cell at the tip of the dorsal branch by limiting the expression domain of branchless (bnl), encoding the FGF ligand, in surrounding muscle cells. Abnormal activation of the Bnl signaling pathway in hairy mutant tracheal cells is exemplified by increased number of dorsal branch cells expressing Bnl receptor, Breathless (Btl) and Pointed, a downstream target of the Bnl/Btl signaling pathway. We also show that hairy genetically interacts with bnl in TC fate restriction and that overexpression of bnl in a subset of the muscle surrounding tracheal cells phenocopied the hairy mutant phenotype. Our studies demonstrate a novel role for Hairy in restriction of the terminal cell fate by limiting the domain of bnl expression in surrounding muscle cells such that only a single dorsal branch cell becomes specified as a terminal cell. These studies provide the first evidence for Hairy in regulation of the FGF signaling pathway during branching morphogenesis.

Simultaneous targeting of insulin-like growth factor-1 receptor and anaplastic lymphoma kinase in embryonal and alveolar rhabdomyosarcoma: a rational choice.

PubMed

van Gaal, J Carlijn; Roeffen, Melissa H S; Flucke, Uta E; van der Laak, Jeroen A W M; van der Heijden, Gwen; de Bont, Eveline S J M; Suurmeijer, Albert J H; Versleijen-Jonkers, Yvonne M H; van der Graaf, Winette T A

2013-11-01

Rhabdomyosarcoma (RMS) is an aggressive soft tissue tumour mainly affecting children and adolescents. Since survival of high-risk patients remains poor, new treatment options are awaited. The aim of this study is to investigate anaplastic lymphoma kinase (ALK) and insulin-like growth factor-1 receptor (IGF-1R) as potential therapeutic targets in RMS. One-hundred-and-twelve primary tumours (embryonal RMS (eRMS)86; alveolar RMS (aRMS)26) were collected. Expression of IGF-1R, ALK and downstream pathway proteins was evaluated by immunohistochemistry. The effect of ALK inhibitor NVP-TAE684 (Novartis), IGF-1R antibody R1507 (Roche) and combined treatment was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in cell lines (aRMS Rh30, Rh41; eRMS Rh18, RD). IGF-1R and ALK expression was observed in 72% and 92% of aRMS and 61% and 39% of eRMS, respectively. Co-expression was observed in 68% of aRMS and 32% of eRMS. Nuclear IGF-1R expression was an adverse prognostic factor in eRMS (5-year survival 46.9 ± 18.7% versus 84.4 ± 5.9%, p=0.006). In vitro, R1507 showed diminished viability predominantly in Rh41. NVP-TAE684 showed diminished viability in Rh41 and Rh30, and to a lesser extent in Rh18 and RD. Simultaneous treatment revealed synergistic activity against Rh41 and Rh30. Co-expression of IGF-1R and ALK is detected in eRMS and particularly in aRMS. As combined inhibition reveals synergistic cytotoxic effects, this combination seems promising and needs further investigation. Copyright © 2013 Elsevier Ltd. All rights reserved.

PHOX2B Is A Reliable Immunomarker in Distinguishing Peripheral Neuroblastic Tumors From CNS Embryonal Tumors.

PubMed

Alexandrescu, Sanda; Paulson, Vera; Dubuc, Adrian; Ligon, Azra; Lidov, Hart G

2018-05-14

The PHOX2B gene regulates neuronal maturation in the brain stem nuclei associated with cardiorespiratory function, and in the autonomic sympathetic and enteric nervous system. PHOX2B expression is a reliable immunomarker for peripheral neuroblastic tumors, however no systematic evaluation of CNS embryonal tumors was included in the studies. We encountered two cases in which the differential diagnosis included neuroblastoma and CNS embryonal tumor, and we hypothesized that PHOX2B immunostain would be helpful establishing the diagnosis. PHOX2B immunostain was performed on 29 pediatric cases, with adequate controls: 1 retroperitoneal embryonal tumor in a child with retinoblastoma (index1), 1 posterior fossa embryonal tumor in a child with a neuroblastoma (index2), 7 medulloblastomas, 4 atypical teratoid/rhabdoid tumors (ATRT), 4 retinoblastomas, 6 pineoblastomas, 4 embryonal tumors with multilayered rosettes (ETMR), and 2 CNS embryonal tumors, NEC. Cell lineage immunomarkers (GFAP, OLIG2, Synaptophysin, NeuN, CRX, PGP9.5), immunosurrogates for molecular alterations (beta-catenin, INI1, Lin28), array CGH and OncoPanel were performed as needed. Medulloblastomas, ATRTs, ETMRs, retinoblastomas and CNS embryonal tumors NOS were essentially negative for PHOX2B. Two (2) of 6 pineoblastomas had significant PHOX2B expression, while the rest were negative. Index1 was negative for PHOX2B and PGP 9.5, and positive for CRX, consistent with retinoblastoma. Index2 had diffuse PHOX2B expression, MYCN amplification and no copy number changes of medulloblastoma, in keeping with neuroblastoma. PHOX2B antibody is helpful in distinguishing between peripheral neuroblastic and CNS embryonal tumors, which are immunonegative, with the caveat that a subset of pineoblastomas has significant expression. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Sensory regulation of spontaneous limb movements in the midstage embryonic chick.

PubMed

Sharp, Andrew A

2015-05-01

It is becoming increasingly apparent that somatosensation plays an important role in regulating prenatal movement and developmental plasticity. Numerous studies performed on embryonic chicks and perinatal rats are beginning to implicate proprioception to be particularly important in modulating motility very soon after afferent connections are made in the spinal cord. In this report, we demonstrate new approaches in the chick embryo to explore the role of sensation in modulating embryonic movement. Force recordings from the legs of chick embryos on E9 and E11, during spontaneous motility, demonstrate changes in sensory regulation consistent with the concept that sensory regulation is functioning one day after sensory synapse formation and that the complexity of this regulation increases by E11. Additionally, we present new video data showing activation of embryonic motility on E5 and E9 in embryos expressing channelrhodopsin in the spinal cord as a novel way to approach the issues of sensorimotor development. © 2015 Wiley Periodicals, Inc.

Patently controversial: markets, morals, and the President's proposal for embryonic stem cell research.

PubMed

Fins, Joseph J; Schachter, Madeleine

2002-09-01

This essay considers the implications of President George W. Bush's proposal for human embryonic stem cell research. Through the perspective of patent law, privacy, and informed consent, we elucidate the ongoing controversy about the moral standing of human embryonic stem cells and their derivatives and consider how the inconsistencies in the president's proposal will affect clinical practice and research.

Effect of antioxidant of bamboo leaves on gene expression associated with mouse embryonic fibroblast reproduction and embryonic development.

PubMed

Yu, Feng; Qian, Xiaowei; Zeng, Zhanghui; Zhao, Xiaoli; Hou, Rong; Zhang, Zhihe; Bian, Hongwu; Han, Ning; Wang, Junhui; Zhu, Muyuan

2017-11-01

Antioxidant of bamboo leaves (AOB) was certified to be a natural antioxidant by the Chinese Ministry of Health in 2003. However, the effects of AOB on animal reproductive and developmental functions remain unclear. The present study aimed to investigate the effects of different concentrations of AOB on mouse embryonic fibroblast (MEF) cells, and to examine the underlying molecular mechanism through which AOB affects the proliferation and apoptosis of MEFs. MEFs prepared from individual embryos were treated with various dosages of AOB. Cell viability and apoptosis were detected by MTT and flow cytometry assays, respectively. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used for the detection of mRNA and protein expression. Functional annotation of differentially‑expressed genes was performed according to the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Compared with the control group, ~50% of MEF cells were inhibited following treatment with a 400 µg/ml concentration of AOB. Treatment with 400 µg/ml AOB for 72 h significantly increased the apoptotic rate of MEF cells compared with the control group. Following treatment with AOB, dehydrogenase/reductase 9, phospholipase A2 group IVE and platelet derived growth factor B were downregulated, while 17 other genes were upregulated in MEF cells. Treatment with AOB markedly increased the expression of phosphorylated extracellular signal‑regulated kinase (ERK), β‑catenin, transcription factor SOX‑17, calcium‑binding tyrosine phosphorylation‑regulated protein, and cholesterol side chain cleavage enzyme mitochondrial (P<0.01). Additionally, the ERK pathway inhibitor U0126 and Wnt pathway inhibitor dickkopf‑related protein 1 markedly suppressed the expression of the above genes (P<0.01). AOB may impact the expression of proteins associated with embryonic fibroblast reproduction and embryonic development through

Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.

PubMed

Veazey, Kylee J; Golding, Michael C

2011-01-01

Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified m

Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia.

PubMed

Alachkar, Houda; Mutonga, Martin B G; Metzeler, Klaus H; Fulton, Noreen; Malnassy, Gregory; Herold, Tobias; Spiekermann, Karsten; Bohlander, Stefan K; Hiddemann, Wolfgang; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2014-12-15

Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients.

Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia

PubMed Central

Alachkar, Houda; Mutonga, Martin B.G.; Metzeler, Klaus H.; Fulton, Noreen; Malnassy, Gregory; Herold, Tobias; Spiekermann, Karsten; Bohlander, Stefan K.; Hiddemann, Wolfgang; Matsuo, Yo; Stock, Wendy; Nakamura, Yusuke

2014-01-01

Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients. PMID:25365263

NFκB signaling regulates embryonic and adult neurogenesis

PubMed Central

ZHANG, Yonggang; HU, Wenhui

2013-01-01

Both embryonic and adult neurogenesis involves the self-renewal/proliferation, survival, migration and lineage differentiation of neural stem/progenitor cells. Such dynamic process is tightly regulated by intrinsic and extrinsic factors and complex signaling pathways. Misregulated neurogenesis contributes much to a large range of neurodevelopmental defects and neurodegenerative diseases. The signaling of NFκB regulates many genes important in inflammation, immunity, cell survival and neural plasticity. During neurogenesis, NFκB signaling mediates the effect of numerous niche factors such as cytokines, chemokines, growth factors, extracellular matrix molecules, but also crosstalks with other signaling pathways such as Notch, Shh, Wnt/β-catenin. This review summarizes current progress on the NFκB signaling in all aspects of neurogenesis, focusing on the novel role of NFκB signaling in initiating early neural differentiation of neural stem cells and embryonic stem cells. PMID:24324484

Fine structures of embryonic discs of in vivo post-hatching porcine blastocysts at the pre-primitive streak stage.

PubMed

Xia, P; Liu, Z; Qin, P

2011-04-01

To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre-primitive streak area of embryonic discs. The cells were 1-2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc. © 2010 Blackwell Verlag GmbH.

Virtual reality imaging techniques in the study of embryonic and early placental health.

PubMed

Rousian, Melek; Koster, Maria P H; Mulders, Annemarie G M G J; Koning, Anton H J; Steegers-Theunissen, Régine P M; Steegers, Eric A P

2018-04-01

Embryonic and placental growth and development in the first trimester of pregnancy have impact on the health of the fetus, newborn, child and even the adult. This emphasizes the importance of this often neglected period in life. The development of three-dimensional transvaginal ultrasonography in combination with virtual reality (VR) opens the possibility of accurate and reliable visualization of embryonic and placental structures with real depth perception. These techniques enable new biometry and volumetry measurements that contribute to the knowledge of the (patho)physiology of embryonic and early placental health. Examples of such measurements are the length of complex structures like the umbilical cord, vitelline duct, limbs and cerebellum or the volume of the whole embryo and brain cavities. Moreover, for the first time, embryos can now be staged in vivo (Carnegie stages) and vasculature volumes of both the embryo and the early placenta can be measured when VR is combined with power Doppler signals. These innovative developments have already been used to study associations between periconceptional maternal factors, such as age, smoking, alcohol use, diet and vitamin status, and embryonic and early placental growth and development. Future studies will also focus on the identification of abnormal embryonic and early placental development already in the earliest weeks of pregnancy, which provides opportunities for early prevention of pregnancy complications. Copyright © 2018 IFPA, Elsevier Ltd. Published by Elsevier Ltd.. All rights reserved.

Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

PubMed

Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

2015-02-25

Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

Is fibroblast growth factor receptor 4 a suitable target of cancer therapy?

PubMed

Heinzle, Christine; Erdem, Zeynep; Paur, Jakob; Grasl-Kraupp, Bettina; Holzmann, Klaus; Grusch, Michael; Berger, Walter; Marian, Brigitte

2014-01-01

Fibroblast growth factors (FGF) and their tyrosine kinase receptors (FGFR) support cell proliferation, survival and migration during embryonic development, organogenesis and tissue maintenance and their deregulation is frequently observed in cancer development and progression. Consequently, increasing efforts are focusing on the development of strategies to target FGF/FGFR signaling for cancer therapy. Among the FGFRs the family member FGFR4 is least well understood and differs from FGFRs1-3 in several aspects. Importantly, FGFR4 deletion does not lead to an embryonic lethal phenotype suggesting the possibility that its inhibition in cancer therapy might not cause grave adverse effects. In addition, the FGFR4 kinase domain differs sufficiently from those of FGFRs1-3 to permit development of highly specific inhibitors. The oncogenic impact of FGFR4, however, is not undisputed, as the FGFR4-mediated hormonal effects of several FGF ligands may also constitute a tissue-protective tumor suppressor activity especially in the liver. Therefore it is the purpose of this review to summarize all relevant aspects of FGFR4 physiology and pathophysiology and discuss the options of targeting this receptor for cancer therapy.

Is Fibroblast Growth Factor Receptor 4 a Suitable Target of Cancer Therapy?

PubMed Central

Heinzle, Christine; Erdem, Zeynep; Paur, Jakob; Grasl-Kraupp, Bettina; Holzmann, Klaus; Grusch, Michael; Berger, Walter; Marian, Brigitte

2017-01-01

Fibroblast growth factors (FGF) and their tyrosine kinase receptors (FGFR) support cell proliferation, survival and migration during embryonic development, organogenesis and tissue maintenance and their deregulation is frequently observed in cancer development and progression. Consequently, increasing efforts are focusing on the development of strategies to target FGF/FGFR signaling for cancer therapy. Among the FGFRs the family member FGFR4 is least well understood and differs from FGFRs1-3 in several aspects. Importantly, FGFR4 deletion does not lead to an embryonic lethal phenotype suggesting the possibility that its inhibition in cancer therapy might not cause grave adverse effects. In addition, the FGFR4 kinase domain differs sufficiently from those of FGFRs1-3 to permit development of highly specific inhibitors. The oncogenic impact of FGFR4, however, is not undisputed, as the FGFR4-mediated hormonal effects of several FGF ligands may also constitute a tissue-protective tumor suppressor activity especially in the liver. Therefore it is the purpose of this review to summarize all relevant aspects of FGFR4 physiology and pathophysiology and discuss the options of targeting this receptor for cancer therapy. PMID:23944363

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

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

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

Approaches for targeting self-renewal pathways in cancer stem cells: implications for hematological treatments.

PubMed

Horne, Gillian A; Copland, Mhairi

2017-05-01

Self-renewal is considered a defining property of stem cells. Self-renewal is essential in embryogenesis and normal tissue repair and homeostasis. However, in cancer, self-renewal pathways, e.g. WNT, NOTCH, Hedgehog and BMP, frequently become de-regulated in stem cells, or more mature progenitor cells acquire self-renewal properties, resulting in abnormal tissue growth and tumorigenesis. Areas covered: This review considers the conserved embryonic self-renewal pathways, including WNT, NOTCH, Hedgehog and BMP. The article describes recent advances in our understanding of these pathways in leukemia and, more specifically, leukemia stem cells (LSC), how these pathways cross-talk and interact with the LSC microenvironment, and discusses the clinical implications and potential therapeutic strategies, both in preclinical and in clinical trials for hematological malignancies. Expert opinion: The conserved embryonic self-renewal pathways are frequently de-regulated in cancer stem cells (CSC), including LSCs. There is significant cross-talk between self-renewal pathways, and their downstream targets, and the microenvironment. Effective targeting of these pathways is challenging due to cross-talk, and importantly, because these pathways are important for normal stem cells as well as CSC, adverse effects on normal tissues may mean a therapeutic window cannot be identified. Nonetheless, several agents targeting these pathways are currently in clinical trials in hematological malignancies.

Imaging of murine embryonic cardiovascular development using optical coherence tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Huang, Yongyang; Degenhardt, Karl R.; Astrof, Sophie; Zhou, Chao

2016-03-01

We have demonstrated the capability of spectral domain optical coherence tomography (SDOCT) system to image full development of mouse embryonic cardiovascular system. Monitoring morphological changes of mouse embryonic heart occurred in different embryonic stages helps identify structural or functional cardiac anomalies and understand how these anomalies lead to congenital heart diseases (CHD) present at birth. In this study, mouse embryo hearts ranging from E9.5 to E15.5 were prepared and imaged in vitro. A customized spectral domain OCT system was used for imaging, with a central wavelength of 1310nm, spectral bandwidth of ~100nm and imaging speed of 47kHz A-scans/s. Axial resolution of this system was 8.3µm in air, and transverse resolution was 6.2 µm with 5X objective. Key features of mouse embryonic cardiovascular development such as vasculature remodeling into circulatory system, separation of atria and ventricles and emergence of valves could be clearly seen in three-dimensional OCT images. Optical clearing was applied to overcome the penetration limit of OCT system. With high resolution, fast imaging speed, 3D imaging capability, OCT proves to be a promising biomedical imaging modality for developmental biology studies, rivaling histology and micro-CT.

Maternal Residential Proximity to Major Roadways and Pediatric Embryonal Tumors in Offspring.

PubMed

Kumar, Shwetha V; Lupo, Philip J; Pompeii, Lisa A; Danysh, Heather E

2018-03-13

The environmental determinants of pediatric embryonal tumors remain unclear. Because of the growing concern over the impact of exposures to traffic-related air pollution on pediatric cancer, we conducted a population-based study evaluating the impact of maternal residential proximity to major roadways on the risk of pediatric embryonal tumors in offspring. We identified children diagnosed with neuroblastoma, Wilms tumor, retinoblastoma, or hepatoblastoma at <5 years of age from the Texas Cancer Registry and selected unaffected controls from birth certificates. Two residential proximity measures were used: (1) distance to the nearest major roadway, and (2) within 500 m of a major roadway. Logistic regression was used to estimate the adjusted odds ratio (aOR) and 95% confidence interval (CI) for each proximity measure on pediatric embryonal tumors. The odds of an embryonal tumor were increased in children born to mothers living within 500 m of a major roadway (aOR = 1.24, 95% CI: 1.00, 1.54). This was consistent for most tumor subtypes, with the strongest associations observed for unilateral retinoblastoma (aOR = 2.57, 95% CI: 1.28, 5.15, for every kilometer closer the mother lived to the nearest major roadway). These findings contribute to the growing evidence that traffic-related air pollution may increase risk for certain pediatric tumors.

A study on embryonic death in goats due to Nicotiana glauca ingestion.

PubMed

Welch, K D; Lee, S T; Panter, K E; Gardner, D R

2014-11-01

Numerous plants are known to be teratogenic in livestock. In addition to causing malformations, several plants can also cause embryonic death. These losses decrease the reproductive efficiency of animals exposed to these plants. The aim of this study was to determine if teratogenic plants such as lupines or tobaccos cause embryonic losses. A goat model using the plant Nicotiana glauca was used in this study, as this model has been used to characterize the mechanism of Lupinus, Conium, and Nicotiana-induced terata. Four groups of goats were dosed from gestational day 1-10, 11-20, 21-30, and 31-40. Goats were evaluated via ultrasound imaging for pregnancy after completion of the dosing regimen and kids were evaluated for malformations at the time of parturition. Overall, there was no evidence from this study that N. glauca (anabasine) at this dose (2 g/kg/day) would cause embryonic losses in goats. However, the dose of N. glauca used in this study was at the lower threshold that would be expected to produce terata. Therefore it is possible that higher doses of anabasine could cause embryonic loss. Further work is also needed to characterize the kinetic profile of anabasine, and other teratogenic alkaloids, in the fetal compartments. Published by Elsevier Ltd.

Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors

PubMed Central

2014-01-01

Background Small membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized. Methods Cytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated. Results CHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T. Conclusions Out of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity. PMID:24779365

Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors.

PubMed

Naujok, Ortwin; Lentes, Jana; Diekmann, Ulf; Davenport, Claudia; Lenzen, Sigurd

2014-04-29

Small membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized. Cytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated. CHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T. Out of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity.

Paternal identity impacts embryonic development for two species of freshwater fish.

PubMed

Siddique, Mohammad Abdul Momin; Linhart, Otomar; Krejszeff, Sławomir; Żarski, Daniel; Pitcher, Trevor E; Politis, Sebastian Nikitas; Butts, Ian Anthony Ernest

2017-05-01

Paternal, compared to maternal, contributions were believed to have only a limited influence on embryonic development and larval fitness traits in fishes. Therefore, the perspective of male influence on early life history traits has come under scrutiny. This study was conducted to determine parental effects on the rate of eyed embryos of Ide Leuciscus idus and Northern pike Esox lucius. Five sires and five dams from each species were crossed using a quantitative genetic breeding design and the resulting 25 sib groups of each species were reared to the embryonic eyed stage. We then partition variation in embryonic phenotypic performance to maternal, paternal, and parental interactions using the Restricted Maximum Likelihood (REML) model. Results showed that paternal, maternal, and the paternal×maternal interaction terms were highly significant for both species; clearly demonstrating that certain family combinations were more compatible than others. Paternal effects explained 20.24% of the total variance, which was 2-fold higher than the maternal effects (10.73%) in Ide, while paternal effects explained 18.9% of the total variance, which was 15-fold higher than the maternal effects (1.3%) in Northern pike. Together, these results indicate that male effects are of major importance during embryonic development for these species. Furthermore, this study demonstrates that genetic compatibility between sires and dams plays an important role and needs to be taken into consideration for reproduction of these and likely other economically important fish species. Copyright © 2016 Elsevier Inc. All rights reserved.

Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

PubMed Central

2013-01-01

Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405

Two- and three-input TALE-based AND logic computation in embryonic stem cells.

PubMed

Lienert, Florian; Torella, Joseph P; Chen, Jan-Hung; Norsworthy, Michael; Richardson, Ryan R; Silver, Pamela A

2013-11-01

Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells. Using a split-intein protein-splicing strategy, we show that a functional TALE can be reconstituted from two inactive parts, thus generating two-input AND logic computation. We further demonstrate three-piece intein splicing in mammalian cells and use it to perform three-input AND computation. Using methods for random as well as targeted insertion of these relatively large genetic circuits, we show that TALE-based logic circuits are functional when integrated into the genome of mouse embryonic stem cells. Comparing construct variants in the same genomic context, we modulated the strength of the TALE-responsive promoter to improve the output of these circuits. Our work establishes split TALEs as a tool for building logic computation with the potential of controlling expression of endogenous genes or transgenes in response to a combination of cellular signals.

Involvement of Rictor/mTORC2 in cardiomyocyte differentiation of mouse embryonic stem cells in vitro

PubMed Central

Zheng, Bei; Wang, Jiadan; Tang, Leilei; Tan, Chao; Zhao, Zhe; Xiao, Yi; Ge, Renshan; Zhu, Danyan

2017-01-01

Rictor is a key regulatory/structural subunit of the mammalian target of rapamycin complex 2 (mTORC2) and is required for phosphorylation of Akt at serine 473. It plays an important role in cell survival, actin cytoskeleton organization and other processes in embryogenesis. However, the role of Rictor/mTORC2 in the embryonic cardiac differentiation has been uncovered. In the present study, we examined a possible link between Rictor expression and cardiomyocyte differentiation of the mouse embryonic stem (mES) cells. Knockdown of Rictor by shRNA significantly reduced the phosphorylation of Akt at serine 473 followed by a decrease in cardiomyocyte differentiation detected by beating embryoid bodies. The protein levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein) and α-Actinin (cardiomyocyte biomarker) decreased in Rictor knockdown group during cardiogenesis. Furthermore, knockdown of Rictor specifically inhibited the ventricular-like cells differentiation of mES cells with reduced level of ventricular-specific protein, MLC-2v. Meanwhile, patch-clamp analysis revealed that shRNA-Rictor significantly increased the number of cardiomyocytes with abnormal electrophysiology. In addition, the expressions and distribution patterns of cell-cell junction proteins (Cx43/Desmoplakin/N-cadherin) were also affected in shRNA-Rictor cardiomyocytes. Taken together, the results demonstrated that Rictor/mTORC2 might play an important role in the cardiomyocyte differentiation of mES cells. Knockdown of Rictor resulted in inhibiting ventricular-like myocytes differentiation and induced arrhythmias symptom, which was accompanied by interfering the expression and distribution patterns of cell-cell junction proteins. Rictor/mTORC2 might become a new target for regulating cardiomyocyte differentiation and a useful reference for application of the induced pluripotent stem cells. PMID:28123351

Adverse Outcome Pathways for Embryonic Vascular Disruption and Alternative Methods to Identify Chemical Vascular Disruptor

EPA Science Inventory

Chemically induced vascular toxicity during embryonic development can result in a wide range of adverse prenatal outcomes. We used information from genetic mouse models linked to phenotypic outcomes and a vascular toxicity knowledge base to construct an embryonic vascular disrupt...

Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules

NASA Technical Reports Server (NTRS)

Killian, C. E.; Wilt, F. H.

1996-01-01

In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

Towards a global human embryonic stem cell bank.

PubMed

Lott, Jason P; Savulescu, Julian

2007-08-01

An increasingly unbridgeable gap exists between the supply and demand of transplantable organs. Human embryonic stem cell technology could solve the organ shortage problem by restoring diseased or damaged tissue across a range of common conditions. However, such technology faces several largely ignored immunological challenges in delivering cell lines to large populations. We address some of these challenges and argue in favor of encouraging contribution or intentional creation of embryos from which widely immunocompatible stem cell lines could be derived. Further, we argue that current immunological constraints in tissue transplantation demand the creation of a global stem cell bank, which may hold particular promise for minority populations and other sub-groups currently marginalized from organ procurement and allocation systems. Finally, we conclude by offering a number of practical and ethically oriented recommendations for constructing a human embryonic stem cell bank that we hope will help solve the ongoing organ shortage problem.

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

PubMed

Qian, Chen; Wong, Carol Wing Yan; Wu, Zhongluan; He, Qiuming; Xia, Huimin; Tam, Paul Kwong Hang; Wong, Kenneth Kak Yuen; Lui, Vincent Chi Hang

2017-01-01

Platelet-derived growth factor receptor alpha (PDGFRα) is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures. To address the temporal requirement of Pdgfra in embryonic development. We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies. Current study showed that (i) conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5) resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii) the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives. Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a) the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b) if mutations / sequence variations of these regulatory elements cause these anomalies.

Embryonic death is linked to maternal identity in the leatherback turtle (Dermochelys coriacea).

PubMed

Rafferty, Anthony R; Santidrián Tomillo, Pilar; Spotila, James R; Paladino, Frank V; Reina, Richard D

2011-01-01

Leatherback turtles have an average global hatching success rate of ~50%, lower than other marine turtle species. Embryonic death has been linked to environmental factors such as precipitation and temperature, although, there is still a lot of variability that remains to be explained. We examined how nesting season, the time of nesting each season, the relative position of each clutch laid by each female each season, maternal identity and associated factors such as reproductive experience of the female (new nester versus remigrant) and period of egg retention between clutches (interclutch interval) affected hatching success and stage of embryonic death in failed eggs of leatherback turtles nesting at Playa Grande, Costa Rica. Data were collected during five nesting seasons from 2004/05 to 2008/09. Mean hatching success was 50.4%. Nesting season significantly influenced hatching success in addition to early and late stage embryonic death. Neither clutch position nor nesting time during the season had a significant affect on hatching success or the stage of embryonic death. Some leatherback females consistently produced nests with higher hatching success rates than others. Remigrant females arrived earlier to nest, produced more clutches and had higher rates of hatching success than new nesters. Reproductive experience did not affect stage of death or the duration of the interclutch interval. The length of interclutch interval had a significant affect on the proportion of eggs that failed in each clutch and the developmental stage they died at. Intrinsic factors such as maternal identity are playing a role in affecting embryonic death in the leatherback turtle.

Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro.

PubMed

Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena

2017-04-14

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos-ETS-embryos-depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos. Copyright © 2017, American Association for the Advancement of Science.

Functional optical coherence tomography for live dynamic analysis of mouse embryonic cardiogenesis

NASA Astrophysics Data System (ADS)

Wang, Shang; Lopez, Andrew L.; Larina, Irina V.

2018-02-01

Blood flow, heart contraction, and tissue stiffness are important regulators of cardiac morphogenesis and function during embryonic development. Defining how these factors are integrated is critically important to advance prevention, diagnostics, and treatment of congenital heart defects. Mammalian embryonic development is taking place deep within the female body, which makes cardiodynamic imaging and analysis during early developmental stages in humans inaccessible. With thousands of mutant lines available and well-established genetic manipulation tools, mouse is a great model to understand how biomechanical factors are integrated with molecular pathways to regulate cardiac function and development. Dynamic imaging and quantitative analysis of the biomechanics of live mouse embryos have become increasingly important, which demands continuous advancements in imaging techniques and live assessment approaches. This has been one of the major drives to keep pushing the frontier of embryonic imaging for better resolution, higher speed, deeper penetration, and more diverse and effective contrasts. Optical coherence tomography (OCT) has played a significant role in addressing such demands, and its features in non-labeling imaging, 3D capability, a large working distance, and various functional derivatives allow OCT to cover a number of specific applications in embryonic imaging. Recently, our group has made several technical improvements in using OCT to probe the biomechanical aspects of live developing mouse embryos at early stages. These include the direct volumetric structural and functional imaging of the cardiodynamics, four-dimensional quantitative Doppler imaging and analysis of the cardiac blood flow, and fourdimensional blood flow separation from the cardiac wall tissue in the beating embryonic heart. Here, we present a short review of these studies together with brief descriptions of the previous work that demonstrate OCT as a valuable and useful imaging tool

Role of leptin in delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

PubMed

Banerjee, A; Meenakumari, K J; Krishna, A

2010-08-01

An adiposity-associated rise in leptin occurs at the time of delayed embryonic development in Cynopterus sphinx. The aim of present study was to examine the mechanism by which leptin may inhibit progesterone, and therefore could be responsible for delayed development. The study showed a significant increase in circulating leptin level during the period of increased fat accumulation, which coincided with significant decrease in serum progesterone level and delayed embryonic development in C. sphinx. The study showed increased Ob-R expression in the corpus luteum and in the utero-embryonic unit during the period of delayed embryonic development. The in vitro study showed suppressive effect of leptin on progesterone synthesis. The effect of high dose of leptin on ovarian steroidogenesis was found to be mediated through decreased expression of StAR and LH-R proteins in the ovary. The treatment with leptin caused increased expression of STAT 3 and iNOS proteins in the ovary, which correlated with decreased expression of StAR protein in the ovary. The inhibitory effects of leptin on progesterone synthesis in the ovary are thus mediated through STAT 3 and iNOS-NO signaling pathways. This study further demonstrated low expression of PCNA coinciding with the increased concentration of the leptin receptor in the utero-embryonic unit and high circulating leptin level during November. In conclusion, adiposity associated increased leptin level during November-December might play role in suppressing progesterone synthesis in the corpus luteum as well as suppressing the rate of cell-proliferation in the utero-embryonic unit thereby causing delayed embryonic development in C. sphinx. Copyright 2010 Elsevier Inc. All rights reserved.

When Is Embryonic Arrest Broken in Turtle Eggs?

PubMed

Williamson, Sean A; Evans, Roger G; Reina, Richard D

Turtle embryos enter a state of arrested development in the oviduct, allowing the mother greater flexibility in her reproductive schedule. Development recommences once eggs transition from the hypoxic oviduct to the normoxic nest. Significant mortality can occur if turtle eggs are moved between 12 h and 20 d after oviposition, and this is linked to the recommencement of embryonic development. To better understand the timing of developmental arrest and to determine how movement-induced mortality might be avoided, we determined the latency (i.e., time elapsed since oviposition) to recommencement of development following oviposition by exposing the eggs of green turtles (Chelonia mydas) to hypoxia (oxygen tension <8 mmHg) for 3 d, commencing 30 min to 48 h after oviposition. Embryonic development-including development of the characteristic opaque white spot on the eggshell-was halted by hypoxic incubation. When the delay before hypoxic incubation was 12 h or less, hatching success did not differ from a control group. If the hypoxic treatment began after 16 h or more in normoxia, then all embryos died. Thus, by returning eggs to a hypoxic environment before they have broken from arrest (i.e., within 12 h of oviposition), it is possible to extend embryonic arrest for at least 3 d, with no apparent detriment to hatching success. Therefore, hypoxic incubation may provide a new approach for avoidance of movement-induced mortality when conservation or research efforts require the relocation of eggs. Our findings also suggest that movement-induced mortality may have constrained the evolution of viviparity in turtles.

Embryonic blood-cerebrospinal fluid barrier formation and function

PubMed Central

Bueno, David; Parvas, Maryam; Hermelo, Ismaïl; Garcia-Fernàndez, Jordi

2014-01-01

During embryonic development and adult life, brain cavities and ventricles are filled with cerebrospinal fluid (CSF). CSF has attracted interest as an active signaling medium that regulates brain development, homeostasis and disease. CSF is a complex protein-rich fluid containing growth factors and signaling molecules that regulate multiple cell functions in the central nervous system (CNS). The composition and substance concentrations of CSF are tightly controlled. In recent years, it has been demonstrated that embryonic CSF (eCSF) has a key function as a fluid pathway for delivering diffusible signals to the developing brain, thus contributing to the proliferation, differentiation and survival of neural progenitor cells, and to the expansion and patterning of the brain. From fetal stages through to adult life, CSF is primarily produced by the choroid plexus. The development and functional activities of the choroid plexus and other blood–brain barrier (BBB) systems in adults and fetuses have been extensively analyzed. However, eCSF production and control of its homeostasis in embryos, from the closure of the anterior neuropore when the brain cavities become physiologically sealed, to the formation of the functional fetal choroid plexus, has not been studied in as much depth and remains open to debate. This review brings together the existing literature, some of which is based on experiments conducted by our research group, concerning the formation and function of a temporary embryonic blood–CSF barrier in the context of the crucial roles played by the molecules in eCSF. PMID:25389383

Measurement of wall shear stress in chick embryonic heart using optical coherence tomography

NASA Astrophysics Data System (ADS)

Ma, Zhenhe; Dou, Shidan; Zhao, Yuqian; Wang, Yi; Suo, Yanyan; Wang, Fengwen

2015-03-01

The cardiac development is a complicated process affected by genetic and environmental factors. Wall shear stress (WSS) is one of the components which have been proved to influence the morphogenesis during early stages of cardiac development. To study the mechanism, WSS measurement is a step with significant importance. WSS is caused by blood flow imposed on the inner surface of the heart wall and it can be determined by calculating velocity gradients of blood flow in a direction perpendicular to the wall. However, the WSS of the early stage embryonic heart is difficult to measure since the embryonic heart is tiny and beating fast. Optical coherence tomography (OCT) is a non-invasive imaging modality with high spatial and temporal resolution, which is uniquely suitable for the study of early stage embryonic heart development. In this paper, we introduce a method to measure the WSS of early stage chick embryonic heart based on high speed spectral domain optical coherence tomography (SDOCT). 4D (x,y,z,t) scan was performed on the outflow tract (OFT) of HH18 (~3 days of incubation) chick embryonic heart. After phase synchronization, OFT boundary segmentation, and OFT center line calculation, Doppler angle of the blood flow in the OFT can be achieved (This method has been described in previous publications). Combining with the Doppler OCT results, we calculate absolute blood flow velocity distribution in the OFT. The boundary of the OFT was segmented at each cross-sectional structural image, then geometrical center of the OFT can be calculated. Thus, the gradients of blood flow in radial direction can be calculated. This velocity gradient near the wall is termed wall shear rate and the WSS value is proportional to the wall shear rate. Based on this method, the WSS at different heart beating phase are compare. The result demonstrates that OCT is capable of early stage chicken embryonic heart WSS study.

Nasal embryonic LHRH factor plays a role in the developmental migration and projection of gonadotropin-releasing hormone 3 neurons in zebrafish.

PubMed

Palevitch, Ori; Abraham, Eytan; Borodovsky, Natalya; Levkowitz, Gil; Zohar, Yonathan; Gothilf, Yoav

2009-01-01

The initiation of puberty and the functioning of the reproductive system depend on proper development of the hypophysiotropic gonadotropin-releasing hormone (GnRH) system. One critical step in this process is the embryonic migration of GnRH neurons from the olfactory area to the hypothalamus. Using a transgenic zebrafish model, Tg(gnrh3:EGFP), in which GnRH3 neurons and axons are fluorescently labeled, we investigated whether zebrafish NELF is essential for the development of GnRH3 neurons. The zebrafish nelf cDNA was cloned and characterized. During embryonic development, nelf is expressed in GnRH3 neurons and in target sites of GnRH3 projections and perikarya, before the initiation of their migration. Nelf knockdown resulted in a disruption of the GnRH3 system which included absence or misguiding of GnRH3 axonal outgrowth and incorrect or arrested migration of GnRH3 perikarya. These results suggest that Nelf is an important factor in the developmental migration and projection of GnRH3 neurons in zebrafish. Copyright (c) 2008 Wiley-Liss, Inc.

Embryonic background risk promotes the survival of tadpoles facing surface predators

PubMed Central

Chivers, Douglas P.; Ferrari, Maud C. O.

2018-01-01

Exposure to intense predation risk can induce morphological and behavioural phenotypes that prepare prey, often at young ages, for surviving attacks from unknown predators. However, previous studies revealed that this survival advantage depended on the predator species. Here, we used alarm cues from injured conspecifics to simulate a period of high predation risk for embryonic wood frogs, Lithobates sylvaticus. Two weeks post-hatching, we tested whether the embryonic risk exposure influenced survival in encounters with two novel predators: (1) a spider (Dolomedes sp.) that ambushes prey exclusively on the surface of the water, and (2) the adult predacious diving beetle (Dytiscus sp.) which displays underwater sit-and-wait posture and pursuit tactics. Tadpoles exposed to embryonic high-risk survived longer when encountering spiders, whereas background risk had no influence on survival with adult beetles. These findings, coupled with survival studies involving other predator types, indicate that a high-risk environment promotes tadpole survival in future encounters with unknown sit-and-wait predators, but at the cost of increased vulnerability to novel predators capable of active pursuit. PMID:29561913

Alterations to embryonic serotonin change aggression and fearfulness

USDA-ARS?s Scientific Manuscript database

Prenatal environment, including maternal hormones, affects the development of the serotonin (5-HT) system, with long-lasting effects on mood and behavioral exhibition in children and adults. The chicken provides a unique animal model to study the effects of embryonic development on childhood and ado...

F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

PubMed

Thompson, Heather L; van Rooijen, Nico; McLelland, Bryce T; Manilay, Jennifer O

2016-01-01

Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin - BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo . Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80 + macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80 + macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro . Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo .

Effect of temperature on embryonic development of Melanotaenia boesemani (Allen and Cross, 1982).

PubMed

Radael, Marcella Costa; Cardoso, Leonardo Demier; de Andrade, Dalcio Ricardo; Ferreira, André Veloso; da Cruz Mattos, Douglas; Vidal, Manuel Vazquez

2016-04-01

The present study aimed to provide data on the time required for Melanotaenia boesemani to complete embryonic development, and to investigate the influence that incubation at different temperatures caused in this species. The effects of temperature on the time and hatching rate are presented, as well as information related to embryonic development stages. After fertilization, the eggs were kept in incubators at 23, 26, 29 or 32°C and observed at predetermined times until the moment of hatching. Stages of development were identified and classified according to morphological and physiological characteristics. Oil droplets were visualized inside the eggs as well as filament adhesion present at the chorion. Embryonic development was similar to that observed in other species of the genus Melanotaenia with hatching and faster development in higher temperatures.

Gene Targeting of Envoplakin, a Cytoskeletal Linker Protein and Precursor of the Epidermal Cornified Envelope

PubMed Central

Määttä, Arto; DiColandrea, Teresa; Groot, Karen; Watt, Fiona M.

2001-01-01

Envoplakin, a member of the plakin family of cytoskeletal linker proteins, is localized in desmosomes of stratified epithelial cells and is a component of the epidermal cornified envelope. Gene targeting in mouse embryonic stem cells was used to generate a null allele of envoplakin. No envoplakin transcripts from the targeted allele could be detected in the skin of newborn mice. Mice homozygous for the targeted allele were born in the normal Mendelian ratio and were fertile. They did not develop any discernible pathological phenotype up to the age of 1 year. The ultrastructural appearance of cornified envelopes from adult epidermis was indistinguishable between wild-type and knockout mice, and there was no evidence that the absence of envoplakin affected the subcellular distribution of periplakin or desmoplakin, two other plakins found in desmosomes. The proportion of immature cornified envelopes in the epidermis of newborn mice was greater in envoplakin-null animals than in heterozygous littermates or wild-type mice, and the envelopes had a larger surface area. This correlated with a slight delay in barrier acquisition during embryonic development. We conclude that although envoplakin is part of the scaffolding on which the cornified envelope is assembled, it is not essential for envelope formation or epidermal barrier function. PMID:11564887

Maternal Residential Proximity to Major Roadways and Pediatric Embryonal Tumors in Offspring

PubMed Central

Kumar, Shwetha V.; Lupo, Philip J.; Pompeii, Lisa A.; Danysh, Heather E.

2018-01-01

The environmental determinants of pediatric embryonal tumors remain unclear. Because of the growing concern over the impact of exposures to traffic-related air pollution on pediatric cancer, we conducted a population-based study evaluating the impact of maternal residential proximity to major roadways on the risk of pediatric embryonal tumors in offspring. We identified children diagnosed with neuroblastoma, Wilms tumor, retinoblastoma, or hepatoblastoma at <5 years of age from the Texas Cancer Registry and selected unaffected controls from birth certificates. Two residential proximity measures were used: (1) distance to the nearest major roadway, and (2) within 500 m of a major roadway. Logistic regression was used to estimate the adjusted odds ratio (aOR) and 95% confidence interval (CI) for each proximity measure on pediatric embryonal tumors. The odds of an embryonal tumor were increased in children born to mothers living within 500 m of a major roadway (aOR = 1.24, 95% CI: 1.00, 1.54). This was consistent for most tumor subtypes, with the strongest associations observed for unilateral retinoblastoma (aOR = 2.57, 95% CI: 1.28, 5.15, for every kilometer closer the mother lived to the nearest major roadway). These findings contribute to the growing evidence that traffic-related air pollution may increase risk for certain pediatric tumors. PMID:29533992

[Cell therapy for Parkinson's disease: III. Neonatal, fetal and embryonic stem cell-based applications].

PubMed

Anisimov, S V

2009-01-01

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

Sp5 induces the expression of Nanog to maintain mouse embryonic stem cell self-renewal.

PubMed

Tang, Ling; Wang, Manman; Liu, Dahai; Gong, Mengting; Ying, Qi-Long; Ye, Shoudong

2017-01-01

Activation of signal transducer and activator of transcription 3 (STAT3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal. Our previous study showed that trans-acting transcription factor 5 (Sp5), an LIF/STAT3 downstream target, supports mESC self-renewal. However, the mechanism by which Sp5 exerts these effects remains elusive. Here, we found that Nanog is a direct target of Sp5 and mediates the self-renewal-promoting effect of Sp5 in mESCs. Overexpression of Sp5 induced Nanog expression, while knockdown or knockout of Sp5 decreased the Nanog level. Moreover, chromatin immunoprecipitation (ChIP) assays showed that Sp5 directly bound to the Nanog promoter. Functional studies revealed that knockdown of Nanog eliminated the mESC self-renewal-promoting ability of Sp5. Finally, we demonstrated that the self-renewal-promoting function of Sp5 was largely dependent on its zinc finger domains. Taken together, our study provides unrecognized functions of Sp5 in mESCs and will expand our current understanding of the regulation of mESC pluripotency.

G protein-coupled estrogen receptor regulates embryonic heart rate in zebrafish

PubMed Central

Romano, Shannon N.; Edwards, Hailey E.; Ryan, Kevin J.

2017-01-01

Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERβ), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required for normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERβ mutant embryos but not in GPER mutants. GPER mutant embryos exhibited reduced basal heart rate, while heart rate was normal in ERα and ERβ mutants. We detected gper transcript in discrete regions of the brain and pituitary but not in the heart, suggesting that GPER acts centrally to regulate heart rate. In the pituitary, we observed gper expression in cells that regulate levels of thyroid hormone triiodothyronine (T3), a hormone known to increase heart rate. Compared to wild type, GPER mutants had reduced levels of T3 and estrogens, suggesting pituitary abnormalities. Exposure to exogenous T3, but not estradiol, rescued the reduced heart rate phenotype in gper mutant embryos, demonstrating that T3 acts downstream of GPER to regulate heart rate. Using genetic and mass spectrometry approaches, we find that GPER regulates maternal estrogen levels, which are required for normal embryonic heart rate. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and suggest that GPER regulates embryonic heart rate by altering maternal estrogen levels and embryonic T3 levels. PMID:29065151

Engineering human cell spheroids to model embryonic tissue fusion in vitro

PubMed Central

Wolf, Cynthia J.; Wood, Carmen; Ren, Hongzu; Grindstaff, Rachel; Padgett, William; Swank, Adam; MacMillan, Denise; Fisher, Anna; Winnik, Witold; Abbott, Barbara D.

2017-01-01

Epithelial-mesenchymal interactions drive embryonic fusion events during development, and perturbations of these interactions can result in birth defects. Cleft palate and neural tube defects can result from genetic defects or environmental exposures during development, yet very little is known about the effect of chemical exposures on fusion events during human development because of a lack of relevant and robust human in vitro assays of developmental fusion behavior. Given the etiology and prevalence of cleft palate and the relatively simple architecture and composition of the embryonic palate, we sought to develop a three-dimensional culture system that mimics the embryonic palate and could be used to study fusion behavior in vitro using human cells. We engineered size-controlled human Wharton’s Jelly stromal cell (HWJSC) spheroids and established that 7 days of culture in osteogenesis differentiation medium was sufficient to promote an osteogenic phenotype consistent with embryonic palatal mesenchyme. HWJSC spheroids supported the attachment of human epidermal keratinocyte progenitor cells (HPEKp) on the outer spheroid surface likely through deposition of collagens I and IV, fibronectin, and laminin by mesenchymal spheroids. HWJSC spheroids coated in HPEKp cells exhibited fusion behavior in culture, as indicated by the removal of epithelial cells from the seams between spheroids, that was dependent on epidermal growth factor signaling and fibroblast growth factor signaling in agreement with palate fusion literature. The method described here may broadly apply to the generation of three-dimensional epithelial-mesenchymal co-cultures to study developmental fusion events in a format that is amenable to predictive toxicology applications. PMID:28898253

G protein-coupled estrogen receptor regulates embryonic heart rate in zebrafish.

PubMed

Romano, Shannon N; Edwards, Hailey E; Souder, Jaclyn Paige; Ryan, Kevin J; Cui, Xiangqin; Gorelick, Daniel A

2017-10-01

Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERβ), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required for normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERβ mutant embryos but not in GPER mutants. GPER mutant embryos exhibited reduced basal heart rate, while heart rate was normal in ERα and ERβ mutants. We detected gper transcript in discrete regions of the brain and pituitary but not in the heart, suggesting that GPER acts centrally to regulate heart rate. In the pituitary, we observed gper expression in cells that regulate levels of thyroid hormone triiodothyronine (T3), a hormone known to increase heart rate. Compared to wild type, GPER mutants had reduced levels of T3 and estrogens, suggesting pituitary abnormalities. Exposure to exogenous T3, but not estradiol, rescued the reduced heart rate phenotype in gper mutant embryos, demonstrating that T3 acts downstream of GPER to regulate heart rate. Using genetic and mass spectrometry approaches, we find that GPER regulates maternal estrogen levels, which are required for normal embryonic heart rate. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and suggest that GPER regulates embryonic heart rate by altering maternal estrogen levels and embryonic T3 levels.

Embryonic development of Ampheres leucopheus and Iporangaia pustulosa (Arachnida: Opiliones: Gonyleptidae).

PubMed

Gnaspini, Pedro; Lerche, Cristiano Frederico

2010-09-15

The first studies concerning the embryonic development of harvestmen started in the late 19th century, and focused mostly on holarctic species, and only three species of the suborder Laniatores (the largest, among the four suborders considered presently) were studied. Moreover, the last studies on embryology of harvestmen were made during the late 1970s. This study focused on the embryonic development of Ampheres leucopheus (Gonyleptidae, Caelopyginae) and Iporangaia pustulosa (Gonyleptidae, Progonyleptoidellinae). The embryonic development was followed in the field, by taking daily photographs of different eggs during about 2 months. When laid, eggs of A. leucopheus and I. pustulosa have approximately 1.13 and 1.30 mm in diameter, respectively, and the second is embedded in a large amount of mucus. The eggs grow, mainly due to water absorption at the beginning of the process, and they reach a diameter of about 1.35 and 1.59 mm, respectively, close to hatching. It took, respectively, 29-56 days and 35-66 days from egg laying to hatching. For the description of the embryonic development, we use photographs from the field, SEM micrographs, and histological analysis. This allowed us, for instance, to document the progression of structures and pigmentation directly from live embryos in the field, and to record microstructures, such as the presence of perforations in the cuticle of the embryo in the place where eyes are developing. Yet, contrary to what was expected in the literature, we record an egg tooth in one of the studied laniatoreans. (c) 2010 Wiley-Liss, Inc.

Embryonic stem cell self-renewal pathways converge on the transcription factor Tfcp2l1

PubMed Central

Ye, Shoudong; Li, Ping; Tong, Chang; Ying, Qi-Long

2013-01-01

Mouse embryonic stem cell (mESC) self-renewal can be maintained by activation of the leukaemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (Stat3) signalling pathway or dual inhibition (2i) of glycogen synthase kinase 3 (Gsk3) and mitogen-activated protein kinase kinase (MEK). Several downstream targets of the pathways involved have been identified that when individually overexpressed can partially support self-renewal. However, none of these targets is shared among the involved pathways. Here, we show that the CP2 family transcription factor Tfcp2l1 is a common target in LIF/Stat3- and 2i-mediated self-renewal, and forced expression of Tfcp2l1 can recapitulate the self-renewal-promoting effect of LIF or either of the 2i components. In addition, Tfcp2l1 can reprogram post-implantation epiblast stem cells to naïve pluripotent ESCs. Tfcp2l1 upregulates Nanog expression and promotes self-renewal in a Nanog-dependent manner. We conclude that Tfcp2l1 is at the intersection of LIF- and 2i-mediated self-renewal pathways and plays a critical role in maintaining ESC identity. Our study provides an expanded understanding of the current model of ground-state pluripotency. PMID:23942238

Embryonic development rates of northern grasshoppers (Orthoptera: Acrididae): implications for climate change and habitat management

USDA-ARS?s Scientific Manuscript database

Temperature-dependent rates of embryonic development are a primary determinant of the life cycle of many species of grasshoppers which, in cold climates, spend two winters in the egg stage. Knowledge of embryonic developmental rates is important for an assessment of the effects of climate change and...

Using the mouse embryonic stem cell test (EST) to evaluate the embryotoxicity of haloacetic acids

EPA Science Inventory

The Embryonic Stem Cell Test (EST) is used to predict the embryotoxic potential of a test compound by combining the data from cytotoxicity assays in undifferentiated mouse embryonic stem (mES) cells and differentiated mouse cells with the data from a differentiation assay in mES ...

Occupancy of tissue-specific cis-regulatory modules by Otx2 and TLE/Groucho for embryonic head specification.

PubMed

Yasuoka, Yuuri; Suzuki, Yutaka; Takahashi, Shuji; Someya, Haruka; Sudou, Norihiro; Haramoto, Yoshikazu; Cho, Ken W; Asashima, Makoto; Sugano, Sumio; Taira, Masanori

2014-07-09

Head specification by the head-selector gene, orthodenticle (otx), is highly conserved among bilaterian lineages. However, the molecular mechanisms by which Otx and other transcription factors (TFs) interact with the genome to direct head formation are largely unknown. Here we employ ChIP-seq and RNA-seq approaches in Xenopus tropicalis gastrulae and find that occupancy of the corepressor, TLE/Groucho, is a better indicator of tissue-specific cis-regulatory modules (CRMs) than the coactivator p300, during early embryonic stages. On the basis of TLE binding and comprehensive CRM profiling, we define two distinct types of Otx2- and TLE-occupied CRMs. Using these devices, Otx2 and other head organizer TFs (for example, Lim1/Lhx1 (activator) or Goosecoid (repressor)) are able to upregulate or downregulate a large battery of target genes in the head organizer. An underlying principle is that Otx marks target genes for head specification to be regulated positively or negatively by partner TFs through specific types of CRMs.

Temporal distribution of endogenous retinoids in the embryonic mouse mandible.

PubMed

Beeman, C S; Kronmiller, J E

1994-09-01

Retinoids play an important part in embryonic pattern formation. They are necessary for normal differentiation of odontogenic tissues and, in excess, disrupt the pattern of tooth formation. Excess retinoids produce supernumerary buds of the dental lamina in the diastema region of the mouse embryonic mandible where teeth do not normally form. This effect is coincident with an increase in epithelial proliferation and an alteration in epidermal growth factor mRNA expression (a gene product necessary for tooth formation). It was found by high-performance liquid chromatography that endogenous retinoids are present in the developing murine mandible and that concentrations of some retinoids reach a peak at the time of the initiation of odontogenesis (dental lamina formation).

Embryonic stem cells improve skeletal muscle recovery after extreme atrophy in mice.

PubMed

Artioli, Guilherme Giannini; De Oliveira Silvestre, João Guilherme; Guilherme, João Paulo Limongi França; Baptista, Igor Luchini; Ramos, Gracielle Vieira; Da Silva, Willian José; Miyabara, Elen Haruka; Moriscot, Anselmo Sigari

2015-03-01

We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy. © 2014 Wiley Periodicals, Inc.

Ca2+ signaling and early embryonic patterning during the blastula and gastrula periods of zebrafish and Xenopus development.

PubMed

Webb, Sarah E; Miller, Andrew L

2006-11-01

It has been proposed that Ca(2+) signaling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern forming events during early vertebrate development [L.F. Jaffe, Organization of early development by calcium patterns, BioEssays 21 (1999) 657-667; M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signaling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11-21; S.E. Webb, A.L. Miller, Calcium signalling during embryonic development, Nat. Rev. Mol. Cell Biol. 4 (2003) 539-551]. With reference to the embryos of zebrafish (Danio rerio) and the frog, Xenopus laevis, we review the Ca(2+) signals reported during the Blastula and Gastrula Periods. This developmental window encompasses the major pattern forming events of epiboly, involution, and convergent extension, which result in the establishment of the basic germ layers and body axes [C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253-310]. Data will be presented to support the suggestion that propagating waves (both long and short range) of Ca(2+) release, followed by sequestration, may play a crucial role in: (1) Coordinating cell movements during these pattern forming events and (2) Contributing to the establishment of the basic embryonic axes, as well as (3) Helping to define the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen [E. Gilland, A.L. Miller, E. Karplus, R. Baker, S.E. Webb, Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation, Proc. Natl. Acad. Sci. USA 96 (1999) 157-161; J.B. Wallingford, A.J. Ewald, R.M. Harland, S.E. Fraser, Calcium signaling during convergent extension in Xenopus, Curr. Biol. 11 (2001) 652-661]. The various potential targets of these Ca(2+) transients will also be discussed, as well as how they might integrate with other known pattern forming

Relationship between delayed embryonic development and metabolic factors and fat deposition in fruit bat Cynopterus sphinx.

PubMed

Banerjee, Arnab; Meenakumari, K J; Krishna, Amitabh

2007-01-01

The present study was undertaken in the fruit bat Cynopterus sphinx, which breeds twice in quick succession at Varanasi, India. Its gestation period varies significantly in the two successive pregnancies of the year owing to delayed embryonic development during the first (winter) pregnancy. The primary aim of the present study was to determine the role of metabolic factors in delayed embryonic development in the fruit bat C. sphinx. Variation in bodyweight, fat deposition, oxygen (O(2)) consumption rate, basal metabolic rate (BMR), body temperature (Tb) and hepatic succinate dehydrogenase (SDH) activity, along with circulating levels of thyroid hormones (tri-iodothyronine and thyroxine), were examined as metabolic factors during the two successive pregnancies in C. sphinx. The increase in bodyweight observed in November was due to accumulation of white adipose tissue in the posterior abdominal region. A significant decline in O(2) consumption rate, BMR, Tb and SDH activity was found in early winter in November-December, which coincides closely with the period of fat accumulation and with the period of delayed embryonic development in C. sphinx. A significantly higher O(2) consumption rate, BMR, Tb and SDH activity was noted during the second pregnancy in, when embryonic development was relatively faster. Thyroid hormone levels were high during the period of embryonic delay compared with levels during the remaining months. The results of the present study suggest that the delayed embryonic development in C. sphinx during early winter may be due to a low O(2) consumption rate, BMR, Tb and SDH activity in November-December. The energy saved by suppressing embryonic development in this species may be advantageous for fat accumulation. Increased thyroid hormone levels during the early winter period might facilitate fat accumulation in C. sphinx.

[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.

Maternal transfer of methimazole and effects on thyroid hormone availability in embryonic tissues.

PubMed

Van Herck, Stijn L J; Geysens, Stijn; Bald, Edward; Chwatko, Grazyna; Delezie, Evelyne; Dianati, Elham; Ahmed, R G; Darras, Veerle M

2013-07-01

Methimazole (MMI) is an anti-thyroid drug used in the treatment of chronic hyperthyroidism. There is, however, some debate about its use during pregnancy as MMI is known to cross the mammalian placenta and reach the developing foetus. A similar problem occurs in birds, where MMI is deposited in the egg and taken up by the developing embryo. To investigate whether maternally derived MMI can have detrimental effects on embryonic development, we treated laying hens with MMI (0.03% in drinking water) and measured total and reduced MMI contents in the tissues of hens and embryos at different stages of development. In hens, MMI was selectively increased in the thyroid gland, while its levels in the liver and especially brain remained relatively low. Long-term MMI treatment induced a pronounced goitre with a decrease in thyroxine (T₄) content but an increase in thyroidal 3,5,3'-triiodothyronine (T₃) content. This resulted in normal T₃ levels in tissues except in the brain. In chicken embryos, MMI levels were similar in the liver and brain. They gradually decreased during development but always remained above those in the corresponding maternal tissues. Contrary to the situation in hens, T₄ availability was only moderately affected in embryos. Peripheral T₃ levels were reduced in 14-day-old embryos but normal in 18-day-old embryos, while brain T₃ content was decreased at all embryonic stages tested. We conclude that all embryonic tissues are exposed to relatively high doses of MMI and its oxidised metabolites. The effect of maternal MMI treatment on embryonic thyroid hormone availability is most pronounced for brain T₃ content, which is reduced throughout the embryonic development period.

SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability

PubMed Central

Kim, Min Jung; Chia, Ian V.; Costantini, Frank

2008-01-01

Axin is a scaffold protein for the β-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-ΔC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.—Kim, M. J., Chia, I. V., Costantini, F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability. PMID:18632848

The embryonic origin of the ampullate silk glands of the spider Cupiennius salei.

PubMed

Hilbrant, Maarten; Damen, Wim G M

2015-05-01

Silk production in spiders is considered a key innovation, and to have been vital for the diversification of the clade. The evolutionary origin of the organs involved in spider silk production, however, and in particular of the silk glands, is poorly understood. Homologies have been proposed between these and other glands found in arachnids, but lacking knowledge of the embryonic development of spider silk glands hampers an evaluation of hypotheses. This study focuses on the embryonic origin of the largest silk glands of the spider Cupiennius salei, the major and minor ampullate glands. We show how the ampullate glands originate from ectodermal invaginations on the embryonic spinneret limb buds, in relation to morphogenesis of these buds. Moreover, we visualize the subsequent growth of the ampullate glands in sections of the early postembryonic stages. The invaginations are shown to correlate with expression of the proneural gene CsASH2, which is remarkable since it has been proposed that spider silk glands and their nozzles originate from sensory bristles. Hence, by confirming the ectodermal origin of spider silk glands, and by describing the (post-)embryonic morphogenesis of the ampullate glands, this work provides a starting point for further investigating into the genetic program that underlies their development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Efficient femtosecond driven SOX 17 delivery into mouse embryonic stem cells: differentiation studies

NASA Astrophysics Data System (ADS)

Thobakgale, Lebogang; Manoto, Sello Lebohang; Lemboumba, Satuurnin Ombinda; Maaza, Malik; Mthunzi-Kufa, Patience

2017-02-01

Embryonic stem cells have great promise in regenerative medicine because of their ability to self-renew and differentiate into various cell types. Delivery of therapeutic genes into cells has already been achieved using of chemical agents and viral vectors with high transfection efficiencies. However, these methods have also been documented as toxic and in the latter case they can cause latent cell infections. In this study we use femtosecond laser pulses to optically deliver genetic material in mouse embryonic stem cells. Femtosecond laser pulses in contrast to the conventional approach, minimises the risk of unwanted side effects because photons are used to create transient pores on the membrane which allow free entry of molecules with no need for delivery agents. Using an Olympus microscope, fluorescence imaging of the samples post irradiation was performed and decreased expression of stage specific embryonic antigen one (SSEA-1) consistent with on-going cellular differentiation was observed. Our results also show that femtosecond laser pulses were effective in delivering SOX 17 plasmid DNA (pSOX17) which resulted in the differentiation of mouse embryonic stem cells into endoderm cells. We thus concluded that laser transfection of stem cells for the purpose of differentiation, holds potential for applications in tissue engineering as a method of generating new cell lines.

Nicotine induces mitochondrial fission through mitofusin degradation in human multipotent embryonic carcinoma cells

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

Hirata, Naoya; Yamada, Shigeru; Asanagi, Miki

Nicotine is considered to contribute to the health risks associated with cigarette smoking. Nicotine exerts its cellular functions by acting on nicotinic acetylcholine receptors (nAChRs), and adversely affects normal embryonic development. However, nicotine toxicity has not been elucidated in human embryonic stage. In the present study, we examined the cytotoxic effects of nicotine in human multipotent embryonal carcinoma cell line NT2/D1. We found that exposure to 10 μM nicotine decreased intracellular ATP levels and inhibited proliferation of NT2/D1 cells. Because nicotine suppressed energy production, which is a critical mitochondrial function, we further assessed the effects of nicotine on mitochondrial dynamics. Stainingmore » with MitoTracker revealed that 10 μM nicotine induced mitochondrial fragmentation. The levels of the mitochondrial fusion proteins, mitofusins 1 and 2, were also reduced in cells exposed to nicotine. These nicotine effects were blocked by treatment with mecamylamine, a nonselective nAChR antagonist. These data suggest that nicotine degrades mitofusin in NT2/D1 cells and thus induces mitochondrial dysfunction and cell growth inhibition in a nAChR-dependent manner. Thus, mitochondrial function in embryonic cells could be used to assess the developmental toxicity of chemicals.« less

Simultaneous cell death and desquamation of the embryonic diffusion barrier during epidermal development.

PubMed

Saathoff, Manuela; Blum, Barbara; Quast, Thomas; Kirfel, Gregor; Herzog, Volker

2004-10-01

The periderm is an epithelial layer covering the emerging epidermis in early embryogenesis of vertebrates. In the chicken embryo, an additional cellular layer, the subperiderm, occurs at later embryonic stages underneath the periderm. The questions arose what is the function of both epithelial layers and, as they are transitory structures, by which mechanism are they removed. By immunocytochemistry, the tight junction (TJ) proteins occludin and claudin-1 were localized in the periderm and in the subperiderm, and sites of close contact between adjacent cells were detected by electron microscopy. Using horseradish peroxidase (HRP) as tracer, these contacts were identified as tight junctions involved in the formation of the embryonic diffusion barrier. This barrier was lost by desquamation at the end of the embryonic period, when the cornified envelope of the emerging epidermis was formed. By TUNEL and DNA ladder assays, we detected simultaneous cell death in the periderm and the subperiderm shortly before hatching. The absence of caspases-3, -6, and -7 activity, key enzymes of apoptosis, and the lack of typical morphological criteria of apoptosis such as cell fragmentation or membrane blebbing point to a special form of programmed cell death (PCD) leading to the desquamation of the embryonic diffusion barrier. Copyright 2004 Elsevier Inc.

Observation of human embryonic behavior in vitro by high-resolution time-lapse cinematography.

PubMed

Iwata, Kyoko; Mio, Yasuyuki

2016-07-01

Assisted reproductive technology (ART) has yielded vast amounts of information and knowledge on human embryonic development in vitro; however, still images provide limited data on dynamic changes in the developing embryos. Using our high-resolution time-lapse cinematography (hR-TLC) system, we were able to describe normal human embryonic development continuously from the fertilization process to the hatched blastocyst stage in detail. Our hR-TLC observation also showed the embryonic abnormality of a third polar body (PB)-like substance likely containing a small pronucleus being extruded and resulting in single-pronucleus (1PN) formation, while our molecular biological investigations suggested the possibility that some 1PN embryos could be diploid, carrying both maternal and paternal genomes. Furthermore, in some embryos the extruded third PB-like substance was eventually re-absorbed into the ooplasm resulting in the formation of an uneven-sized, two-PN zygote. In addition, other hR-TLC observations showed that cytokinetic failure was correlated with equal-sized, multi-nucleated blastomeres that were also observed in the embryo showing early initiation of compaction. Assessment combining our hR-TLC with molecular biological techniques enables a better understanding of embryonic development and potential improvements in ART outcomes.

Correlation of Versican Expression, Accumulation, and Degradation during Embryonic Development by Quantitative Immunohistochemistry

PubMed Central

Snyder, Jessica M.; Washington, Ida M.; Birkland, Timothy; Chang, Mary Y.; Frevert, Charles W.

2015-01-01

Versican, a chondroitin sulfate proteoglycan, is important in embryonic development, and disruption of the versican gene is embryonically lethal in the mouse. Although several studies show that versican is increased in various organs during development, a focused quantitative study on versican expression and distribution during lung and central nervous system development in the mouse has not previously been performed. We tracked changes in versican (Vcan) gene expression and in the accumulation and degradation of versican. Vcan expression and quantitative immunohistochemistry performed from embryonic day (E) 11.5 to E15.5 showed peak Vcan expression at E13.5 in the lungs and brain. Quantitative mRNA analysis and versican immunohistochemistry showed differences in the expression of the versican isoforms in the embryonic lung and head. The expression of Vcan mRNA and accumulation of versican in tissues was complementary. Immunohistochemistry demonstrated co-localization of versican accumulation and degradation, suggesting distinct roles of versican deposition and degradation in embryogenesis. Very little versican mRNA or protein was found in the lungs of 12- to 16-week-old mice but versican accumulation was significantly increased in mice with Pseudomonas aeruginosa lung infection. These data suggest that versican plays an important role in fundamental, overlapping cellular processes in lung development and infection. PMID:26385570

Quantitation of two endogenous lactose-inhibitable lectins in embryonic and adult chicken tissues

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

Beyer, E.C.; Barondes, S.H.

Two lactose-binding lectins from chicken tissues, chicken-lactose-lectin-I (CLL-I) and chicken-lactose-lectin-II (CLL-II) were quantified with a radioimmunoassay in extracts of a number of developing and adult chicken tissues. Both lectins could be measured in the same extract without separation, because they showed no significant immunological cross- reactivity. Many embryonic and adult tissues, including brain, heart, intestine, kidney, liver, lung, muscle, pancreas, and spleen, contained one or both lectins, although their concentrations differed markedly. For example, embryonic muscle, the richest source of CLL-I contained only traces of CLL-II whereas embryonic kidney, a very rich source of CLL-II contained substantial CLL-I. In bothmore » muscle and kidney, lectin levels in adulthood were much lower than in the embryonic state. In contrast, CLL-I in liver and CLL-II in intestine were 10-fold to 30-fold more concentrated in the adult than in the 15-d embryo. CLL-I and CLL-II from several tissues were purified by affinity chromatography and their identity in the various tissues was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping. The results suggest that these lectins might have different functions in the many developing and adult tissues in which they are found.« less

Bio-engineering inslulin-secreting cells from embryonic stem cells: a review of progress.

PubMed

Roche, E; Sepulcre, M P; Enseñat-Waser, R; Maestre, I; Reig, J A; Soria, B

2003-07-01

According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.

[Embryos and embryo-like entities: problem of definition in the draft of the Swiss embryonic research law].

PubMed

Bürgin, M T; Bürkli, P

2002-11-01

At the end of May 2002, the draft of the Swiss "Federal Act on Research on Surplus Embryos and Embryonic Stem Cells" (EFG, Embryonic Research Act) reached the pre-legislative consultation stage. Under certain conditions, it would allow research on "surplus" embryos from in-vitro fertilization, and the derivation of embryonic stem cells from surplus embryos for research purposes. The EFG draft defines an embryo as "the developing organism from the point of nuclear fusion until the completion of organ development". New technological developments show that embryo-like entities can also be created without nuclear fusion having taken place. It remains unclear how to treat embryonic entities that don't fall under the draft's narrow definition of an embryo. Expanding this definition would be a welcome improvement.

In vitro thermal effects on embryonic cells of endangered hawksbill turtle Eretmochelys imbricata.

PubMed

Takeshita, Satoshi; Matsuda, Naoki; Kodama, Seiji; Suzuki, Keiji; Watanabe, Masami

2013-12-01

The hawksbill turtle is an ectotherm, whose sex is determined by temperature during embryonic development. This study aimed to determine whether embryonic hawksbill turtle cells respond differently to temperature than mammalian cells. Embryonic hawksbill turtle cells were established in culture, and thermal effects on these cells were investigated in vitro. Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with non-essential amino acids, vitamin solution, sodium pyruvate, and 10% fetal bovine serum at 33°C and cell proliferation occurred at 25-33°C. When cells were incubated at 37°C (the temperature of mammalian cell culture) for 24 h, cell growth was completely inhibited. This growth inhibition was evidently recovered by changing the incubation temperature back to 33°C. Expression of heat shock protein was found to increase with elevating culture temperature from 25 to 33°C.

Abnormal differentiation, hyperplasia and embryonic/perinatal lethality in BK5-T/t transgenic mice

PubMed Central

Chen, Xin; Schneider-Broussard, Robin; Hollowell, Debra; McArthur, Mark; Jeter, Collene R.; Benavides, Fernando; DiGiovanni, John; Tang, Dean G.

2009-01-01

The cell-of-origin has a great impact on the types of tumors that develop and the stem/progenitor cells have long been considered main targets of malignant transformation. The SV40 large T and small t antigens (T/t), have been targeted to multiple differentiated cellular compartments in transgenic mice. In most of these studies, transgenic animals develop tumors without apparent defects in animal development. In this study, we used the bovine keratin 5 (BK5) promoter to target the T/t antigens to stem/progenitor cell-containing cytokeratin 5 (CK5) cellular compartment. A transgene construct, BK5-T/t, was made and microinjected into the male pronucleus of FVB/N mouse oocytes. After implanting ∼1700 embryos, only 7 transgenics were obtained, including 4 embryos (E9.5, E13, E15, and E20) and 3 postnatal animals, which died at P1, P2, and P18, respectively. Immunohistological analysis revealed aberrant differentiation and prominent hyperplasia in several transgenic CK5 tissues, especially the upper digestive organs (tongue, oral mucosa, esophagus, and forestomach) and epidermis, the latter of which also showed focal dysplasia. Altogether, these results indicate that constitutive expression of the T/t antigens in CK5 cellular compartment results in abnormal epithelial differentiation and leads to embryonic/perinatal animal lethality. PMID:19272531

Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes.

PubMed

Wang, Bangmei; Li, Kunyu; Wang, Amy; Reiser, Michelle; Saunders, Thom; Lockey, Richard F; Wang, Jia-Wang

2015-10-01

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-29

...; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH... Embryonic Stem Cell Line to be Approved for Use in NIH-Funded Research, 0925-0601, Expiration Date 04/30... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be...

High- and low-temperature manipulation during late incubation: effects on embryonic development, the hatching process, and metabolism in broilers.

PubMed

Willemsen, H; Kamers, B; Dahlke, F; Han, H; Song, Z; Ansari Pirsaraei, Z; Tona, K; Decuypere, E; Everaert, N

2010-12-01

Temperatures continuously higher and lower than the standard incubation temperature by 3°C from embryonic d 16 until embryonic d 18.5 result in differential effects on embryonic development, the hatching process, and embryonic metabolism. Embryos in the high-temperature group were forced into a state of malnutrition by the temperature treatment, as reflected by reduced embryo growth and yolk consumption, resulting in a significantly lower chick weight at hatch. In addition, altered air cell and blood gases as well as a retarded hatching process further indicated reduced growth of embryos exposed to higher incubation temperatures during the latter part of incubation. In addition, hatchability was significantly reduced by the high-temperature treatment due to higher embryonic mortality during the treatment period and the hatching process. Levels of blood glucose, lactate, liver glycogen, plasma triglycerides, and nonesterified fatty acids indicated an altered carbohydrate and lipid metabolism for the high-temperature group. Although the hatching process of embryos exposed to lower incubation temperatures was also significantly retarded, their embryonic development and growth were strikingly similar to those of the control group.

Early embryonic sensitivity to cyclophosphamide in cardiac differentiation from human embryonic stem cells.

PubMed

Zhu, Ming-Xia; Zhao, Jin-Yuan; Chen, Gui-An; Guan, Li

2011-09-01

hESCs (human embryonic stem cells) can differentiate into tissue derivatives of all three germ layers in vitro and mimic the development of the embryo in vivo. In this study, we have investigated the potential of an hESC-based assay for the detection of toxicity to cardiac differentiation in embryonic development. First of all, we developed the protocol of cardiac induction from hESCs according to our previous work and distinguished cardiac precursor cells and late mature cardiomyocytes from differentiated cells, demonstrated by the Q-PCR (quantitative real-time PCR), immunocytochemistry and flow cytometry analysis. In order to test whether CPA (cyclophosphamide) induces developmental and cellular toxicity in the human embryo, we exposed the differentiating cells from hESCs to CPA (a well-known proteratogen) at different stages. We have found that a high concentration of CPA could inhibit cardiac differentiation of hESCs. Two separate exposure intervals were used to determine the effects of CPA on cardiac precursor cells and late mature cardiomyocytes respectively. The cardiac precursor cells were sensitive to CPA in non-cytotoxic concentrations for the expression of the cardiac-specific mRNA markers Nkx2.5 (NK2 transcription factor related, locus 5), GATA-4 (GATA binding protein 4 transcription factor) and TNNT2 (troponin T type 2). Non-cytotoxic CPA concentrations did not affect the mRNA markers' expression in late mature cardiomyocytes, indicating that cardiac precursors were more sensitive to CPA than late cardiomyocytes in cardiogenesis. We set up the in vitro developmental toxicity test model so as to reduce the number of test animals and expenses without compromising the safety of consumers and patients. Furthermore, such in vitro methods may be possibly suited to test a large number of chemicals than the classical employed in vivo tests.

Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

PubMed

Hertsenberg, Andrew J; Funderburgh, James L

2016-01-01

Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

Arabidopsis LEAFY COTYLEDON1 controls cell fate determination during post-embryonic development

PubMed Central

Huang, Mingkun; Hu, Yilong; Liu, Xu; Li, Yuge; Hou, Xingliang

2015-01-01

Arabidopsis LEAFY COTYLEDON1 (LEC1) transcription factor is a master regulator that shapes plant embryo development and post-embryonic seedling establishment. Loss-of-function of LEC1 alters the cotyledon identity, causing the formation of ectopic trichomes, which does not occur in wild-type seedlings, implying that LEC1 might regulate embryonic cell fate determination during post-embryonic development. To test this hypothesis, we compared the expression of trichome development-related genes between the wild-type and the lec1 mutant. We observed that transcripts of GLABROUS1 (GL1), GL2, and GL3, genes encoding the positive regulators in trichome development, were significantly upregulated, while the TRICHOMELESS1 (TCL2), ENHANCER OF TRY AND CPC1 (ETC1), and ETC2 genes, encoding the negative regulators in trichome development, were downregulated in the lec1 mutant. Furthermore, overexpression of LEC1 activated the expressions of TCL2, CAPPICE (CPC), and ETC1, resulting in production of cotyledonary leaves with no or fewer trichomes during vegetative development. In addition, we demonstrated that LEC1 interacts with TCL2 in yeast and in vitro. A genetic experiment showed that loss-of-function of GL2 rescued the ectopic trichome formation in the lec1 mutant. These findings strongly support that LEC1 regulates trichome development, providing direct evidence for the role of LEC1 in cell fate determination during post-embryonic development. PMID:26579186

An Optimized and Simplified System of Mouse Embryonic Stem Cell Cardiac Differentiation for the Assessment of Differentiation Modifiers

PubMed Central

Hartman, Matthew E.; Librande, Jason R.; Medvedev, Ivan O.; Ahmad, Rabiah N.; Moussavi-Harami, Farid; Gupta, Pritha P.; Chien, Wei-Ming; Chin, Michael T.

2014-01-01

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is

Expression pattern of pluripotent markers in different embryonic developmental stages of buffalo (Bubalus bubalis) embryos and putative embryonic stem cells generated by parthenogenetic activation.

PubMed

Singh, Karn P; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K; Chauhan, Manmohan S

2012-12-01

In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.

Identification and Characterization of Long Non-Coding RNAs Related to Mouse Embryonic Brain Development from Available Transcriptomic Data

PubMed Central

He, Hongjuan; Xiu, Youcheng; Guo, Jing; Liu, Hui; Liu, Qi; Zeng, Tiebo; Chen, Yan; Zhang, Yan; Wu, Qiong

2013-01-01

Long non-coding RNAs (lncRNAs) as a key group of non-coding RNAs have gained widely attention. Though lncRNAs have been functionally annotated and systematic explored in higher mammals, few are under systematical identification and annotation. Owing to the expression specificity, known lncRNAs expressed in embryonic brain tissues remain still limited. Considering a large number of lncRNAs are only transcribed in brain tissues, studies of lncRNAs in developmental brain are therefore of special interest. Here, publicly available RNA-sequencing (RNA-seq) data in embryonic brain are integrated to identify thousands of embryonic brain lncRNAs by a customized pipeline. A significant proportion of novel transcripts have not been annotated by available genomic resources. The putative embryonic brain lncRNAs are shorter in length, less spliced and show less conservation than known genes. The expression of putative lncRNAs is in one tenth on average of known coding genes, while comparable with known lncRNAs. From chromatin data, putative embryonic brain lncRNAs are associated with active chromatin marks, comparable with known lncRNAs. Embryonic brain expressed lncRNAs are also indicated to have expression though not evident in adult brain. Gene Ontology analysis of putative embryonic brain lncRNAs suggests that they are associated with brain development. The putative lncRNAs are shown to be related to possible cis-regulatory roles in imprinting even themselves are deemed to be imprinted lncRNAs. Re-analysis of one knockdown data suggests that four regulators are associated with lncRNAs. Taken together, the identification and systematic analysis of putative lncRNAs would provide novel insights into uncharacterized mouse non-coding regions and the relationships with mammalian embryonic brain development. PMID:23967161

Hypoxia promotes production of neural crest cells in the embryonic head.

PubMed

Scully, Deirdre; Keane, Eleanor; Batt, Emily; Karunakaran, Priyadarssini; Higgins, Debra F; Itasaki, Nobue

2016-05-15

Hypoxia is encountered in either pathological or physiological conditions, the latter of which is seen in amniote embryos prior to the commencement of a functional blood circulation. During the hypoxic stage, a large number of neural crest cells arise from the head neural tube by epithelial-to-mesenchymal transition (EMT). As EMT-like cancer dissemination can be promoted by hypoxia, we investigated whether hypoxia contributes to embryonic EMT. Using chick embryos, we show that the hypoxic cellular response, mediated by hypoxia-inducible factor (HIF)-1α, is required to produce a sufficient number of neural crest cells. Among the genes that are involved in neural crest cell development, some genes are more sensitive to hypoxia than others, demonstrating that the effect of hypoxia is gene specific. Once blood circulation becomes fully functional, the embryonic head no longer produces neural crest cells in vivo, despite the capability to do so in a hypoxia-mimicking condition in vitro, suggesting that the oxygen supply helps to stop emigration of neural crest cells in the head. These results highlight the importance of hypoxia in normal embryonic development. © 2016. Published by The Company of Biologists Ltd.

Embryonic death in goats caused by the ingestion of Mimosa tenuiflora.

PubMed

Dantas, Antônio Flávio M; Riet-Correa, Franklin; Medeiros, Rosane M T; Lopes, José Radmácyo; Gardner, Dale R; Panter, Kip; Mota, Rinaldo A

2012-04-01

To determine the teratogenic effect of Mimosa tenuiflora, the green fresh plant was administered ad libitum to 12 goats (Group 1) from day 1 to day 30 of gestation. Upon ultrasonographic examination, on day 30, not one of these goats was pregnant, demonstrating that M. tenuiflora causes embryonic death. Six goats (Group 2) ingested M. tenuiflora from day 30 to day 60 of pregnancy. Four goats delivered seven healthy kids and two were not pregnant based on ultrasonographic examination on day 45 suggesting late embryonic death. Three other groups of six goats each received the plant on days 60-90 (Group 3), 90-120 (Group 4), and 120-150 (Group 5) of gestation and a control group (Group 6) all delivered normal kids, except one goat in Group 4 that aborted and one adult goat from Group 5 that was found dead. It is concluded that M. tenuiflora causes embryonic death. The failure to induce malformations might have resulted from a high dose of an unknown active principle of the plant causing fetal death. Copyright © 2012. Published by Elsevier Ltd.

Patterns of Interspecific Variation in the Heart Rates of Embryonic Reptiles

PubMed Central

Du, Wei-Guo; Ye, Hua; Zhao, Bo; Pizzatto, Ligia; Ji, Xiang; Shine, Richard

2011-01-01

New non-invasive technologies allow direct measurement of heart rates (and thus, developmental rates) of embryos. We applied these methods to a diverse array of oviparous reptiles (24 species of lizards, 18 snakes, 11 turtles, 1 crocodilian), to identify general influences on cardiac rates during embryogenesis. Heart rates increased with ambient temperature in all lineages, but (at the same temperature) were faster in lizards and turtles than in snakes and crocodilians. We analysed these data within a phylogenetic framework. Embryonic heart rates were faster in species with smaller adult sizes, smaller egg sizes, and shorter incubation periods. Phylogenetic changes in heart rates were negatively correlated with concurrent changes in adult body mass and residual incubation period among the lizards, snakes (especially within pythons) and crocodilians. The total number of embryonic heart beats between oviposition and hatching was lower in squamates than in turtles or the crocodilian. Within squamates, embryonic iguanians and gekkonids required more heartbeats to complete development than did embryos of the other squamate families that we tested. These differences plausibly reflect phylogenetic divergence in the proportion of embryogenesis completed before versus after laying. PMID:22174948

Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

PubMed

Winzi, Maria K; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

2011-11-01

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

Isolation and Characterization of Node/Notochord-Like Cells from Mouse Embryonic Stem Cells

PubMed Central

Winzi, Maria K.; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

2014-01-01

The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP+ cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen’s node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures. PMID:21351873

Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow

PubMed Central

Zhu, Jie; Wang, Hong; Yang, Shuo; Guo, Liqiao; Li, Zhen; Wang, Wei; Wang, Suhan; Huang, Wenting; Wang, Liping; Yang, Tan; Ma, Qiang; Bi, Yongyi

2013-01-01

Benzene is an occupational toxicant and an environmental pollutant that potentially causes hematotoxicity and leukemia in exposed populations. Epidemiological studies suggest an association between an increased incidence of childhood leukemia and benzene exposure during the early stages of pregnancy. However, experimental evidence supporting the association is lacking at the present time. It is believed that benzene and its metabolites target hematopoietic stem cells (HSCs) to cause toxicity and cancer in the hematopoietic system. In the current study, we compared the effects of hydroquinone (HQ), a major metabolite of benzene in humans and animals, on mouse embryonic yolk sac hematopoietic stem cells (YS-HSCs) and adult bone marrow hematopoietic stem cells (BM-HSCs). YS-HSCs and BM-HSCs were isolated and enriched, and were exposed to HQ at increasing concentrations. HQ reduced the proliferation and the differentiation and colony formation, but increased the apoptosis of both YS-HSCs and BM-HSCs. However, the cytotoxic and apoptotic effects of HQ were more apparent and reduction of colony formation by HQ was more severe in YS-HSCs than in BM-HSCs. Differences in gene expression profiles were observed in HQ-treated YS-HSCs and BM-HSCs. Cyp4f18 was induced by HQ both in YS-HSCs and BM-HSCs, whereas DNA-PKcs was induced in BM-HSCs only. The results revealed differential effects of benzene metabolites on embryonic and adult HSCs. The study established an experimental system for comparison of the hematopoietic toxicity and leukemogenicity of benzene and metabolites during mouse embryonic development and adulthood. PMID:23940708

Laser ablation of Drosophila embryonic motoneurons causes ectopic innervation of target muscle fibers

NASA Technical Reports Server (NTRS)

Chang, T. N.; Keshishian, H.

1996-01-01

We have tested the effects of neuromuscular denervation in Drosophila by laser-ablating the RP motoneurons in intact embryos before synaptogenesis. We examined the consequences of this ablation on local synaptic connectivity in both 1st and 3rd instar larvae. We find that the partial or complete loss of native innervation correlates with the appearance of alternate inputs from neighboring motor endings and axons. These collateral inputs are found at ectopic sites on the denervated target muscle fibers. The foreign motor endings are electrophysiologically functional and are observed on the denervated muscle fibers by the 1st instar larval stage. Our data are consistent with the existence of a local signal from the target environment, which is regulated by innervation and influences synaptic connectivity. Our results show that, despite the stereotypy of Drosophila neuromuscular connections, denervation can induce local changes in connectivity in wild-type Drosophila, suggesting that mechanisms of synaptic plasticity may also be involved in normal Drosophila neuromuscular development.

Loss of embryonic MET signaling alters profiles of hippocampal interneurons.

PubMed

Martins, Gabriela J; Plachez, Céline; Powell, Elizabeth M

2007-01-01

Hippocampal interneurons arise in the ventral forebrain and migrate dorsally in response to cues, including hepatocyte growth factor/scatter factor which signals via its receptor MET. Examination of the hippocampus in adult mice in which MET had been inactivated in the embryonic proliferative zones showed an increase in parvalbumin-expressing cells in the dentate gyrus, but a loss of these cells in the CA3 region. An overall loss of calretinin-expressing cells was seen throughout the hippocampus. A similar CA3 deficit of parvalbumin and calretinin cells was observed when MET was eliminated only in postmitotic cells. These data suggest that MET is required for the proper hippocampal development, and embryonic perturbations lead to long-term anatomical defects with possible learning and memory dysfunction.

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.

Embryonic ablation of neuronal VGF increases energy expenditure and reduces body weight

PubMed Central

Jiang, Cheng; Lin, Wei-Jye; Sadahiro, Masato; Shin, Andrew C.; Buettner, Christoph; Salton, Stephen R.

2016-01-01

Germline ablation of VGF, a secreted neuronal, neuroendocrine, and endocrine peptide precursor, results in lean, hypermetabolic, and infertile adult mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes (Hahm et al., 1999, 2002). To assess whether this phenotype is predominantly driven by reduced VGF expression in developing and/or adult neurons, or in peripheral endocrine and neuroendocrine tissues, we generated and analyzed conditional VGF knockout mice, obtained by mating loxP-flanked (floxed) Vgf mice with either pan-neuronal Synapsin-Cre- or forebrain alpha-CaMKII-Cre-recombinase-expressing transgenic mice. Adult male and female mice, with conditional ablation of the Vgf gene in embryonic neurons had significantly reduced body weight, increased energy expenditure, and were resistant to diet-induced obesity. Conditional forebrain postnatal ablation of VGF in male mice, primarily in adult excitatory neurons, had no measurable effect on body weight nor on energy expenditure, but led to a modest increase in adiposity, partially overlapping the effect of AAV-Cre-mediated targeted ablation of VGF in the adult ventromedial hypothalamus and arcuate nucleus of floxed Vgf mice (Foglesong et al., 2016), and also consistent with results of icv delivery of the VGF-derived peptide TLQP-21 to adult mice, which resulted in increased energy expenditure and reduced adiposity (Bartolomucci et al., 2006). Because the lean, hypermetabolic phenotype of germline VGF knockout mice is to a great extent recapitulated in Syn-Cre+/−,Vgfflpflox/flpflox mice, we conclude that the metabolic profile of germline VGF knockout mice is largely the result of VGF ablation in embryonic CNS neurons, rather than peripheral endocrine and/or neuroendocrine cells, and that in forebrain structures such as hypothalamus, VGF and/or VGF-derived peptides play uniquely different roles in the developing and adult nervous system. PMID:28024880

Embryonic ablation of neuronal VGF increases energy expenditure and reduces body weight.

PubMed

Jiang, Cheng; Lin, Wei-Jye; Sadahiro, Masato; Shin, Andrew C; Buettner, Christoph; Salton, Stephen R

2017-08-01

Germline ablation of VGF, a secreted neuronal, neuroendocrine, and endocrine peptide precursor, results in lean, hypermetabolic, and infertile adult mice that are resistant to diet-, lesion-, and genetically-induced obesity and diabetes (Hahm et al., 1999, 2002). To assess whether this phenotype is predominantly driven by reduced VGF expression in developing and/or adult neurons, or in peripheral endocrine and neuroendocrine tissues, we generated and analyzed conditional VGF knockout mice, obtained by mating loxP-flanked (floxed) Vgf mice with either pan-neuronal Synapsin-Cre- or forebrain alpha-CaMKII-Cre-recombinase-expressing transgenic mice. Adult male and female mice, with conditional ablation of the Vgf gene in embryonic neurons had significantly reduced body weight, increased energy expenditure, and were resistant to diet-induced obesity. Conditional forebrain postnatal ablation of VGF in male mice, primarily in adult excitatory neurons, had no measurable effect on body weight nor on energy expenditure, but led to a modest increase in adiposity, partially overlapping the effect of AAV-Cre-mediated targeted ablation of VGF in the adult ventromedial hypothalamus and arcuate nucleus of floxed Vgf mice (Foglesong et al., 2016), and also consistent with results of icv delivery of the VGF-derived peptide TLQP-21 to adult mice, which resulted in increased energy expenditure and reduced adiposity (Bartolomucci et al., 2006). Because the lean, hypermetabolic phenotype of germline VGF knockout mice is to a great extent recapitulated in Syn-Cre +/- ,Vgf flpflox/flpflox mice, we conclude that the metabolic profile of germline VGF knockout mice is largely the result of VGF ablation in embryonic CNS neurons, rather than peripheral endocrine and/or neuroendocrine cells, and that in forebrain structures such as hypothalamus, VGF and/or VGF-derived peptides play uniquely different roles in the developing and adult nervous system. Copyright © 2016 Elsevier Ltd. All rights

Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

PubMed

Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

2011-01-01

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

Pontin functions as an essential coactivator for Oct4-dependent lincRNA expression in mouse embryonic stem cells.

PubMed

Boo, Kyungjin; Bhin, Jinhyuk; Jeon, Yoon; Kim, Joomyung; Shin, Hi-Jai R; Park, Jong-Eun; Kim, Kyeongkyu; Kim, Chang Rok; Jang, Hyonchol; Kim, In-Hoo; Kim, V Narry; Hwang, Daehee; Lee, Ho; Baek, Sung Hee

2015-04-10

The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.

NG2 glia are required for vessel network formation during embryonic development

PubMed Central

Minocha, Shilpi; Valloton, Delphine; Brunet, Isabelle; Eichmann, Anne

2015-01-01

The NG2+ glia, also known as polydendrocytes or oligodendrocyte precursor cells, represent a new entity among glial cell populations in the central nervous system. However, the complete repertoire of their roles is not yet identified. The embryonic NG2+ glia originate from the Nkx2.1+ progenitors of the ventral telencephalon. Our analysis unravels that, beginning from E12.5 until E16.5, the NG2+ glia populate the entire dorsal telencephalon. Interestingly, their appearance temporally coincides with the establishment of blood vessel network in the embryonic brain. NG2+ glia are closely apposed to developing cerebral vessels by being either positioned at the sprouting tip cells or tethered along the vessel walls. Absence of NG2+ glia drastically affects the vascular development leading to severe reduction of ramifications and connections by E18.5. By revealing a novel and fundamental role for NG2+ glia, our study brings new perspectives to mechanisms underlying proper vessels network formation in embryonic brains. DOI: http://dx.doi.org/10.7554/eLife.09102.001 PMID:26651999

Integration of immunological aspects in the European Human Embryonic Stem Cell Registry.

PubMed

Borstlap, Joeri; Kurtz, Andreas

2008-05-01

The immunological properties of stem cells are of increasing importance in regenerative medicine. Immunomodulatory mechanisms seem to play an important role not only with respect to the understanding of underlying mechanisms of autologous versus allogenic therapeutic approaches, but also for endogeneous tissue regeneration. The newly established European human embryonic stem cell registry (hESCreg) offers an international database for the registration, documentation and characterisation of human embryonic stem cells (hESC) and their use. By doing so, hESCreg aims to develop a model procedure for further standardisation efforts in the field of stem cell research and regenerative medicine, and eventually the registry may lead to a repository of therapy-related information. Currently the stem cell characterisation data acquired by the registry are divided into several categories such as cell derivation, culture conditions, genetic constitution, stem cell marker expression and degree of modification. This article describes immunological aspects of stem cell characterisation and explores the layout and relevance of a possible additional section to the hESCreg repository to include immunological characteristics of human embryonic stem cells.

In vitro organogenesis of gut-like structures from mouse embryonic stem cells.

PubMed

Kuwahara, M; Ogaeri, T; Matsuura, R; Kogo, H; Fujimoto, T; Torihashi, S

2004-04-01

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

High-Frequency Ultrasound for the Study of Early Mouse Embryonic Cardiovascular System.

PubMed

Greco, Adelaide; Coda, Anna Rita Daniela; Albanese, Sandra; Ragucci, Monica; Liuzzi, Raffaele; Auletta, Luigi; Gargiulo, Sara; Lamagna, Francesco; Salvatore, Marco; Mancini, Marcello

2015-12-01

An accurate diagnosis of congenital heart defects during fetal development is critical for interventional planning. Mice can be used to generate animal models with heart defects, and high-frequency ultrasound (HFUS) imaging enables in utero imaging of live mouse embryos. A wide range of physiological measurements is possible using Doppler-HFUS imaging; limitations of any single measurement warrant a multiparameter approach to characterize cardiovascular function. Doppler-HFUS was used to explore the embryonic (heart, aorta) and extraembryonic (umbilical blood flow) circulatory systems to create a database in normal mouse embryos between 9.5 and 16.5 days of gestation. Multivariate analyses were performed to explore correlations between gestational age and embryo echocardiographic parameters. Heart rate and peak velocity in the aorta were positively correlated with gestational time, whereas cardiac cycle length, isovolumetric relaxation time, myocardial performance index, and arterial deceleration time of the umbilical cord were negatively correlated with it. Doppler-HFUS facilitated detailed characterization of the embryonic mouse circulation and represents a useful tool for investigation of the early mouse embryonic cardiovascular system. © The Author(s) 2015.

Predictive computation of genomic logic processing functions in embryonic development

PubMed Central

Peter, Isabelle S.; Faure, Emmanuel; Davidson, Eric H.

2012-01-01

Gene regulatory networks (GRNs) control the dynamic spatial patterns of regulatory gene expression in development. Thus, in principle, GRN models may provide system-level, causal explanations of developmental process. To test this assertion, we have transformed a relatively well-established GRN model into a predictive, dynamic Boolean computational model. This Boolean model computes spatial and temporal gene expression according to the regulatory logic and gene interactions specified in a GRN model for embryonic development in the sea urchin. Additional information input into the model included the progressive embryonic geometry and gene expression kinetics. The resulting model predicted gene expression patterns for a large number of individual regulatory genes each hour up to gastrulation (30 h) in four different spatial domains of the embryo. Direct comparison with experimental observations showed that the model predictively computed these patterns with remarkable spatial and temporal accuracy. In addition, we used this model to carry out in silico perturbations of regulatory functions and of embryonic spatial organization. The model computationally reproduced the altered developmental functions observed experimentally. Two major conclusions are that the starting GRN model contains sufficiently complete regulatory information to permit explanation of a complex developmental process of gene expression solely in terms of genomic regulatory code, and that the Boolean model provides a tool with which to test in silico regulatory circuitry and developmental perturbations. PMID:22927416

Embryonic development of pleuropodia of the cicada, Magicicada cassini

PubMed Central

Strauß, Johannes; Lakes-Harlan, Reinhard

2006-01-01

In many insects the first abdominal segment possesses embryonic appendages called pleuropodia. Here we show the embryogenesis of pleuropodial cells of the periodical cicada, Magicicada cassini (Fisher 1851) (Insecta, Homoptera, Cicadidae). An antibody, anti-horseradish perioxidase (HRP), that is usually neuron-specific strongly marked the pleuropodial anlagen and revealed their ectodermal origin shortly after limb bud formation. Thereafter the cells sank into the epidermis and their apical parts enlarged. A globular part protruded from the body wall. Filamentous structures were marked at the stem region and into the apical dilation. In later embryonic stages the pleuropodia degenerated. Despite the binding of anti-HRP the cells had no morphological neuronal characters and cannot be regarded as neurons. The binding indicates that glycosylated cell surface molecules contribute to the adhesion between the presumably glandular pleuropodial cells. In comparison, anti-HRP does not mark the pleuropodia of Orthoptera. PMID:19537987

Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

PubMed Central

Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

2012-01-01

Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

Recombinant rabbit leukemia inhibitory factor and rabbit embryonic fibroblasts support the derivation and maintenance of rabbit embryonic stem cells.

PubMed

Xue, Fei; Ma, Yinghong; Chen, Y Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang; Xu, Jie

2012-08-01

The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs.

Development and production of good manufacturing practice grade human embryonic stem cell lines as source material for clinical application.

PubMed

De Sousa, P A; Downie, J M; Tye, B J; Bruce, K; Dand, P; Dhanjal, S; Serhal, P; Harper, J; Turner, M; Bateman, M

2016-09-01

From 2006 to 2011, Roslin Cells Ltd derived 17 human embryonic stem cells (hESC) while developing (RCM1, RC-2 to -8, -10) and implementing (RC-9, -11 to -17) quality assured standards of operation in a facility operating in compliance with European Union (EU) directives and United Kingdom (UK) licensure for procurement, processing and storage of human cells as source material for clinical application, and targeted to comply with an EU Good Manufacturing Practice specification. Here we describe the evolution and specification of the facility, its operation and outputs, complementing hESC resource details communicated in Stem Cell Research Lab Resources. Copyright © 2016. Published by Elsevier B.V.

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

PubMed

Calder, Michele D; Watson, Patricia H; Watson, Andrew J

2011-11-01

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

Innovative virtual reality measurements for embryonic growth and development.

PubMed

Verwoerd-Dikkeboom, C M; Koning, A H J; Hop, W C; van der Spek, P J; Exalto, N; Steegers, E A P

2010-06-01

Innovative imaging techniques, using up-to-date ultrasonic equipment, necessitate specific biometry. The aim of our study was to test the possibility of detailed human embryonic biometry using a virtual reality (VR) technique. In a longitudinal study, three-dimensional (3D) measurements were performed from 6 to 14 weeks gestational age in 32 pregnancies (n = 16 spontaneous conception, n = 16 IVF/ICSI). A total of 125 3D volumes were analysed in the I-Space VR system, which allows binocular depth perception, providing a realistic 3D illusion. Crown-rump length (CRL), biparietal diameter (BPD), occipito-frontal diameter (OFD), head circumference (HC) and abdominal circumference (AC) were measured as well as arm length, shoulder width, elbow width, hip width and knee width. CRL, BPD, OFD and HC could be measured in more than 96% of patients, and AC in 78%. Shoulder width, elbow width, hip width and knee width could be measured in more than 95% of cases, and arm length in 82% of cases. Growth curves were constructed for all variables. Ear and foot measurements were only possible beyond 9 weeks gestation. This study provides a detailed, longitudinal description of normal human embryonic growth, facilitated by a VR system. Growth curves were created for embryonic biometry of the CRL, BPD, HC and AC early in pregnancy and also of several 'new' biometric measurements. Applying virtual embryoscopy will enable us to diagnose growth and/or developmental delay earlier and more accurately. This is especially important for pregnancies at risk of severe complications, such as recurrent late miscarriage and early growth restriction.

Development of an invitro technique to use mouse embryonic stem cell in evaluating effects of xenobiotics

EPA Science Inventory

Our goal has been to develop a high-throughput, in vitro technique for evaluating the effects of xenobiotics using mouse embryonic stem cells (mESCs). We began with the Embryonic Stem Cell Test (EST), which is used to predict the embryotoxic potential of a test compound by combin...

Rat embryonic palatal shelves respond to TCDD in organ culture

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

Abbott, B.D.; Birnbaum, L.S.

1990-05-01