Mammalian Cardiovascular Patterning as Determined by Hemodynamic Forces and Blood Vessel Genetics

NASA Astrophysics Data System (ADS)

Anderson, Gregory Arthur

Cardiovascular development is a process that involves the timing of multiple molecular events, and numerous subtle three-dimensional conformational changes. Traditional developmental biology techniques have provided large quantities of information as to how these complex organ systems develop. However, the major drawback of the majority of current developmental biological imaging is that they are two-dimensional in nature. It is now well recognized that circulation of blood is required for normal patterning and remodeling of blood vessels. Normal blood vessel formation is dependent upon a complex network of signaling pathways, and genetic mutations in these pathways leads to impaired vascular development, heart failure, and lethality. As such, it is not surprising that mutant mice with aberrant cardiovascular patterning are so common, since normal development requires proper coordination between three systems: the heart, the blood, and the vasculature. This thesis describes the implementation of a three-dimensional imaging technique, optical projection tomography (OPT), in conjunction with a computer-based registration algorithm to statistically analyze developmental differences in groups of wild-type mouse embryos. Embryos that differ by only a few hours' gestational time are shown to have developmental differences in blood vessel formation and heart development progression that can be discerned. This thesis describes how we analyzed mouse models of cardiovascular perturbation by OPT to detect morphological differences in embryonic development in both qualitative and quantitative ways. Both a blood vessel specific mutation and a cardiac specific mutation were analyzed, providing evidence that developmental defects of these types can be quantified. Finally, we describe the implementation of OPT imaging to identify statistically significant phenotypes from three different mouse models of cardiovascular perturbation across a range of developmental time points. Image registration methods, combined with intensity- and deformation-based analyses are described and utilized to fully characterize myosin light chain 2a (Mlc2a), delta-like ligand 4 (Dll4), and Endoglin (Eng) mutant mouse embryos. We show that Eng mutant embryos are statistically similar to the Mlc2a phenotype, confirming that these mouse mutants suffer from a primary cardiac developmental defect. Thus, a loss of hemodynamic force caused by defective pumping of the heart is the primary developmental defect affecting these mice.

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.

Development and matching of binocular orientation preference in mouse V1.

PubMed

Bhaumik, Basabi; Shah, Nishal P

2014-01-01

Eye-specific thalamic inputs converge in the primary visual cortex (V1) and form the basis of binocular vision. For normal binocular perceptions, such as depth and stereopsis, binocularly matched orientation preference between the two eyes is required. A critical period of binocular matching of orientation preference in mice during normal development is reported in literature. Using a reaction diffusion model we present the development of RF and orientation selectivity in mouse V1 and investigate the binocular orientation preference matching during the critical period. At the onset of the critical period the preferred orientations of the modeled cells are mostly mismatched in the two eyes and the mismatch decreases and reaches levels reported in juvenile mouse by the end of the critical period. At the end of critical period 39% of cells in binocular zone in our model cortex is orientation selective. In literature around 40% cortical cells are reported as orientation selective in mouse V1. The starting and the closing time for critical period determine the orientation preference alignment between the two eyes and orientation tuning in cortical cells. The absence of near neighbor interaction among cortical cells during the development of thalamo-cortical wiring causes a salt and pepper organization in the orientation preference map in mice. It also results in much lower % of orientation selective cells in mice as compared to ferrets and cats having organized orientation maps with pinwheels.

Examination of diagnostic features in multiphoton microscopy and optical coherence tomography images of ovarian tumorigenesis in a mouse model

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.

Ovarian cancer is a deadly disease owing to the non-specific symptoms and suspected rapid progression, leading to frequent late stage detection and poor prognosis. Medical imaging methods such as CT, MRI and ultrasound as well as serum testing for cancer markers have had extremely poor performance for early disease detection. Due to the poor performance of available screening methods, and the impracticality and ineffectiveness of taking tissue biopsies from the ovary, women at high risk for developing ovarian cancer are often advised to undergo prophylactic salpingo-oophorectomy. This surgery results in many side effects and is most often unnecessary since only a fraction of high risk women go on to develop ovarian cancer. Better understanding of the early development of ovarian cancer and characterization of morphological changes associated with early disease could lead to the development of an effective screening test for women at high risk. Optical imaging methods including optical coherence tomography (OCT) and multiphoton microscopy (MPM) are excellent tools for studying disease progression owing to the high resolution and depth sectioning capabilities. Further, these techniques are excellent for optical biopsy because they can image in situ non-destructively. In the studies described in this dissertation OCT and MPM are used to identify cellular and tissue morphological changes associated with early tumor development in a mouse model of ovarian cancer. This work is organized into three specific aims. The first aim is to use the images from the MPM phenomenon of second harmonic generation to quantitatively examine the morphological differences in collagen structure in normal mouse ovarian tissue and mouse ovarian tumors. The second aim is to examine the differences in endogenous two-photon excited fluorescence in normal mouse ovarian tissue and mouse ovarian tumors. The third and final aim is to identify changes in ovarian microstructure resulting from early disease development by imaging animals in vivo at three time points during a long-term survival study.

Quantitative volumetric imaging of normal, neoplastic and hyperplastic mouse prostate using ultrasound.

PubMed

Singh, Shalini; Pan, Chunliu; Wood, Ronald; Yeh, Chiuan-Ren; Yeh, Shuyuan; Sha, Kai; Krolewski, John J; Nastiuk, Kent L

2015-09-21

Genetically engineered mouse models are essential to the investigation of the molecular mechanisms underlying human prostate pathology and the effects of therapy on the diseased prostate. Serial in vivo volumetric imaging expands the scope and accuracy of experimental investigations of models of normal prostate physiology, benign prostatic hyperplasia and prostate cancer, which are otherwise limited by the anatomy of the mouse prostate. Moreover, accurate imaging of hyperplastic and tumorigenic prostates is now recognized as essential to rigorous pre-clinical trials of new therapies. Bioluminescent imaging has been widely used to determine prostate tumor size, but is semi-quantitative at best. Magnetic resonance imaging can determine prostate volume very accurately, but is expensive and has low throughput. We therefore sought to develop and implement a high throughput, low cost, and accurate serial imaging protocol for the mouse prostate. We developed a high frequency ultrasound imaging technique employing 3D reconstruction that allows rapid and precise assessment of mouse prostate volume. Wild-type mouse prostates were examined (n = 4) for reproducible baseline imaging, and treatment effects on volume were compared, and blinded data analyzed for intra- and inter-operator assessments of reproducibility by correlation and for Bland-Altman analysis. Examples of benign prostatic hyperplasia mouse model prostate (n = 2) and mouse prostate implantation of orthotopic human prostate cancer tumor and its growth (n =  ) are also demonstrated. Serial measurement volume of the mouse prostate revealed that high frequency ultrasound was very precise. Following endocrine manipulation, regression and regrowth of the prostate could be monitored with very low intra- and interobserver variability. This technique was also valuable to monitor the development of prostate growth in a model of benign prostatic hyperplasia. Additionally, we demonstrate accurate ultrasound image-guided implantation of orthotopic tumor xenografts and monitoring of subsequent tumor growth from ~10 to ~750 mm(3) volume. High frequency ultrasound imaging allows precise determination of normal, neoplastic and hyperplastic mouse prostate. Low cost and small image size allows incorporation of this imaging modality inside clean animal facilities, and thereby imaging of immunocompromised models. 3D reconstruction for volume determination is easily mastered, and both small and large relative changes in volume are accurately visualized. Ultrasound imaging does not rely on penetration of exogenous imaging agents, and so may therefore better measure poorly vascularized or necrotic diseased tissue, relative to bioluminescent imaging (IVIS). Our method is precise and reproducible with very low inter- and intra-observer variability. Because it is non-invasive, mouse models of prostatic disease states can be imaged serially, reducing inter-animal variability, and enhancing the power to detect small volume changes following therapeutic intervention.

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

PubMed Central

Ohno, Misa; Togashi, Yuto; Tsuda, Kyoko; Okawa, Kazuaki; Kamaya, Minori; Sakaguchi, Masayoshi; Sugahara, Yasusato; Oyama, Fumitaka

2013-01-01

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific. PMID:23826286

Automated Cell Detection and Morphometry on Growth Plate Images of Mouse Bone

PubMed Central

Ascenzi, Maria-Grazia; Du, Xia; Harding, James I; Beylerian, Emily N; de Silva, Brian M; Gross, Ben J; Kastein, Hannah K; Wang, Weiguang; Lyons, Karen M; Schaeffer, Hayden

2014-01-01

Microscopy imaging of mouse growth plates is extensively used in biology to understand the effect of specific molecules on various stages of normal bone development and on bone disease. Until now, such image analysis has been conducted by manual detection. In fact, when existing automated detection techniques were applied, morphological variations across the growth plate and heterogeneity of image background color, including the faint presence of cells (chondrocytes) located deeper in tissue away from the image’s plane of focus, and lack of cell-specific features, interfered with identification of cell. We propose the first method of automated detection and morphometry applicable to images of cells in the growth plate of long bone. Through ad hoc sequential application of the Retinex method, anisotropic diffusion and thresholding, our new cell detection algorithm (CDA) addresses these challenges on bright-field microscopy images of mouse growth plates. Five parameters, chosen by the user in respect of image characteristics, regulate our CDA. Our results demonstrate effectiveness of the proposed numerical method relative to manual methods. Our CDA confirms previously established results regarding chondrocytes’ number, area, orientation, height and shape of normal growth plates. Our CDA also confirms differences previously found between the genetic mutated mouse Smad1/5CKO and its control mouse on fluorescence images. The CDA aims to aid biomedical research by increasing efficiency and consistency of data collection regarding arrangement and characteristics of chondrocytes. Our results suggest that automated extraction of data from microscopy imaging of growth plates can assist in unlocking information on normal and pathological development, key to the underlying biological mechanisms of bone growth. PMID:25525552

Caspase inhibition supports proper gene expression in ex vivo mouse limb cultures.

PubMed

De Valck, D; Luyten, F P

2001-10-01

We standardized conditions for ex vivo mouse limb culture to study cartilage maturation and joint formation. We compared 12.5 d.p.c. mouse forelimbs that were cultured either mounted or freely rotating for up to 72 h. Limb outgrowth progressed ex vivo at a variable rate as compared to its development in vivo, spanning approximately 48 h. Although cartilage maturation and joint formation developed grossly normal, aberrant expression of skeletal marker genes was seen. Interestingly, no regression of the interdigital webs took place in mounted cultures, in contrast to limited webbing under freely rotating conditions. Caspase inhibition, by addition of zVAD-fmk to the culture medium of freely rotating limbs, supported proper gene expression associated with skeletal development, and prevented interdigital web regression. Taken together, a freely rotating ex vivo culture for mouse limb outgrowth that is combined with caspase inhibition provides a good model to study cartilage maturation and joint formation.

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line.

PubMed Central

Cavard, C; Grimber, G; Dubois, N; Chasse, J F; Bennoun, M; Minet-Thuriaux, M; Kamoun, P; Briand, P

1988-01-01

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated. Images PMID:3162766

Hes1 Is Required for Appropriate Morphogenesis and Differentiation during Mouse Thyroid Gland Development

PubMed Central

Carre, Aurore; Rachdi, Latif; Tron, Elodie; Richard, Bénédicte; Castanet, Mireille; Schlumberger, Martin; Bidart, Jean-Michel

2011-01-01

Notch signalling plays an important role in endocrine development, through its target gene Hes1. Hes1, a bHLH transcriptional repressor, influences progenitor cell proliferation and differentiation. Recently, Hes1 was shown to be expressed in the thyroid and regulate expression of the sodium iodide symporter (Nis). To investigate the role of Hes1 for thyroid development, we studied thyroid morphology and function in mice lacking Hes1. During normal mouse thyroid development, Hes1 was detected from E9.5 onwards in the median anlage, and at E11.5 in the ultimobranchial bodies. Hes1 −/− mouse embryos had a significantly lower number of Nkx2-1-positive progenitor cells (p<0.05) at E9.5 and at E11.5. Moreover, Hes1 −/− mouse embryos showed a significantly smaller total thyroid surface area (−40 to −60%) compared to wild type mice at all study time points (E9.5−E16.5). In both Hes1 −/− and wild type mouse embryos, most Nkx2-1-positive thyroid cells expressed the cell cycle inhibitor p57 at E9.5 in correlation with low proliferation index. In Hes1 −/− mouse embryos, fusion of the median anlage with the ultimobranchial bodies was delayed by 3 days (E16.5 vs. E13.5 in wild type mice). After fusion of thyroid anlages, hypoplastic Hes1 −/− thyroids revealed a significantly decreased labelling area for T4 (−78%) and calcitonin (−65%) normalized to Nkx2-1 positive cells. Decreased T4-synthesis might be due to reduced Nis labelling area (−69%). These findings suggest a dual role of Hes1 during thyroid development: first, control of the number of both thyrocyte and C-cell progenitors, via a p57-independent mechanism; second, adequate differentiation and endocrine function of thyrocytes and C-cells. PMID:21364918

The Effect of Radiation on the Normal and Oestrone-treated Mouse Vagina Grown in vitro

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

Lasnitzki, Ilse

1961-06-01

The effect of radiation on the differentiation of normal and oestrone- treated organ cultures of the mouse vagina was studied during growth in natural and in chemically defined medium. After explanation into either type of medium without addition of the hormone the vaginal epithelium spontaneously forms new squamous keratinising epithelium which displaces the original secretory epithelium. Addition of oestrone to the medium stimulates the growth of the new cells and hastens and increases keratin synthesis. Exposure to 200 r of x rays given one day after explanation inhibits the squamous development, suppresses keratin formation and preserves the original epithelium inmore » both normal and oestrone-treated explants grown in natural medium. Radiation of cultures kept in defined medium fails to influence the normal squamous differentiation and keratinisation. The mechanism of radiation action is discussed in the light of the differnet results obtained under the two conditions. (auth)« less

Magnetic resonance imaging study of eye congenital birth defects in mouse model

PubMed Central

Tucker, Zachary; Mongan, Maureen; Meng, Qinghang; Xia, Ying

2017-01-01

Purpose Embryonic eyelid closure is a well-documented morphogenetic episode in mammalian eye development. Detection of eyelid closure defect in humans is a major challenge because eyelid closure and reopen occur entirely in utero. As a consequence, congenital eye defects that are associated with failure of embryonic eyelid closure remain unknown. To fill the gap, we developed a mouse model of defective eyelid closure. This preliminary work demonstrates that the magnetic resonance imaging (MRI) approach can be used for the detection of extraocular muscle abnormalities in the mouse model. Methods Mice with either normal (Map3k1+/−) or defective (Map3k1−/−) embryonic eyelid closure were used in this study. Images of the extraocular muscles were obtained with a 9.4 T high resolution microimaging MRI system. The extraocular muscles were identified, segmented, and measured in each imaging slice using an in-house program. Results In agreement with histological findings, the imaging data show that mice with defective embryonic eyelid closure develop less extraocular muscle than normal mice. In addition, the size of the eyeballs was noticeably reduced in mice with defective embryonic eyelid closure. Conclusions We demonstrated that MRI can potentially be used for the study of extraocular muscle in the mouse model of the eye open-at-birth defect, despite the lack of specificity of muscle group provided by the current imaging resolution. PMID:28848319

High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting.

PubMed

Ackermann, Amanda M; Zhang, Jia; Heller, Aryel; Briker, Anna; Kaestner, Klaus H

2017-03-01

α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER "knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreER T2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. We utilized CRISPR-Cas9 technology to insert an IRES-CreER T2 sequence into the 3' UTR of the Glucagon ( Gcg ) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreER T2 mice. Recombination efficiency in GCG + pancreatic α-cells and glucagon-like peptide 1 positive (GLP1 + ) enteroendocrine L-cells was measured in Gcg-CreER T2 ; Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Tamoxifen injection of Gcg-CreER T2 ; Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreER T2 allele were phenotypically normal. We successfully derived a Gcg-CreER T2 mouse line that expresses CreER T2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.

Development and matching of binocular orientation preference in mouse V1

PubMed Central

Bhaumik, Basabi; Shah, Nishal P.

2014-01-01

Eye-specific thalamic inputs converge in the primary visual cortex (V1) and form the basis of binocular vision. For normal binocular perceptions, such as depth and stereopsis, binocularly matched orientation preference between the two eyes is required. A critical period of binocular matching of orientation preference in mice during normal development is reported in literature. Using a reaction diffusion model we present the development of RF and orientation selectivity in mouse V1 and investigate the binocular orientation preference matching during the critical period. At the onset of the critical period the preferred orientations of the modeled cells are mostly mismatched in the two eyes and the mismatch decreases and reaches levels reported in juvenile mouse by the end of the critical period. At the end of critical period 39% of cells in binocular zone in our model cortex is orientation selective. In literature around 40% cortical cells are reported as orientation selective in mouse V1. The starting and the closing time for critical period determine the orientation preference alignment between the two eyes and orientation tuning in cortical cells. The absence of near neighbor interaction among cortical cells during the development of thalamo-cortical wiring causes a salt and pepper organization in the orientation preference map in mice. It also results in much lower % of orientation selective cells in mice as compared to ferrets and cats having organized orientation maps with pinwheels. PMID:25104927

[Muscle regeneration in mdx mouse, and a trial of normal myoblast transfer into regenerating dystrophic muscle].

PubMed

Takemitsu, M; Arahata, K; Nonaka, I

1990-10-01

The most ideal therapeutic trial on Duchenne muscular dystrophy (DMD) is a transfer of normal myoblasts into dystrophic muscle which has been attempted on animal models in several institutes. In the process of muscle regeneration, the transferred normal myoblasts are expected to incorporate into the regenerating fibers in host dystrophic mouse. To know the capacity of muscle regeneration in dystrophic muscle, we compared the regenerating process of the normal muscle with that of the dystrophic muscle after myonecrosis induced by 0.25% bupivacaine hydrochloride (BPVC) chronologically. In the present study, C57BL/10ScSn-mdx (mdx) mouse was used as an animal model of DMD and C57BL/10ScSn (B10) mouse as a control. There was no definite difference in the behavior of muscle fiber regeneration between normal and dystrophic muscles. The dystrophic muscle regenerated rapidly at the similar tempo to the normal as to their size and fiber type differentiation. The variation in fiber size diameter of dystrophic muscle, however, was more obvious than that of normal. To promote successful myoblast transfer from B10 mouse into dystrophic mdx mouse at higher ratio, cultured normal myoblasts were transferred into the regenerating dystrophic muscle on the first and the second day after myonecrosis induced by BPVC. Two weeks after the myoblast injection, the muscles were examined with immunohistochemical stain using anti dystrophin antibody. Although dystrophin-positive fibers appeared in dystrophic muscle, the positive fibers were unexpectedly small in number (3.86 +/- 1.50%).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer

PubMed Central

Yamazaki, Yukiko; Makino, Hatsune; Hamaguchi-Hamada, Kayoko; Hamada, Shun; Sugino, Hidehiko; Kawase, Eihachiro; Miyata, Takaki; Ogawa, Masaharu; Yanagimachi, Ryuzo; Yagi, Takeshi

2001-01-01

When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5–17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells. PMID:11698647

Gene expression of Hsp70, Hsp90 and Hsp110 families in normal palate and cleft palate during mouse embryogenesis.

PubMed

Zhu, Yongfei; Ren, Chuanlu; Wan, Xuying; Zhu, Yuping; Zhu, Jiangbo; Zhou, Hongyuan; Zhang, Tianbao

2013-11-01

Most previous studies focused on a small number of heat shock proteins (Hsps) and their relationships with embryogenesis, and the actual roles of these Hsps in normal and abnormal embryonic development remain unclear. It was found in the present systemic study that except for Grp170, whose expression was not detectable at GD18, all 19 Hsps of Hsp70, Hsp90 and Hsp110 families were expressed in the normal development of embryonic palate tissue in mice, but their expression patterns varied with different Hsps, presenting as a correlation with the developmental phases. In the treatment group by all-trans retinoic acid (atRA), the messenger RNA (mRNA) abundance of HspA1A, HspA1L, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14, Hsp90AA1, Hsp90AB1, Grp94, Trap1, Hsp105, Hsp110 and Grp170 was higher in the palates at GD11 (the beginning of palate development), the mRNA abundance of HspA1A, HspA12A and HspA12B was higher at GD18 (before birth) and an mRNA expression peak of HspA1L, HspA8, HspA9, Hsp90AA1, Grp94, Hsp110 and Grp170 was observed at GD17. The mRNA abundance of most genes in atRA-induced cleft palates of the treatment group was different from that of the control group. Grp78, HspA14 and Hsp105 were closely associated with the normal palate development and cleft palate in mouse embryo, possibly as palate development-related genes. Except Grp170, the other genes may be closely associated with the development of mouse palates through participating in the stress response process and/or the antiapoptosis process.

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

PubMed

Haram, Kerstyn M; Peltier, Heidi J; Lu, Bin; Bhasin, Manoj; Otu, Hasan H; Choy, Bob; Regan, Meredith; Libermann, Towia A; Latham, Gary J; Sanda, Martin G; Arredouani, Mohamed S

2008-10-01

Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: influence of the maternally inherited components.

PubMed

Mognetti, B; Leppens, G; Sakkas, D

1996-04-01

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In-view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose.

Long-term imaging of mouse embryos using adaptive harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Thayil, Anisha; Watanabe, Tomoko; Jesacher, Alexander; Wilson, Tony; Srinivas, Shankar; Booth, Martin

2011-04-01

We present a detailed description of an adaptive harmonic generation (HG) microscope and culture techniques that permit long-term, three-dimensional imaging of mouse embryos. HG signal from both pre- and postimplantation stage (0.5-5.5 day-old) mouse embryos are fully characterized. The second HG images reveal central spindles during cytokinesis whereas third HG images show several features, such as lipid droplets, nucleoli, and plasma membranes. The embryos are found to develop normally during one-day-long discontinuous HG imaging, permitting the observation of several dynamic events, such as morula compaction and blastocyst formation.

Zebrafish pit1 mutants lack three pituitary cell types and develop severe dwarfism.

PubMed

Nica, Gabriela; Herzog, Wiebke; Sonntag, Carmen; Hammerschmidt, Matthias

2004-05-01

The Pou domain transcription factor Pit-1 is required for lineage determination and cellular commitment processes during mammalian adenohypophysis development. Here we report the cloning and mutational analysis of a pit1 homolog from zebrafish. Compared with mouse, zebrafish pit1 starts to be expressed at a much earlier stage of adenohypophysis development. However, as in the mouse, expression is restricted to a subset of pituitary cell types, excluding proopiomelanocortin (pomc)-expressing cells (corticotropes, melanotropes) and possibly gonadotropes. We could identify two N-ethyl-N-nitrosourea-induced zebrafish pit1 null mutants. Most mutants die during larval stages, whereas survivors develop severe dwarfism. Mutant larvae lack lactotropes, somatotropes, and thyrotropes, although the adenohypophysis is of normal size, without any sign of increased apoptosis rates. Instead, mutant embryos initiate ectopic expression of pomc in pit1-positive cells, leading to an expansion of the Pomc lineage. Similarly, the number of gonadotropes seems increased, as indicated by the expression of gsualpha, a marker for thyrotropes and gonadotropes. In pit1 mutants, the total number of gsualpha-positive cells is normal despite the loss of gsualpha and tshbeta coexpressing cells. Together, these data suggest a transfating of the Pit1 lineage to the Pomc and possibly the gonadotroph lineages in the mutant, and a pomc- and gonadotropin-repressive role of Pit1 during normal zebrafish development. This is different from mouse, for which a repressive role of Pit-1 has only been reported for the gonadotropin Lhbeta, but not for Pomc. In sum, our data point to both conserved and class-specific aspects of Pit1 function during pituitary development in different vertebrate species.

Near-infrared laser irradiation improves the development of mouse pre-implantation embryos.

PubMed

Yokoo, Masaki; Mori, Miho

2017-05-27

The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing of blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P < 0.05). When 195 irradiated blastocysts were transferred to 18 pseudopregnant mice, all became pregnant and 92 (47.2%) normal-looking pups were born alive. When 182 control blastocysts were transferred to 17 pseudopregnant mice, 14 (82.4%) became pregnant and 54 (29.7%) normal-looking pups were born alive. The growth trajectories (up to 5 weeks) of offspring from irradiated blastocysts were similar to those from control blastocysts. Second generation offspring from transplanted animals were all fertile. These results indicate that near-infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

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).

Mutants in the mouse NuRD/Mi2 component P66alpha are embryonic lethal.

PubMed

Marino, Susan; Nusse, Roel

2007-06-13

The NuRD/Mi2 chromatin complex is involved in histone modifications and contains a large number of subunits, including the p66 protein. There are two mouse and human p66 paralogs, p66alpha and p66beta. The functions of these genes are not clear, in part because there are no mutants available, except in invertebrate model systems. We made loss of function mutants in the mouse p66alpha gene (mp66alpha, official name Gatad2a, MGI:2384585). We found that mp66alpha is essential for development, as mutant embryos die around day 10 of embryogenesis. The gene is not required for normal blastocyst development or for implantation. The phenotype of mutant embryos and the pattern of gene expression in mutants are consistent with a role of mp66alpha in gene silencing. mp66alpha is an essential gene, required for early mouse development. The lethal phenotype supports a role in execution of methylated DNA silencing.

In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes.

PubMed

Quinlan, Jonathan M; Yu, Wei-Yuan; Hornsey, Mark A; Tosh, David; Slack, Jonathan M W

2006-05-25

Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable.

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

PubMed Central

Komatsu, Yoshihiro

2014-01-01

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

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

PubMed

Komatsu, Yoshihiro; Mishina, Yuji

2013-12-01

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

System parameters for erythropoiesis control model: Comparison of normal values in human and mouse model

NASA Technical Reports Server (NTRS)

1979-01-01

The computer model for erythropoietic control was adapted to the mouse system by altering system parameters originally given for the human to those which more realistically represent the mouse. Parameter values were obtained from a variety of literature sources. Using the mouse model, the mouse was studied as a potential experimental model for spaceflight. Simulation studies of dehydration and hypoxia were performed. A comparison of system parameters for the mouse and human models is presented. Aside from the obvious differences expected in fluid volumes, blood flows and metabolic rates, larger differences were observed in the following: erythrocyte life span, erythropoietin half-life, and normal arterial pO2.

Single-shot dimension measurements of the mouse eye using SD-OCT.

PubMed

Jiang, Minshan; Wu, Pei-Chang; Fini, M Elizabeth; Tsai, Chia-Ling; Itakura, Tatsuo; Zhang, Xiangyang; Jiao, Shuliang

2012-01-01

The authors demonstrate the feasibility and advantage of spectral-domain optical coherence tomography (SD-OCT) for single-shot ocular biometric measurement during the development of the mouse eye. A high-resolution SD-OCT system was built for single-shot imaging of the whole mouse eye in vivo. The axial resolution and imaging depth of the system are 4.5 μm (in tissue) and 5.2 mm, respectively. The system is capable of acquiring a cross-sectional OCT image consisting of 2,048 depth scans in 85 ms. The imaging capability of the SD-OCT system was validated by imaging the normal ocular growth and experimental myopia model using C57BL/6J mice. The biometric dimensions of the mouse eye can be calculated directly from one snapshot of the SD-OCT image. The biometric parameters of the mouse eye including axial length, corneal thickness, anterior chamber depth, lens thickness, vitreous chamber depth, and retinal thickness were successfully measured by the SD-OCT. In the normal ocular growth group, the axial length increased significantly from 28 to 82 days of age (P < .001). The lens thickness increased and the vitreous chamber depth decreased significantly during this period (P < .001 and P = .001, respectively). In the experimental myopia group, there were significant increases in vitreous chamber depth and axial length in comparison to the control eyes (P = .040 and P < .001, respectively). SD-OCT is capable of providing single-shot direct, fast, and high-resolution measurements of the dimensions of young and adult mouse eyes. As a result, SD-OCT is a potentially powerful tool that can be easily applied to research in eye development and myopia using small animal models. Copyright 2012, SLACK Incorporated.

Three-dimensional imaging of the developing mouse female reproductive organs with optical coherence tomography

NASA Astrophysics Data System (ADS)

Burton, Jason C.; Wang, Shang; Behringer, Richard R.; Larina, Irina V.

2016-03-01

Infertility is a known major health concern and is estimated to impact ~15% of couples in the U.S. The majority of failed pregnancies occur before or during implantation of the fertilized embryo into the uterus. Understanding the mechanisms regulating development by studying mouse reproductive organs could significantly contribute to an improved understanding of normal development of reproductive organs and developmental causes of infertility in humans. Towards this goal, we report a three-dimensional (3D) imaging study of the developing mouse reproductive organs (ovary, oviduct, and uterus) using optical coherence tomography (OCT). In our study, OCT was used for 3D imaging of reproductive organs without exogenous contrast agents and provides micro-scale spatial resolution. Experiments were conducted in vitro on mouse reproductive organs ranging from the embryonic day 14.5 to adult stages. Structural features of the ovary, oviduct, and uterus are presented. Additionally, a comparison with traditional histological analysis is illustrated. These results provide a basis for a wide range of infertility studies in mouse models. Through integration with traditional genetic and molecular biology approaches, this imaging method can improve understanding of ovary, oviduct, and uterus development and function, serving to further contribute to our understanding of fertility and infertility.

Near-infrared laser irradiation improves the development of mouse pre-implantation embryos

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

Yokoo, Masaki; Mori, Miho

The aim of the present study was to assess the effects of near-infrared laser irradiation on the in vitro development of mouse embryos. Female ICR mice were superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG), and mated with male mice. Two-cell stage embryos were collected 40 h after administering hCG and cultured in M16 medium. Two-cell embryos (0 h after culture), 8-cell embryos (approx. 30 h after culture), morula (approx. 48 h after culture), and blastocysts (approx. 73 h after culture) were irradiated at 904 nm for 60 s. These embryos were cultured in a time-lapse monitoring system and the timing ofmore » blastocyst hatching was evaluated. Some of the irradiated blastocysts were transferred to the uterine horns of pseudopregnant recipients immediately after irradiation. Pregnancy rates, and offspring growth and fertility, were evaluated. Near-infrared laser irradiation increased the speed of in vitro mouse embryo development. In irradiated blastocysts, hatching was faster than in control (non-irradiated) blastocysts (18.4 vs. 28.2 h, P < 0.05). When 195 irradiated blastocysts were transferred to 18 pseudopregnant mice, all became pregnant and 92 (47.2%) normal-looking pups were born alive. When 182 control blastocysts were transferred to 17 pseudopregnant mice, 14 (82.4%) became pregnant and 54 (29.7%) normal-looking pups were born alive. The growth trajectories (up to 5 weeks) of offspring from irradiated blastocysts were similar to those from control blastocysts. Second generation offspring from transplanted animals were all fertile. These results indicate that near-infrared laser irradiation improves the quality of mouse embryo development in vitro, and increases the live birth rate without affecting the normality of the offspring. Thus, the near-infrared laser method may enhance the quality of embryos and contribute to improvements in reproductive technologies in mammals. - Highlights: • Irradiation of blastocysts with a near-infrared laser improves embryo development. • Irradiation of blastocysts increases the live birth rate after embryo transfer. • Irradiation of blastocysts did not affect the normality of the pups. • Near-infrared laser irradiation may be useful to enhance the quality of embryos. • This study may contribute to improvements in reproductive technologies in mammals.« less

Characterization and Separation of Cancer Cells with a Wicking Fiber Device.

PubMed

Tabbaa, Suzanne M; Sharp, Julia L; Burg, Karen J L

2017-12-01

Current cancer diagnostic methods lack the ability to quickly, simply, efficiently, and inexpensively screen cancer cells from a mixed population of cancer and normal cells. Methods based on biomarkers are unreliable due to complexity of cancer cells, plasticity of markers, and lack of common tumorigenic markers. Diagnostics are time intensive, require multiple tests, and provide limited information. In this study, we developed a novel wicking fiber device that separates cancer and normal cell types. To the best of our knowledge, no previous work has used vertical wicking of cells through fibers to identify and isolate cancer cells. The device separated mouse mammary tumor cells from a cellular mixture containing normal mouse mammary cells. Further investigation showed the device separated and isolated human cancer cells from a heterogeneous mixture of normal and cancerous human cells. We report a simple, inexpensive, and rapid technique that has potential to identify and isolate cancer cells from large volumes of liquid samples that can be translated to on-site clinic diagnosis.

Mouse Models as Tools to Identify Genetic Pathways for Retinal Degeneration, as Exemplified by Leber's Congenital Amaurosis.

PubMed

Chang, Bo

2016-01-01

Leber's congenital amaurosis (LCA) is an inherited retinal degenerative disease characterized by severe loss of vision in the first year of life. In addition to early vision loss, a variety of other eye-related abnormalities including roving eye movements, deep-set eyes, and sensitivity to bright light also occur with this disease. Many animal models of LCA are available and the study them has led to a better understanding of the pathology of the disease, and has led to the development of therapeutic strategies aimed at curing or slowing down LCA. Mouse models, with their well-developed genetics and similarity to human physiology and anatomy, serve as powerful tools with which to investigate the etiology of human LCA. Such mice provide reproducible, experimental systems for elucidating pathways of normal development, function, designing strategies and testing compounds for translational research and gene-based therapies aimed at delaying the diseases progression. In this chapter, I describe tools used in the discovery and evaluation of mouse models of LCA including a Phoenix Image-Guided Optical Coherence Tomography (OCT) and a Diagnosys Espion Visual Electrophysiology System. Three mouse models are described, the rd3 mouse model for LCA12 and LCA1, the rd12 mouse model for LCA2, and the rd16 mouse model for LCA10.

Insulin-Like growth factor 1 related pathways and high-fat diet promotion of transgenic adenocarcinoma mouse prostate (TRAMP) cancer progression.

PubMed

Xu, H; Jiang, H W; Ding, Q

2015-04-01

We aimed to investigate the role of IGF-1 related pathway in high-fat diet (HFD) promotion of TRAMP mouse PCa progression. TRAMP mice were randomly divided into two groups: HFD group and normal diet group. TRAMP mice of both groups were sacrificed and sampled on the 20th, 24th and 28th week respectively. Serum levels of insulin, IGF-1 and IGF-2 were tested by ELISA. Prostate tissue of TRAMP mice was used for both HE staining and immunohistochemical staining of IGF-1 related pathway proteins, including IGF-1Rα, IGF -1Rβ, IGFBPs and AKT. The mortality of TRAMP mice from HFD group was significantly higher than that of normal diet group (23.81% and 7.14%, p=.035). The tumor incidence of HFD TRAMP mice at 20(th) week was significantly higher than normal diet group (78.57% and 35.71%, p=.022). Serum IGF-1 level of HFD TRAMP mice was significantly higher than that of normal diet TRAMP mice. Serum IGF-1 level tended to increase with HFD TRAMP mice's age. HFD TRAMP mice had higher positive staining rate of IGF-1Rα, IGF-1Rβ, IGFBP3 and Akt than normal diet TRAMP mice. IGF-1 related pathway played an important role in high-fat diet promotion of TRAMP mouse PCa development and progression. Copyright © 2014 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

A PML/Slit Axis Controls Physiological Cell Migration and Cancer Invasion in the CNS.

PubMed

Amodeo, Valeria; A, Deli; Betts, Joanne; Bartesaghi, Stefano; Zhang, Ying; Richard-Londt, Angela; Ellis, Matthew; Roshani, Rozita; Vouri, Mikaella; Galavotti, Sara; Oberndorfer, Sarah; Leite, Ana Paula; Mackay, Alan; Lampada, Aikaterini; Stratford, Eva Wessel; Li, Ningning; Dinsdale, David; Grimwade, David; Jones, Chris; Nicotera, Pierluigi; Michod, David; Brandner, Sebastian; Salomoni, Paolo

2017-07-11

Cell migration through the brain parenchyma underpins neurogenesis and glioblastoma (GBM) development. Since GBM cells and neuroblasts use the same migratory routes, mechanisms underlying migration during neurogenesis and brain cancer pathogenesis may be similar. Here, we identify a common pathway controlling cell migration in normal and neoplastic cells in the CNS. The nuclear scaffold protein promyelocytic leukemia (PML), a regulator of forebrain development, promotes neural progenitor/stem cell (NPC) and neuroblast migration in the adult mouse brain. The PML pro-migratory role is active also in transformed mouse NPCs and in human primary GBM cells. In both normal and neoplastic settings, PML controls cell migration via Polycomb repressive complex 2 (PRC2)-mediated repression of Slits, key regulators of axon guidance. Finally, a PML/SLIT1 axis regulates sensitivity to the PML-targeting drug arsenic trioxide in primary GBM cells. Taken together, these findings uncover a drug-targetable molecular axis controlling cell migration in both normal and neoplastic cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Alanine–glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer

PubMed Central

Salido, Eduardo C.; Li, Xiao M.; Lu, Yang; Wang, Xia; Santana, Alfredo; Roy-Chowdhury, Namita; Torres, Armando; Shapiro, Larry J.; Roy-Chowdhury, Jayanta

2006-01-01

Mutations in the alanine–glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt−/− mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria. PMID:17110443

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.

PubMed

Herrlinger, Stephanie A; Shao, Qiang; Ma, Li; Brindley, Melinda; Chen, Jian-Fu

2018-04-26

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

NASA Astrophysics Data System (ADS)

Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

2015-11-01

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in hypergravity conditions does not affect the normal development and actin filament structures of mouse embryos.

Gasdermin D (Gsdmd) is dispensable for mouse intestinal epithelium development.

PubMed

Fujii, Tomoaki; Tamura, Masaru; Tanaka, Shigekazu; Kato, Yoriko; Yamamoto, Hiromi; Mizushina, Youichi; Shiroishi, Toshihiko

2008-08-01

Members of the novel gene family Gasdermin (Gsdm) are exclusively expressed in a highly tissue-specific manner in the epithelium of skin and the gastrointestinal tract. Based on their expression patterns and the phenotype of the Gsdma3 spontaneous mutations, it is inferred that the Gsdm family genes are involved in epithelial cell growth and/or differentiations in different tissues. To investigate possible roles of the Gsdm gene family in the development of intestinal tracts, we generated a Gsdmd mutant mouse, which is a solitary member of the Gsdmd subfamily and which is predominantly expressed in the intestinal tract by means of targeted disruption. In the mutant homozygotes, we found no abnormality of intestinal tract morphology. Moreover, in mutant mice, there was normal differentiation of all constituent cell types of the intestinal epithelium. Thus, this study clearly shows that Gsdmd is not essential for development of mouse intestinal tract or epithelial cell differentiation.

Histological and reference system for the analysis of mouse intervertebral disc.

PubMed

Tam, Vivian; Chan, Wilson C W; Leung, Victor Y L; Cheah, Kathryn S E; Cheung, Kenneth M C; Sakai, Daisuke; McCann, Matthew R; Bedore, Jake; Séguin, Cheryle A; Chan, Danny

2018-01-01

A new scoring system based on histo-morphology of mouse intervertebral disc (IVD) was established to assess changes in different mouse models of IVD degeneration and repair. IVDs from mouse strains of different ages, transgenic mice, or models of artificially induced IVD degeneration were assessed. Morphological features consistently observed in normal, and early/later stages of degeneration were categorized into a scoring system focused on nucleus pulposus (NP) and annulus fibrosus (AF) changes. "Normal NP" exhibited a highly cellularized cell mass that decreased with natural ageing and in disc degeneration. "Normal AF" consisted of distinct concentric lamellar structures, which was disrupted in severe degeneration. NP/AF clefts indicated more severe changes. Consistent scores were obtained between experienced and new users. Altogether, our scoring system effectively differentiated IVD changes in various strains of wild-type and genetically modified mice and in induced models of IVD degeneration, and is applicable from the post-natal stage to the aged mouse. This scoring tool and reference resource addresses a pressing need in the field for studying IVD changes and cross-study comparisons in mice, and facilitates a means to normalize mouse IVD assessment between different laboratories. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:233-243, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Development of (99m)Tc-Labeled Pyridyl Benzofuran Derivatives To Detect Pancreatic Amylin in Islet Amyloid Model Mice.

PubMed

Yoshimura, Masashi; Ono, Masahiro; Watanabe, Hiroyuki; Kimura, Hiroyuki; Saji, Hideo

2016-06-15

While islet amyloid deposition comprising amylin is one of pathological hallmarks of type 2 diabetes mellitus (T2DM), no useful amylin-imaging probe has been reported. In this study, we evaluated two (99m)Tc-labeled pyridyl benzofuran derivatives as novel amylin-imaging probes using the newly established islet amyloid model mouse. Binding experiments in vitro demonstrated that [(99m)Tc]1 displayed a higher affinity for amylin aggregates than [(99m)Tc]2. Autoradiographic studies using human pancreas sections with T2DM revealed that [(99m)Tc]1 clearly labeled islet amyloid in T2DM pancreatic sections, while [(99m)Tc]2 did not. Although the initial uptake of [(99m)Tc]1 by the normal mouse pancreas was low (0.74%ID/g at 2 min post-injection), [(99m)Tc]1 showed higher retention in the model mouse pancreas than that of the normal mouse, and exhibited strong binding to amylin aggregates in the living pancreas of the model mice. These results suggest that [(99m)Tc]1 is a potential imaging probe targeting islet amyloids in the T2DM pancreas.

Botulinic toxin detection by counterimmunoelectrophoresis.

PubMed

Nuñez, L; Amato de Lagarde, E A; d'Aquino, M

1990-03-01

A counterimmunoelectrophoretic (CIE) technique was developed to detect botulinic toxin type A, and the method was compared with the mouse bioassay. A 100 LD50 concentration was detected within 2 h. Crossed reactions were observed with antitoxins of types B and F. As regards other Clostridium species, precipitin lines were seen between C. sporogenes and antitoxin type A. Foodstuffs artificially contaminated with C. botulinum type A were assayed by means of CIE and mouse bioassay, without recording interference by substances normally contained in foodstuffs.

Mutants in the Mouse NuRD/Mi2 Component P66α Are Embryonic Lethal

PubMed Central

Marino, Susan; Nusse, Roel

2007-01-01

Background The NuRD/Mi2 chromatin complex is involved in histone modifications and contains a large number of subunits, including the p66 protein. There are two mouse and human p66 paralogs, p66α and p66β. The functions of these genes are not clear, in part because there are no mutants available, except in invertebrate model systems. Methodology We made loss of function mutants in the mouse p66α gene (mp66α, official name Gatad2a, MGI:2384585). We found that mp66α is essential for development, as mutant embryos die around day 10 of embryogenesis. The gene is not required for normal blastocyst development or for implantation. The phenotype of mutant embryos and the pattern of gene expression in mutants are consistent with a role of mp66α in gene silencing. Conclusion mp66α is an essential gene, required for early mouse development. The lethal phenotype supports a role in execution of methylated DNA silencing. PMID:17565372

Aberrant pulmonary lymphatic development in the nitrofen mouse model of congenital diaphragmatic hernia

PubMed Central

Shue, Eveline; Wu, Jianfeng; Schecter, Samuel; Miniati, Doug

2013-01-01

Purpose Many infants develop a postsurgical chylothorax after diaphragmatic hernia repair. The pathogenesis remains elusive but may be due to dysfunctional lymphatic development. This study characterizes pulmonary lymphatic development in the nitrofen mouse model of CDH. Methods CD1 pregnant mice were fed nitrofen/bisdiamine (N/B) or olive oil at E8.5. At E14.5 and E15.5, lung buds were categorized by phenotype: normal, N/B without CDH (N/B−CDH), or N/B with CDH (N/B+CDH). Anti-CD31 was used to localize all endothelial cells, while anti-LYVE-1 was used to identify lymphatic endothelial cells in lung buds using immunofluorescence. Differential protein expression of lymphatic-specific markers was analyzed. Results Lymphatic endothelial cells localized to the mesenchyme surrounding the airway epithelium at E15.5. CD31 and LYVE-1 colocalization identified lymphatic endothelial cells. LYVE-1 expression was upregulated in N/B+CDH lung buds in comparison to N/B−CDH and normal lung buds by immunofluorescence. Western blotting shows that VEGF-D, LYVE-1, Prox-1, and VEGFR-3 expression was upregulated in N/B+CDH lung buds in comparison to N/B−CDH or control lung buds at E14.5. Conclusions Lung lymphatics are hyperplastic in N/B+CDH. Upregulation of lymphatic-specific genes suggest that lymphatic hyperplasia plays an important role in dysfunctional lung lymphatic development in the nitrofen mouse model of CDH. PMID:23845607

Aberrant pulmonary lymphatic development in the nitrofen mouse model of congenital diaphragmatic hernia.

PubMed

Shue, Eveline; Wu, Jianfeng; Schecter, Samuel; Miniati, Doug

2013-06-01

Many infants develop a postsurgical chylothorax after diaphragmatic hernia repair. The pathogenesis remains elusive but may be owing to dysfunctional lymphatic development. This study characterizes pulmonary lymphatic development in the nitrofen mouse model of CDH. CD1 pregnant mice were fed nitrofen/bisdiamine (N/B) or olive oil at E8.5. At E14.5 and E15.5, lung buds were categorized by phenotype: normal, N/B without CDH (N/B - CDH), or N/B with CDH (N/B+CDH). Anti-CD31 was used to localize all endothelial cells, while anti-LYVE-1 was used to identify lymphatic endothelial cells in lung buds using immunofluorescence. Differential protein expression of lymphatic-specific markers was analyzed. Lymphatic endothelial cells localized to the mesenchyme surrounding the airway epithelium at E15.5. CD31 and LYVE-1 colocalization identified lymphatic endothelial cells. LYVE-1 expression was upregulated in N/B+CDH lung buds in comparison to N/B - CDH and normal lung buds by immunofluorescence. Western blotting shows that VEGF-D, LYVE-1, Prox-1, and VEGFR-3 expression was upregulated in N/B+CDH lung buds in comparison to N/B - CDH or control lung buds at E14.5. Lung lymphatics are hyperplastic in N/B+CDH. Upregulation of lymphatic-specific genes suggests that lymphatic hyperplasia plays an important role in dysfunctional lung lymphatic development in the nitrofen mouse model of CDH. Copyright © 2013 Elsevier Inc. All rights reserved.

Canonical Wnt Signaling as a Specific Mark of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2011-02-01

aggressive mammary tumors. 15. SUBJECT TERMS Breast cancer stem cells, Wnt signaling, canonical Wnt signaling, B-catenin, normal stem cells, adult stem...Wnt pathway is associated with abnormal mouse mammary development, tumorigenesis, and human breast cancer. In addition, increasing evidence suggests...activation occurs in human breast cancer and is required for proliferation of various other stem cell compartments, addressing how Wnt signaling promotes

Early detection of disease program: Evaluation of the cellular immune response

NASA Technical Reports Server (NTRS)

Criswell, B. S.; Knight, V.; Martin, R. R.; Kasel, J. A.

1975-01-01

Surfaces of normal, cultured, and mitogen-stimulated mouse lymphoid cells were examined by scanning electron microscopy (SEM). Lymphocytes with smooth, highly villous and intermediate surfaces were observed in cell suspensions from both spleens and thymuses of normal mice and from spleens of congenitally athymic (nude) mice. Several strain-specific surface features were noted, including the spine-like appearance of microvilli on C57B1/6 lymphocytes. Although thymus cell suspensions contained somewhat more smooth cells than did spleen cell preparations, lymphocyte derivation could not be inferred from SEM examination. Studies of cells stimulated with mitogenic agents for thymus-derived lymphocytes (concanavalin A) or for bone marrow-derived lymphocytes (lipopolysaccharide) suggested that, in the mouse, development of a complex villous surface is a general concomitant of lymphocyte activation and transformation.

Microenvironmental Regulation of Mammary Carcinogenesis

DTIC Science & Technology

2008-06-01

cells. These models share many of the hallmarks of multistage human breast cancer development including histological disease progression and immune cell... developed by Muller and colleagues20, represents a reasonable recapitulation of late-stage human breast cancer as determined by histological progression ...Annual Progress Report d. Develop a profile of proteolytic activities in normal and neoplastic mammary tissues from mouse models of mammary

Collagen in organ development

NASA Technical Reports Server (NTRS)

Hardman, P.; Spooner, B. S.

1992-01-01

It is important to know whether microgravity will adversely affect developmental processes. Collagens are macromolecular structural components of the extracellular matrix (ECM) which may be altered by perturbations in gravity. Interstitial collagens have been shown to be necessary for normal growth and morphogenesis in some embryonic organs, and in the mouse salivary gland, the biosynthetic pattern of these molecules changes during development. Determination of the effects of microgravity on epithelial organ development must be preceded by crucial ground-based studies. These will define control of normal synthesis, secretion, and deposition of ECM macromolecules and the relationship of these processes to morphogenesis.

A novel mouse running wheel that senses individual limb forces: biomechanical validation and in vivo testing

PubMed Central

Roach, Grahm C.; Edke, Mangesh

2012-01-01

Biomechanical data provide fundamental information about changes in musculoskeletal function during development, adaptation, and disease. To facilitate the study of mouse locomotor biomechanics, we modified a standard mouse running wheel to include a force-sensitive rung capable of measuring the normal and tangential forces applied by individual paws. Force data were collected throughout the night using an automated threshold trigger algorithm that synchronized force data with wheel-angle data and a high-speed infrared video file. During the first night of wheel running, mice reached consistent running speeds within the first 40 force events, indicating a rapid habituation to wheel running, given that mice generated >2,000 force-event files/night. Average running speeds and peak normal and tangential forces were consistent throughout the first four nights of running, indicating that one night of running is sufficient to characterize the locomotor biomechanics of healthy mice. Twelve weeks of wheel running significantly increased spontaneous wheel-running speeds (16 vs. 37 m/min), lowered duty factors (ratio of foot-ground contact time to stride time; 0.71 vs. 0.58), and raised hindlimb peak normal forces (93 vs. 115% body wt) compared with inexperienced mice. Peak normal hindlimb-force magnitudes were the primary force component, which were nearly tenfold greater than peak tangential forces. Peak normal hindlimb forces exceed the vertical forces generated during overground running (50-60% body wt), suggesting that wheel running shifts weight support toward the hindlimbs. This force-instrumented running-wheel system provides a comprehensive, noninvasive screening method for monitoring gait biomechanics in mice during spontaneous locomotion. PMID:22723628

Integrated expression analysis identifies transcription networks in mouse and human gastric neoplasia.

PubMed

Chen, Zheng; Soutto, Mohammed; Rahman, Bushra; Fazili, Muhammad W; Peng, DunFa; Blanca Piazuelo, Maria; Chen, Heidi; Kay Washington, M; Shyr, Yu; El-Rifai, Wael

2017-07-01

Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. The Tff1 knockout (KO) mouse model develops gastric lesions that include low-grade dysplasia (LGD), high-grade dysplasia (HGD), and adenocarcinomas. In this study, we used Affymetrix microarrays gene expression platforms for analysis of molecular signatures in the mouse stomach [Tff1-KO (LGD) and Tff1 wild-type (normal)] and human gastric cancer tissues and their adjacent normal tissue samples. Combined integrated bioinformatics analysis of mouse and human datasets indicated that 172 genes were consistently deregulated in both human gastric cancer samples and Tff1-KO LGD lesions (P < .05). Using Ingenuity pathway analysis, these genes mapped to important transcription networks that include MYC, STAT3, β-catenin, RELA, NFATC2, HIF1A, and ETS1 in both human and mouse. Further analysis demonstrated activation of FOXM1 and inhibition of TP53 transcription networks in human gastric cancers but not in Tff1-KO LGD lesions. Using real-time RT-PCR, we validated the deregulated expression of several genes (VCAM1, BGN, CLDN2, COL1A1, COL1A2, COL3A1, EpCAM, IFITM1, MMP9, MMP12, MMP14, PDGFRB, PLAU, and TIMP1) that map to altered transcription networks in both mouse and human gastric neoplasia. Our study demonstrates significant similarities in deregulated transcription networks in human gastric cancer and gastric tumorigenesis in the Tff1-KO mouse model. The data also suggest that activation of MYC, STAT3, RELA, and β-catenin transcription networks could be an early molecular step in gastric carcinogenesis. © 2017 Wiley Periodicals, Inc.

BBSome function is required for both the morphogenesis and maintenance of the photoreceptor outer segment

PubMed Central

Hsu, Ying; Kim, Gunhee; Zhang, Qihong; Datta, Poppy; Seo, Seongjin

2017-01-01

Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of Bbs8, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of Bbs8 in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting Bbs8 gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated. PMID:29049287

Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development.

PubMed

Gong, Zu-Kang; Wang, Shuang-Jie; Huang, Yong-Qi; Zhao, Rui-Qiang; Zhu, Qi-Fang; Lin, Wen-Zhen

2014-12-01

RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes (Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C(t) method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis.

Scube3 Is Expressed in Multiple Tissues during Development but Is Dispensable for Embryonic Survival in the Mouse

PubMed Central

Xavier, Guilherme M.; Panousopoulos, Leonidas; Cobourne, Martyn T.

2013-01-01

The vertebrate Scube family consists of three independent members Scube1-3; which encode secreted cell surface-associated membrane glycoproteins that share a domain organization of at least five recognizable motifs and the ability to both homo- and heterodimerize. There is recent biochemical evidence to suggest that Scube2 is directly involved in Hedgehog signaling, acting co-operatively with Dispatched to mediate the release in soluble form of cholesterol and palmitate-modified Hedgehog ligand during long-range activity. Indeed, in the zebrafish myotome, all three Scube proteins can subtly promote Hedgehog signal transduction in a non-cell autonomous manner. In order to further investigate the role of Scube genes during development, we have generated mice with targeted inactivation of Scube3. Despite a dynamic developmental expression pattern, with transcripts present in neuroectoderm, endoderm and endochondral tissues, particularly within the craniofacial region; an absence of Scube3 function results in no overt embryonic phenotype in the mouse. Mutant mice are born at expected Mendelian ratios, are both viable and fertile, and seemingly retain normal Hedgehog signaling activity in craniofacial tissues. These findings suggest that in the mouse, Scube3 is dispensable for normal development; however, they do not exclude the possibility of a co-operative role for Scube3 with other Scube members during embryogenesis or a potential role in adult tissue homeostasis over the long-term. PMID:23383134

Mouse vocal communication system: are ultrasounds learned or innate?

PubMed Central

Arriaga, Gustavo; Jarvis, Erich D.

2013-01-01

Mouse ultrasonic vocalizations (USVs) are often used as behavioral readouts of internal states, to measure effects of social and pharmacological manipulations, and for behavioral phenotyping of mouse models for neuropsychiatric and neurodegenerative disorders. However, little is known about the neurobiological mechanisms of rodent USV production. Here we discuss the available data to assess whether male mouse song behavior and the supporting brain circuits resemble those of known vocal non-learning or vocal learning species. Recent neurobiology studies have demonstrated that the mouse USV brain system includes motor cortex and striatal regions, and that the vocal motor cortex sends a direct sparse projection to the brainstem vocal motor nucleus ambiguous, a projection thought be unique to humans among mammals. Recent behavioral studies have reported opposing conclusions on mouse vocal plasticity, including vocal ontogeny changes in USVs over early development that might not be explained by innate maturation processes, evidence for and against a role for auditory feedback in developing and maintaining normal mouse USVs, and evidence for and against limited vocal imitation of song pitch. To reconcile these findings, we suggest that the trait of vocal learning may not be dichotomous but encompass a broad set of behavioral and neural traits we call the continuum hypothesis, and that mice possess some of the traits associated with a capacity for limited vocal learning. PMID:23295209

Refractive index measurement of the mouse crystalline lens using optical coherence tomography

PubMed Central

Chakraborty, Ranjay; Lacy, Kip D.; Tan, Christopher C.; Park, Han na; Pardue, Machelle T.

2014-01-01

In recent years, there has been a growing interest for using mouse models in refractive development and myopia research. The crystalline lens is a critical optical component of the mouse eye that occupies greater than 50% of the ocular space, and significant increases in thickness with age. However, changes in refractive index of the mouse crystalline lens are less known. In this study, we examined the changes in thickness and refractive index of the mouse crystalline lens for two different strains, wild-type (WT) and a nyx mutant (nob) over the course of normal visual development or after form deprivation. Refractive index and lens thickness measurements were made on ex vivo lens using spectral domain optical coherence tomography (SD-OCT). Comparison of refractive index measurements on 5 standard ball lenses using the SD-OCT and their known refractive indices (manufacturer provided) indicated good precision (intra-class correlation coefficient, 0.998 and Bland-Altman coefficient of repeatability, 0.116) of the SD-OCT to calculate mouse lens refractive index ex vivo. During normal visual development, lens thickness increased significantly with age for three different cohorts of mice, aged 4 (average thickness from both eyes; WT: 1.78 ± 0.03, nob: 1.79 ± 0.08 mm), 10 (WT: 2.02 ± 0.05, nob: 2.01 ± 0.04 mm) and 16 weeks (WT: 2.12 ± 0.06, nob: 2.09 ± 0.06 mm, p<0.001). Lens thickness was not significantly different between the two strains at any age (p=0.557). For mice with normal vision, refractive index for isolated crystalline lenses in nob mice was significantly greater than WT mice (mean for all ages; WT: 1.42 ± 0.01, nob: 1.44 ± 0.001, p<0.001). After 4 weeks of form deprivation to the right eye using a skull-mounted goggling apparatus, a thinning of the crystalline lens was observed in both right and left eyes of goggled animals compared to their naïve controls (average from both the right and the left eye) for both strains (p=0.052). In form deprived mice, lens refractive index was significantly different between the goggled animals and non-goggled naïve controls in nob mice, but not in WT mice (p=0.009). Both eyes of goggled nob mice had significantly greater lens refractive index (goggled, 1.49 ± 0.01; opposite, 1.47 ± 0.03) compared to their naïve controls (1.45 ± 0.02, p<0.05). The results presented here suggest that there are genetic differences in the crystalline lens refractive index of the mouse eye, and that the lens refractive index in mice significantly increase with form deprivation. Research applications requiring precise optical measurements of the mouse eye should take these lens refractive indices into account when interpreting SD-OCT data. PMID:24939747

The Oncogenic Role of RhoGAPs in Basal-Like Breast Cancer

DTIC Science & Technology

2015-02-01

cell lines, and mouse models . c) In vivo tumorigenesis and metastasis assays. Milestones: Identify whether ArhGAP11A and RacGAP1 can promote tumor growth...also upregulated in basal (C3(I)-Tag) but not luminal (MMTV-Neu) genetically- engineered mouse models (Fig. 1B). At the protein level, RacGAP1 was...hypothesis that these RhoGAPs are indeed playing an oncogenic role in these cells. Human Tumors Mouse Model Tumors Normal Luminal A Basal-like Normal

Sox2 and Jagged1 Expression in Normal and Drug-Damaged Adult Mouse Inner Ear

PubMed Central

Campbell, Sean; Taylor, Ruth R.; Forge, Andrew; Hume, Clifford R.

2007-01-01

Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear. PMID:18157569

Pipette-based Method to Study Embryoid Body Formation Derived from Mouse and Human Pluripotent Stem Cells Partially Recapitulating Early Embryonic Development Under Simulated Microgravity Conditions

NASA Astrophysics Data System (ADS)

Shinde, Vaibhav; Brungs, Sonja; Hescheler, Jürgen; Hemmersbach, Ruth; Sachinidis, Agapios

2016-06-01

The in vitro differentiation of pluripotent stem cells partially recapitulates early in vivo embryonic development. More recently, embryonic development under the influence of microgravity has become a primary focus of space life sciences. In order to integrate the technique of pluripotent stem cell differentiation with simulated microgravity approaches, the 2-D clinostat compatible pipette-based method was experimentally investigated and adapted for investigating stem cell differentiation processes under simulated microgravity conditions. In order to keep residual accelerations as low as possible during clinorotation, while also guaranteeing enough material for further analysis, stem cells were exposed in 1-mL pipettes with a diameter of 3.5 mm. The differentiation of mouse and human pluripotent stem cells inside the pipettes resulted in the formation of embryoid bodies at normal gravity (1 g) after 24 h and 3 days. Differentiation of the mouse pluripotent stem cells on a 2-D pipette-clinostat for 3 days also resulted in the formation of embryoid bodies. Interestingly, the expression of myosin heavy chain was downregulated when cultivation was continued for an additional 7 days at normal gravity. This paper describes the techniques for culturing and differentiation of pluripotent stem cells and exposure to simulated microgravity during culturing or differentiation on a 2-D pipette clinostat. The implementation of these methodologies along with -omics technologies will contribute to understand the mechanisms regulating how microgravity influences early embryonic development.

Ablation of TrkB expression in RGS9-2 cells leads to hyperphagic obesity★

PubMed Central

Liao, Guey-Ying; Li, Yuqing; Xu, Baoji

2013-01-01

Brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB (tropomyosin receptor kinase B), are widely expressed in the brain where they regulate a wide variety of biological processes, including energy homeostasis. However, the specific population(s) of TrkB-expressing neurons through which BDNF governs energy homeostasis remain(s) to be determined. Using the Cre-loxP recombination system, we deleted the mouse TrkB gene in RGS9-2-expressing cells. In this mouse mutant, TrkB expression was abolished in several hypothalamic nuclei, including arcuate nucleus, dorsomedial hypothalamus, and lateral hypothalamus. TrkB expression was also abolished in a small number of cells in other brain regions, including the cerebral cortex and striatum. The mutant animals developed hyperphagic obesity with normal energy expenditure. Despite hyperglycemia under fed conditions, these animals exhibited normal fasting blood glucose levels and normal glucose tolerance. These results suggest that BDNF regulates energy homeostasis in part through TrkB-expressing neurons in the hypothalamus. PMID:24327964

Influence of prenatal EGCG treatment and Dyrk1a dosage reduction on craniofacial features associated with Down syndrome.

PubMed

McElyea, Samantha D; Starbuck, John M; Tumbleson-Brink, Danika M; Harrington, Emily; Blazek, Joshua D; Ghoneima, Ahmed; Kula, Katherine; Roper, Randall J

2016-11-15

Trisomy 21 (Ts21) affects craniofacial precursors in individuals with Down syndrome (DS). The resultant craniofacial features in all individuals with Ts21 may significantly affect breathing, eating and speaking. Using mouse models of DS, we have traced the origin of DS-associated craniofacial abnormalities to deficiencies in neural crest cell (NCC) craniofacial precursors early in development. Hypothetically, three copies of Dyrk1a (dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A), a trisomic gene found in most humans with DS and mouse models of DS, may significantly affect craniofacial structure. We hypothesized that we could improve DS-related craniofacial abnormalities in mouse models using a Dyrk1a inhibitor or by normalizing Dyrk1a gene dosage. In vitro and in vivo treatment with Epigallocatechin-3-gallate (EGCG), a Dyrk1a inhibitor, modulated trisomic NCC deficiencies at embryonic time points. Furthermore, prenatal EGCG treatment normalized some craniofacial phenotypes, including cranial vault in adult Ts65Dn mice. Normalization of Dyrk1a copy number in an otherwise trisomic Ts65Dn mice normalized many dimensions of the cranial vault, but did not correct all craniofacial anatomy. These data underscore the complexity of the gene–phenotype relationship in trisomy and suggest that changes in Dyrk1a expression play an important role in morphogenesis and growth of the cranial vault. These results suggest that a temporally specific prenatal therapy may be an effective way to ameliorate some craniofacial anatomical changes associated with DS.

R-spondin3 is associated with basal-progenitor behavior in normal and tumor mammary cells.

PubMed

Tocci, Johanna Melisa; Felcher, Carla María; García Solá, Martín E; Goddio, María Victoria; Zimberlin, María Noel; Rubinstein, Natalia; Srebrow, Anabella; Coso, Omar Adrián; Abba, Martín C; Meiss, Roberto P; Kordon, Edith C

2018-05-10

R-spondin3 (RSPO3) is a member of a family of secreted proteins that enhance Wnt signaling pathways in diverse processes including cancer. However, the role of RSPO3 in mammary gland and breast cancer development remains unclear. In this study, we show that RSPO3 is expressed in the basal stem cell-enriched compartment of normal mouse mammary glands but is absent from committed mature luminal cells in which exogenous RSPO3 impairs lactogenic differentiation. RSPO3 knockdown in basal-like mouse mammary tumor cells reduced canonical Wnt signaling, epithelial-to-mesenchymal transition-like features, migration capacity, and tumor formation in vivo. Conversely, RSPO3 overexpression, which was associated with some LGR and RUNX factors, highly correlated with the basal-like subtype among breast cancer patients. Thus we identified RSPO3 as a novel key modulator of breast cancer development and a potential target for treatment of basal-like breast cancers. Copyright ©2018, American Association for Cancer Research.

Glucose dysregulation and response to common anti-diabetic agents in the FATZO/Pco mouse.

PubMed

Peterson, Richard G; Jackson, Charles Van; Zimmerman, Karen M; Alsina-Fernandez, Jorge; Michael, M Dodson; Emmerson, Paul J; Coskun, Tamer

2017-01-01

The FATZO/Pco mouse is the result of a cross of the C57BL/6J and AKR/J strains. The crossing of these two strains and the selective inbreeding for obesity, insulin resistance and hyperglycemia has resulted in an inbred strain exhibiting obesity in the presumed presence of an intact leptin pathway. Routinely used rodent models for obesity and diabetes research have a monogenic defect in leptin signaling that initiates obesity. Given that obesity and its sequelae in humans are polygenic in nature and not associated with leptin signaling defects, the FATZO mouse may represent a more translatable rodent model for study of obesity and its associated metabolic disturbances. The FATZO mouse develops obesity spontaneously when fed a normal chow diet. Glucose intolerance with increased insulin levels are apparent in FATZO mice as young as 6 weeks of age. These progress to hyperglycemia/pre-diabetes and frank diabetes with decreasing insulin levels as they age. The disease in these mice is multi-faceted, similar to the metabolic syndrome apparent in obese individuals, and thus provides a long pre-diabetic state for determining the preventive value of new interventions. We have assessed the utility of this new model for the pre-clinical screening of agents to stop or slow progression of the metabolic syndrome to severe diabetes. Our assessment included: 1) characterization of the spontaneous development of disease, 2) comparison of metabolic disturbances of FATZO mice to control mice and 3) validation of the model with regard to the effectiveness of current and emerging anti-diabetic agents; rosiglitazone, metformin and semaglutide. Male FATZO mice spontaneously develop significant metabolic disease when compared to normal controls while maintaining hyperglycemia in the presence of high leptin levels and hyperinsulinemia. The disease condition responds to commonly used antidiabetic agents.

New Insights on the Morphology of Adult Mouse Penis1

PubMed Central

Rodriguez, Esequiel; Weiss, Dana A.; Yang, Jennifer H.; Menshenina, Julia; Ferretti, Max; Cunha, Tristan J.; Barcellos, Dale; Chan, Lok Yun; Risbridger, Gail; Cunha, Gerald R.; Baskin, Laurence S.

2011-01-01

ABSTRACT The adult mouse penis represents the end point of masculine sex differentiation of the embryonic genital tubercle and contains bone, cartilage, the urethra, erectile bodies, several types of epithelium, and many individual cell types arrayed into specific anatomical structures. Using contemporary high-resolution imaging techniques, we sought to provide new insights to the current description of adult mouse penile morphology to enable understanding of penile abnormalities, including hypospadias. Examination of serial transverse and longitudinal sections, scanning electron microscopy, and three-dimensional (3D) reconstruction provided a new appreciation of the individual structures in the adult mouse penis and their 3D interrelationships. In so doing, we discovered novel paired erectile bodies, the male urogenital mating protuberance (MUMP), and more accurately described the urethral meatus. These morphological observations were quantified by morphometric analysis and now provide accurate morphological end points of sex differentiation of mouse penis that will be the foundation of future studies to identify normal and abnormal penile development. PMID:21918128

Reconstituted normal human breast in nude mice: effect of host pregnancy environment and human chorionic gonadotropin on proliferation.

PubMed

Popnikolov, N; Yang, J; Liu, A; Guzman, R; Nandi, S

2001-03-01

The proliferation of normal human breast epithelial cells in women is highest during the first trimester of pregnancy. In an attempt to analyze this hormonal environment in a model system, the effect of host mouse pregnancy and the administration of human chorionic gonadotropin (hCG) were assessed in normal human breast epithelial cells transplanted into athymic nude mice. Human breast epithelial cells, dissociated from reduction mammoplasty specimens and embedded inside the extracellular matrices comprised of collagen gel and Matrigel, were transplanted into nude mice. Proliferation was measured in vivo by BrdU labeling followed by immunostaining of sections from recovered gels in response to an altered hormonal environment of the host animal. The host animal was mated to undergo pregnancy and the complex hormonal environment of the host animal pregnancy stimulated growth of transplanted human cells. This effect increased with progression of pregnancy and reached the maximum during late pregnancy prior to parturition. In order to determine whether additional stimulation could be achieved, the transplanted human cells were exposed to a second cycle of host mouse pregnancy by immediately mating the animal after parturition. This additional exposure of host mouse pregnancy did not result in further increase of proliferation. The effect of hCG administration on transplanted human cells was also tested, since hCG level is highest during the first trimester of human pregnancy and coincides with the maximal breast cell proliferation. Administration of hCG alone stimulated proliferation of human cells in a dose-dependent manner, and could further enhance stimulation achieved with estrogen. The host mouse mammary gland also responded to hCG treatment resulting in increased branching and lobulo-alveolar development. However, the hCG effect on both human and mouse cells was dependent on intact ovary since the stimulation did not occur in ovariectomized animals. Although hCG receptor transcripts were detected in human breast epithelial cells, raising the possibility of a direct mitogenic action, the hCG effect observed in this study may have been mediated via the ovary by increased secretion of ovarian steroids. In summary, using our in vivo nude mice system, the proliferation of normal human breast epithelial cells could be stimulated by host mouse pregnancy and by administration of hCG.

RBP-Jκ-Dependent Notch Signaling Is Dispensable for Mouse Early Embryonic Development

PubMed Central

Souilhol, Céline; Cormier, Sarah; Tanigaki, Kenji; Babinet, Charles; Cohen-Tannoudji, Michel

2006-01-01

The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jκ-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion. PMID:16782866

RBP-Jkappa-dependent notch signaling is dispensable for mouse early embryonic development.

PubMed

Souilhol, Céline; Cormier, Sarah; Tanigaki, Kenji; Babinet, Charles; Cohen-Tannoudji, Michel

2006-07-01

The Notch signaling pathway is an evolutionarily conserved signaling system which has been shown to be essential in cell fate specification and in numerous aspects of embryonic development in all metazoans thus far studied. We recently demonstrated that several components of the Notch signaling pathway, including the four Notch receptors and their five ligands known in mammals, are expressed in mouse oocytes, in mouse preimplantation embryos, or both. This suggested a possible implication of the Notch pathway in the first cell fate specification of the dividing mouse embryo, which results in the formation of the blastocyst. To address this issue directly, we generated zygotes in which both the maternal and the zygotic expression of Rbpsuh, a key element of the core Notch signaling pathway, were abrogated. We find that such zygotes give rise to blastocysts which implant and develop normally. Nevertheless, after gastrulation, these embryos die around midgestation, similarly to Rbpsuh-null mutants. This demonstrates that the RBP-Jkappa-dependent pathway, otherwise called the canonical Notch pathway, is dispensable for blastocyst morphogenesis and the establishment of the three germ layers, ectoderm, endoderm, and mesoderm. These results are discussed in the light of recent observations which have challenged this conclusion.

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.

PubMed

Kilpatrick, L A; Li, Q; Yang, J; Goddard, J C; Fekete, D M; Lang, H

2011-06-01

Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear

PubMed Central

Kilpatrick, Lauren A.; Li, Qian; Yang, John; Goddard, John C; Fekete, Donna M.; Lang, Hainan

2010-01-01

Murine models are ideal for studying cochlear gene transfer as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, due to its small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAV) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear and allows for near-complete preservation of low and middle frequency hearing. In the present study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6, and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus (CMV) promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells (IHCs) were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness. PMID:21209625

Absence of diabetes and pancreatic exocrine dysfunction in a transgenic model of carboxyl-ester lipase-MODY (maturity-onset diabetes of the young).

PubMed

Ræder, Helge; Vesterhus, Mette; El Ouaamari, Abdelfattah; Paulo, Joao A; McAllister, Fiona E; Liew, Chong Wee; Hu, Jiang; Kawamori, Dan; Molven, Anders; Gygi, Steven P; Njølstad, Pål R; Kahn, C Ronald; Kulkarni, Rohit N

2013-01-01

CEL-MODY is a monogenic form of diabetes with exocrine pancreatic insufficiency caused by mutations in CARBOXYL-ESTER LIPASE (CEL). The pathogenic processes underlying CEL-MODY are poorly understood, and the global knockout mouse model of the CEL gene (CELKO) did not recapitulate the disease. We therefore aimed to create and phenotype a mouse model specifically over-expressing mutated CEL in the pancreas. We established a monotransgenic floxed (flanking LOX sequences) mouse line carrying the human CEL mutation c.1686delT and crossed it with an elastase-Cre mouse to derive a bitransgenic mouse line with pancreas-specific over-expression of CEL carrying this disease-associated mutation (TgCEL). Following confirmation of murine pancreatic expression of the human transgene by real-time quantitative PCR, we phenotyped the mouse model fed a normal chow and compared it with mice fed a 60% high fat diet (HFD) as well as the effects of short-term and long-term cerulein exposure. Pancreatic exocrine function was normal in TgCEL mice on normal chow as assessed by serum lipid and lipid-soluble vitamin levels, fecal elastase and fecal fat absorption, and the normoglycemic mice exhibited normal pancreatic morphology. On 60% HFD, the mice gained weight to the same extent as controls, had normal pancreatic exocrine function and comparable glucose tolerance even after resuming normal diet and follow up up to 22 months of age. The cerulein-exposed TgCEL mice gained weight and remained glucose tolerant, and there were no detectable mutation-specific differences in serum amylase, islet hormones or the extent of pancreatic tissue inflammation. In this murine model of human CEL-MODY diabetes, we did not detect mutation-specific endocrine or exocrine pancreatic phenotypes, in response to altered diets or exposure to cerulein.

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

PubMed

Yu, Shengqiang; Yeh, Chiuan-Ren; Niu, Yuanjie; Chang, Hong-Chiang; Tsai, Yu-Chieh; Moses, Harold L; Shyr, Chih-Rong; Chang, Chawnshang; Yeh, Shuyuan

2012-03-01

Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF-1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear. To further probe the stromal fibroblast AR roles in prostate development, we generated tissue-selective knockout mice with the AR gene deleted in stromal fibroblasts (FSP-ARKO). We also used primary culture stromal cells to confirm the in vivo data and investigate mechanisms related to prostate development. The results showed cellular alterations in the FSP-ARKO mouse prostate with decreased epithelial proliferation, increased apoptosis, and decreased collagen composition. Further mechanistic studies demonstrated that FSP-ARKO mice have defects in the expression of prostate stromal growth factors. To further confirm these in vivo findings, we prepared primary cultured mouse prostate stromal cells and found knocking down the stromal AR could result in growth retardation of prostate stromal cells and co-cultured prostate epithelial cells, as well as decrease of some stromal growth factors. Our FSP-ARKO mice not only provide the first in vivo evidence in Cre-loxP knockout system for the requirement of stromal fibroblast AR to maintain the normal development of the prostate, but may also suggest the selective knockdown of stromal AR might become a potential therapeutic approach to battle prostate hyperplasia and cancer. Copyright © 2011 Wiley Periodicals, Inc.

Saccharomyces cerevisiae-derived virus-like particle parvovirus B19 vaccine elicits binding and neutralizing antibodies in a mouse model for sickle cell disease.

PubMed

Penkert, Rhiannon R; Young, Neal S; Surman, Sherri L; Sealy, Robert E; Rosch, Jason; Dormitzer, Philip R; Settembre, Ethan C; Chandramouli, Sumana; Wong, Susan; Hankins, Jane S; Hurwitz, Julia L

2017-06-22

Parvovirus B19 infections are typically mild in healthy individuals, but can be life threatening in individuals with sickle cell disease (SCD). A Saccharomyces cerevisiae-derived B19 VLP vaccine, now in pre-clinical development, is immunogenic in wild type mice when administered with the adjuvant MF59. Because SCD alters the immune response, we evaluated the efficacy of this vaccine in a mouse model for SCD. Vaccinated mice with SCD demonstrated similar binding and neutralizing antibody responses to those of heterozygous littermate controls following a prime-boost-boost regimen. Due to the lack of a mouse parvovirus B19 challenge model, we employed a natural mouse pathogen, Sendai virus, to evaluate SCD respiratory tract responses to infection. Normal mucosal and systemic antibody responses were observed in these mice. Results demonstrate that mice with SCD can respond to a VLP vaccine and to a respiratory virus challenge, encouraging rapid development of the B19 vaccine for patients with SCD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tetraploid Embryonic Stem Cells Maintain Pluripotency and Differentiation Potency into Three Germ Layers.

PubMed

Imai, Hiroyuki; Kano, Kiyoshi; Fujii, Wataru; Takasawa, Ken; Wakitani, Shoichi; Hiyama, Masato; Nishino, Koichiro; Kusakabe, Ken Takeshi; Kiso, Yasuo

2015-01-01

Polyploid amphibians and fishes occur naturally in nature, while polyploid mammals do not. For example, tetraploid mouse embryos normally develop into blastocysts, but exhibit abnormalities and die soon after implantation. Thus, polyploidization is thought to be harmful during early mammalian development. However, the mechanisms through which polyploidization disrupts development are still poorly understood. In this study, we aimed to elucidate how genome duplication affects early mammalian development. To this end, we established tetraploid embryonic stem cells (TESCs) produced from the inner cell masses of tetraploid blastocysts using electrofusion of two-cell embryos in mice and studied the developmental potential of TESCs. We demonstrated that TESCs possessed essential pluripotency and differentiation potency to form teratomas, which differentiated into the three germ layers, including diploid embryonic stem cells. TESCs also contributed to the inner cell masses in aggregated chimeric blastocysts, despite the observation that tetraploid embryos fail in normal development soon after implantation in mice. In TESCs, stability after several passages, colony morphology, and alkaline phosphatase activity were similar to those of diploid ESCs. TESCs also exhibited sufficient expression and localization of pluripotent markers and retained the normal epigenetic status of relevant reprogramming factors. TESCs proliferated at a slower rate than ESCs, indicating that the difference in genomic dosage was responsible for the different growth rates. Thus, our findings suggested that mouse ESCs maintained intrinsic pluripotency and differentiation potential despite tetraploidization, providing insights into our understanding of developmental elimination in polyploid mammals.

DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos.

PubMed

Bakhtari, Azizollah; Ross, Pablo J

2014-09-01

Dppa3 has been described in mice as an important maternal factor contributed by the oocyte that participates in protecting the maternal genome from oxidation of methylated cytosines (5mC) to hydroxymethylated cytosines (5hmC). Dppa3 is also required for normal mouse preimplantation development. This gene is poorly conserved across mammalian species, with less than 32% of protein sequence shared between mouse, cow and human. RNA-seq analysis of bovine oocytes and preimplantation embryos revealed that DPPA3 transcripts are some of the most highly abundant mRNAs in the oocyte, and their levels gradually decrease toward the time of embryonic genome activation (EGA). Knockdown of DPPA3 by injection of siRNA in germinal vesicle (GV) stage oocytes was used to assess its role in epigenetic remodeling and embryo development. DPPA3 knockdown resulted in increased intensity of 5hmC staining in the maternal pronucleus (PN), demonstrating a role for this factor in the asymmetric remodeling of the maternal and paternal PN in bovine zygotes. Also, DPPA3 knockdown decreased the developmental competence of parthenogenetic and in vitro fertilized embryos. Finally, DPPA3 knockdown embryos that reached the blastocyst stage had significantly fewer ICM cells as compared with control embryos. We conclude that DPPA3 is a maternal factor important for correct epigenetic remodeling and normal embryonic development in cattle, indicating that the role of DPPA3 during early development is conserved between species.

Defective natural killer cell activity in a mouse model of eczema herpeticum.

PubMed

Kawakami, Yuko; Ando, Tomoaki; Lee, Jong-Rok; Kim, Gisen; Kawakami, Yu; Nakasaki, Tae; Nakasaki, Manando; Matsumoto, Kenji; Choi, Youn Soo; Kawakami, Toshiaki

2017-03-01

Patients with atopic dermatitis (AD) are susceptible to several viruses, including herpes simplex virus (HSV). Some patients experience 1 or more episodes of a severe skin infection caused by HSV termed eczema herpeticum (EH). There are numerous mouse models of AD, but no established model exists for EH. We sought to establish and characterize a mouse model of EH. We infected AD-like skin lesions with HSV1 to induce severe skin lesions in a dermatitis-prone mouse strain of NC/Nga. Gene expression was investigated by using a microarray and quantitative PCR; antibody titers were measured by means of ELISA; and natural killer (NK) cell, cytotoxic T-cell, regulatory T-cell, and follicular helper T-cell populations were evaluated by using flow cytometry. The role of NK cells in HSV1-induced development of severe skin lesions was examined by means of depletion and adoptive transfer. Inoculation of HSV1 induced severe erosive skin lesions in eczematous mice, which had an impaired skin barrier, but milder lesions in small numbers of normal mice. Eczematous mice exhibited lower NK cell activity but similar cytotoxic T-cell activity and humoral immune responses compared with normal mice. The role of NK cells in controlling HSV1-induced skin lesions was demonstrated by experiments depleting or transferring NK cells. A murine model of EH with an impaired skin barrier was established in this study. We demonstrated a critical role of defective NK activities in the development of HSV1-induced severe skin lesions in eczematous mice. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. All rights reserved.

An association between Schistosoma mansoni worms and an enzymatically-active protease/peptidase in mouse blood.

PubMed

Darani, H Y; Doenhoff, M J

2008-04-01

An enzyme found previously in extracts of adult Schistosoma mansoni worms, that hydrolysed the chromogenic substrate N-acetyl-DL-phenylalanine beta-naphthyl-ester, has here been further investigated and characterized. Evidence that the molecule found in the parasite was antigenically and enzymatically homologous with a constituent of normal mouse plasma has been consolidated using a monospecific serum in immunoelectrophoresis and Western immunoblotting. The molecular size of the enzyme was found to be approximately 70 kDa and it was inhibited by a serine protease inhibitor, but not by inhibitors of other classes of protease. The enzymatic activity found in normal mouse serum was also found in normal rat serum, but not in sera from several other mammalian species.

Targeted Deletion of the Antisilencer/Enhancer (ASE) Element from Intron 1 of the Myelin Proteolipid Protein Gene (Plp1) in Mouse Reveals that the Element Is Dispensable for Plp1 Expression in Brain during Development and Remyelination

PubMed Central

Pereira, Glauber B.; Meng, Fanxue; Kockara, Neriman T.; Yang, Baoli; Wight, Patricia A.

2012-01-01

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. While removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is nonfunctional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. PMID:23157328

Development and Function of CD94-Deficient Natural Killer Cells

PubMed Central

Orr, Mark T.; Wu, Jun; Fang, Min; Sigal, Luis J.; Spee, Pieter; Egebjerg, Thomas; Dissen, Erik; Fossum, Sigbjørn; Phillips, Joseph H.; Lanier, Lewis L.

2010-01-01

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions. PMID:21151939

Development and function of CD94-deficient natural killer cells.

PubMed

Orr, Mark T; Wu, Jun; Fang, Min; Sigal, Luis J; Spee, Pieter; Egebjerg, Thomas; Dissen, Erik; Fossum, Sigbjørn; Phillips, Joseph H; Lanier, Lewis L

2010-12-03

The CD94 transmembrane-anchored glycoprotein forms disulfide-bonded heterodimers with the NKG2A subunit to form an inhibitory receptor or with the NKG2C or NKG2E subunits to assemble a receptor complex with activating DAP12 signaling proteins. CD94 receptors expressed on human and mouse NK cells and T cells have been proposed to be important in NK cell tolerance to self, play an important role in NK cell development, and contribute to NK cell-mediated immunity to certain infections including human cytomegalovirus. We generated a gene-targeted CD94-deficient mouse to understand the role of CD94 receptors in NK cell biology. CD94-deficient NK cells develop normally and efficiently kill NK cell-susceptible targets. Lack of these CD94 receptors does not alter control of mouse cytomegalovirus, lymphocytic choriomeningitis virus, vaccinia virus, or Listeria monocytogenes. Thus, the expression of CD94 and its associated NKG2A, NKG2C, and NKG2E subunits is dispensable for NK cell development, education, and many NK cell functions.

Minireview: Progesterone Regulation of Proliferation in the Normal Human Breast and in Breast Cancer: A Tale of Two Scenarios?

PubMed Central

Graham, J. Dinny; Clarke, Christine L.

2015-01-01

Progesterone (P), which signals through the P receptor (PR), is critical in normal development of the breast, but its signaling axis is also a major driver of breast cancer risk. Here we review recent advances in the understanding of P signaling in the normal human breast, with a focus on the importance of the balance between autocrine and paracrine signaling. To date, most data (which derive largely from mouse models or human breast cancer cell line studies) have demonstrated that the vast majority of PR+ cells appear to act as “sensor” cells, which respond to P stimulation by translating these hormonal cues into paracrine signals. However, growing evidence suggests that, dependent on the cellular context, P may also signal in an autocrine manner in a subset of cells in the normal mouse mammary gland and human breast. It has been suggested that it may be dysregulation of this autocrine signaling, resulting in a “switch” from a predominance of paracrine signaling to autocrine signaling in PR+ cells, which is an early event during breast tumorigenesis. This review summarizes current evidence in the literature that demonstrates the mechanisms through which P acts in the normal human breast, as well as highlighting the important questions that remain unanswered. PMID:26266959

Analysis of second-harmonic-generation microscopy in a mouse model of ovarian carcinoma

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.; Rice, Photini F.; Marion, Samuel L.; Brewer, Molly A.; Davis, John R.; Rodriguez, Jeffrey J.; Utzinger, Urs; Hoyer, Patricia B.; Barton, Jennifer K.

2012-07-01

Second-harmonic-generation (SHG) imaging of mouse ovaries ex vivo was used to detect collagen structure changes accompanying ovarian cancer development. Dosing with 4-vinylcyclohexene diepoxide and 7,12-dimethylbenz[a]anthracene resulted in histologically confirmed cases of normal, benign abnormality, dysplasia, and carcinoma. Parameters for each SHG image were calculated using the Fourier transform matrix and gray-level co-occurrence matrix (GLCM). Cancer versus normal and cancer versus all other diagnoses showed the greatest separation using the parameters derived from power in the highest-frequency region and GLCM energy. Mixed effects models showed that these parameters were significantly different between cancer and normal (P<0.008). Images were classified with a support vector machine, using 25% of the data for training and 75% for testing. Utilizing all images with signal greater than the noise level, cancer versus not-cancer specimens were classified with 81.2% sensitivity and 80.0% specificity, and cancer versus normal specimens were classified with 77.8% sensitivity and 79.3% specificity. Utilizing only images with greater than of 75% of the field of view containing signal improved sensitivity and specificity for cancer versus normal to 81.5% and 81.1%. These results suggest that using SHG to visualize collagen structure in ovaries could help with early cancer detection.

Activation of the Hedgehog Signaling Pathway in the Developing Lens Stimulates Ectopic FoxE3 Expression and Disruption in Fiber Cell Differentiation

PubMed Central

Kerr, Christine L.; Huang, Jian; Williams, Trevor; West-Mays, Judith A.

2012-01-01

Purpose. The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. Methods. We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. Results. Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. Conclusions. Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3. PMID:22491411

Characterization of adenoviral transduction profile in prostate cancer cells and normal prostate tissue.

PubMed

Ai, Jianzhong; Tai, Phillip W L; Lu, Yi; Li, Jia; Ma, Hong; Su, Qin; Wei, Qiang; Li, Hong; Gao, Guangping

2017-09-01

Prostate diseases are common in males worldwide with high morbidity. Gene therapy is an attractive therapeutic strategy for prostate diseases, however, it is currently underdeveloped. As well known, adeno virus (Ad) is the most widely used gene therapy vector. The aims of this study are to explore transduction efficiency of Ad in prostate cancer cells and normal prostate tissue, thus further providing guidance for future prostate pathophysiological studies and therapeutic development of prostate diseases. We produced Ad expressing enhanced green fluorescence protein (EGFP), and characterized the transduction efficiency of Ad in both human and mouse prostate cancer cell lines in vitro, as well as prostate tumor xenograft, and wild-type mouse prostate tissue in vivo. Ad transduction efficiency was determined by EGFP fluorescence using microscopy and flow cytometry. Cell type-specific transduction was examined by immunofluorescence staining of cell markers. Our data showed that Ad efficiently transduced human and mouse prostate cancer cells in vitro in a dose dependent manner. Following intratumoral and intraprostate injection, Ad could efficiently transduce prostate tumor xenograft and the major prostatic cell types in vivo, respectively. Our findings suggest that Ad can efficiently transduce prostate tumor cells in vitro as well as xenograft and normal prostate tissue in vivo, and further indicate that Ad could be a potentially powerful toolbox for future gene therapy of prostate diseases. © 2017 Wiley Periodicals, Inc.

Reactivity of mouse antibodies against bromelain-treated mouse erythrocytes with thrombin-treated mouse platelets.

PubMed Central

Kawaguchi, S

1989-01-01

The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with mouse platelets before and after thrombin treatment was assessed by flow cytometry. Anti-BrMRBC antibodies could bind to thrombin-treated platelets, although normal platelets were also weakly reactive with the antibodies. The binding of anti-BrMRBC antibodies to platelets was confirmed by complement-dependent lysis. It is suggested that thrombin-activated platelets may be a real target for anti-BrMRBC antibodies. PMID:2467876

Normal and Abnormal Development of the Intrapericardial Arterial Trunks in Man and Mouse

PubMed Central

Anderson, Robert H.; Chaudhry, Bill; Mohun, Timothy J.; Bamforth, Simon D.; Hoyland, Darren; Phillips, Helen M.; Webb, Sandra; Moorman, Antoon F.J.; Brown, Nigel A.; Henderson, Deborah J.

2014-01-01

Aims The definitive cardiac outflow channels have three components: the intrapericardial arterial trunks; the arterial roots with valves; and the ventricular outflow tracts. We studied the normal and abnormal development of the most distal of these, the arterial trunks, comparing findings in mouse and man. Methods and Results Using lineage tracing and three-dimensional visualization by episcopic reconstruction and scanning electron microscopy, we studied embryonic day 9.5 to 12.5 mouse hearts, clarifying the development of the outflow tracts distal to the primordia of the arterial valves. We characterize a transient aortopulmonary foramen, located between the leading edge of a protrusion from the dorsal wall of the aortic sac and the distal margins of the two outflow cushions. The foramen is closed by fusion of the protrusion, with its cap of neural crest cells, with the neural crest cell-filled cushions; the resulting structure then functioning transiently as an aortopulmonary septum. Only subsequent to this closure is it possible to recognize, more proximally, the previously described aortopulmonary septal complex. The adjacent walls of the intrapericardial trunks are derived from the protrusion and distal parts of the outflow cushions, while the lateral walls are formed from intrapericardial extensions of pharyngeal mesenchyme derived from the second heart field. Conclusions We provide, for the first time, objective evidence of the mechanisms of closure of an aortopulmonary foramen that exists distally between the lumens of the developing intrapericardial arterial trunks. Our findings provide insights into the formation of aortopulmonary windows and the variants of common arterial trunk. PMID:22499773

Dysregulation of the PDGFRA gene causes inflow tract anomalies including TAPVR: integrating evidence from human genetics and model organisms

PubMed Central

Bleyl, Steven B.; Saijoh, Yukio; Bax, Noortje A.M.; Gittenberger-de Groot, Adriana C.; Wisse, Lambertus J.; Chapman, Susan C.; Hunter, Jennifer; Shiratori, Hidetaka; Hamada, Hiroshi; Yamada, Shigehito; Shiota, Kohei; Klewer, Scott E.; Leppert, Mark F.; Schoenwolf, Gary C.

2010-01-01

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect inherited via complex genetic and/or environmental factors. We report detailed mapping in extended TAPVR kindreds and mutation analysis in TAPVR patients that implicate the PDGFRA gene in the development of TAPVR. Gene expression studies in mouse and chick embryos for both the Pdgfra receptor and its ligand Pdgf-a show temporal and spatial patterns consistent with a role in pulmonary vein (PV) development. We used an in ovo function blocking assay in chick and a conditional knockout approach in mouse to knock down Pdgfra expression in the developing venous pole during the period of PV formation. We observed that loss of PDGFRA function in both organisms causes TAPVR with low penetrance (∼7%) reminiscent of that observed in our human TAPVR kindreds. Intermediate inflow tract anomalies occurred in a higher percentage of embryos (∼30%), suggesting that TAPVR occurs at one end of a spectrum of defects. We show that the anomalous pulmonary venous connection seen in chick and mouse is highly similar to TAPVR discovered in an abnormal early stage embryo from the Kyoto human embryo collection. Whereas the embryology of the normal venous pole and PV is becoming understood, little is known about the embryogenesis or molecular pathogenesis of TAPVR. These models of TAPVR provide important insight into the pathogenesis of PV defects. Taken together, these data from human genetics and animal models support a role for PDGF-signaling in normal PV development, and in the pathogenesis of TAPVR. PMID:20071345

Convergent synaptic and circuit substrates underlying autism genetic risks.

PubMed

McGee, Aaron; Li, Guohui; Lu, Zhongming; Qiu, Shenfeng

2014-02-01

There has been a surge of diagnosis of autism spectrum disorders (ASD) over the past decade. While large, high powered genome screening studies of children with ASD have identified numerous genetic risk factors, research efforts to understanding how each of these risk factors contributes to the development autism has met with limited success. Revealing the mechanisms by which these genetic risk factors affect brain development and predispose a child to autism requires mechanistic understanding of the neurobiological changes underlying this devastating group of developmental disorders at multifaceted molecular, cellular and system levels. It has been increasingly clear that the normal trajectory of neurodevelopment is compromised in autism, in multiple domains as much as aberrant neuronal production, growth, functional maturation, patterned connectivity, and balanced excitation and inhibition of brain networks. Many autism risk factors identified in humans have been now reconstituted in experimental mouse models to allow mechanistic interrogation of the biological role of the risk gene. Studies utilizing these mouse models have revealed that underlying the enormous heterogeneity of perturbed cellular events, mechanisms directing synaptic and circuit assembly may provide a unifying explanation for the pathophysiological changes and behavioral endophenotypes seen in autism, although synaptic perturbations are far from being the only alterations relevant for ASD. In this review, we discuss synaptic and circuit abnormalities obtained from several prevalent mouse models, particularly those reflecting syndromic forms of ASD that are caused by single gene perturbations. These compiled results reveal that ASD risk genes contribute to proper signaling of the developing gene networks that maintain synaptic and circuit homeostasis, which is fundamental to normal brain development.

Injurious Effects of Emodin on Maturation of Mouse Oocytes, Fertilization and Fetal Development via Apoptosis

PubMed Central

Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

2012-01-01

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20–40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process. PMID:23203041

Injurious effects of emodin on maturation of mouse oocytes, fertilization and fetal development via apoptosis.

PubMed

Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

2012-10-29

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20-40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process.

Refractive index measurement of the mouse crystalline lens using optical coherence tomography.

PubMed

Chakraborty, Ranjay; Lacy, Kip D; Tan, Christopher C; Park, Han Na; Pardue, Machelle T

2014-08-01

In recent years, there has been a growing interest for using mouse models in refractive development and myopia research. The crystalline lens is a critical optical component of the mouse eye that occupies greater than 50% of the ocular space, and significant increases in thickness with age. However, changes in refractive index of the mouse crystalline lens are less known. In this study, we examined the changes in thickness and refractive index of the mouse crystalline lens for two different strains, wild-type (WT) and a nyx mutant (nob) over the course of normal visual development or after form deprivation. Refractive index and lens thickness measurements were made on ex vivo lenses using spectral domain optical coherence tomography (SD-OCT). Comparison of refractive index measurements on 5 standard ball lenses using the SD-OCT and their known refractive indices (manufacturer provided) indicated good precision (intra-class correlation coefficient, 0.998 and Bland-Altman coefficient of repeatability, 0.116) of the SD-OCT to calculate mouse lens refractive index ex vivo. During normal visual development, lens thickness increased significantly with age for three different cohorts of mice, aged 4 (average thickness from both eyes; WT: 1.78 ± 0.03, nob: 1.79 ± 0.08 mm), 10 (WT: 2.02 ± 0.05, nob: 2.01 ± 0.04 mm) and 16 weeks (WT: 2.12 ± 0.06, nob: 2.09 ± 0.06 mm, p < 0.001). Lens thickness was not significantly different between the two strains at any age (p = 0.557). For mice with normal vision, refractive index for isolated crystalline lenses in nob mice was significantly greater than WT mice (mean for all ages; WT: 1.42 ± 0.01, nob: 1.44 ± 0.001, p < 0.001). After 4 weeks of form deprivation to the right eye using a skull-mounted goggling apparatus, a thinning of the crystalline lens was observed in both right and left eyes of goggled animals compared to their naïve controls (average from both the right and the left eye) for both strains (p = 0.052). In form deprived mice, lens refractive index was significantly different between the goggled animals and non-goggled naïve controls in nob mice, but not in WT mice (p = 0.009). Both eyes of goggled nob mice had significantly greater lens refractive index (goggled, 1.49 ± 0.01; opposite, 1.47 ± 0.03) compared to their naïve controls (1.45 ± 0.02, p < 0.05). The results presented here suggest that there are genetic differences in the crystalline lens refractive index of the mouse eye, and that the lens refractive index in mice significantly increase with form deprivation. Research applications requiring precise optical measurements of the mouse eye should take these lens refractive indices into account when interpreting SD-OCT data. Published by Elsevier Ltd.

New technique for mouse oocyte injection via a modified holding pipette.

PubMed

Lyu, Q F; Deng, L; Xue, S G; Cao, S F; Liu, X Y; Jin, W; Wu, L Q; Kuang, Y P

2010-11-01

To improve mouse oocyte survival from intracytoplasmic sperm injection, the sharp tip of the injection pipette has been modified to have a flat end. Here, for the same goal but for a more convenient manipulation, a sharp injection pipette was kept whereas the holding pipette was modified to have a trumpet-shaped opening, which allows deeper injection into the oocyte as it is held. Mouse oocyte injection with mouse and human spermatozoa was performed at 37°C. For the injection of mouse oocyte with mouse sperm head, a significantly higher survival rate (83%) was achieved by utilizing the modified holding pipette than the conventional one (21%; P<0.001) and the fertilization rates were normal and comparable for both methods (82% versus 81%). A superior survival rate (82%) and acceptable normal fertilization rate (71%) were also achieved by utilizing the modified holding pipette for interspecies ICSI (injecting mouse oocyte with human spermatozoon). Taken together, by utilizing a holding pipette with a trumpet-shaped opening, acceptable rates of mouse oocyte survival and fertilization can be achieved using a sharp injection pipette under conditions usual for human oocyte injection. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Ultraviolet-C Light for Treatment of Candida albicans Burn Infection in Mice

PubMed Central

Dai, Tianhong; Kharkwal, Gitika B; Zhao, Jie; St. Denis, Tyler G; Wu, Qiuhe; Xia, Yumin; Huang, Liyi; Sharma, Sulbha K; d’Enfert, Christophe; Hamblin, Michael R

2011-01-01

Burn patients are at high risk of invasive fungal infections, which are a leading cause of morbidity, mortality, and related expense exacerbated by the emergence of drug resistant fungal strains. In this study, we investigated the use of UVC light (254-nm) for the treatment of Candida albicans infection in mouse third degree burns. In-vitro studies demonstrated that UVC could selectively kill the pathogenic yeast C. albicans compared to a normal keratinocyte cell line in a light exposure dependent manner. A mouse model of chronic C. albicans infection in non-lethal 3rd degree burns was developed. The C. albicans strain was stably transformed with a version of the Gaussia princeps luciferase gene that allowed real-time bioluminescence imaging of the progression of C. albicans infection. UVC treatment with a single exposure carried out on day 0 (30 minutes post-infection) gave an average 2.16-log10-unit (99.2%) loss of fungal luminescence when 2.92 J/cm2 UVC had been delivered, while UVC 24-hours post-infection gave 1.94-log10-unit (95.8%) reduction of fungal luminescence after 6.48 J/cm2. Statistical analysis demonstrated that UVC treatment carried out both on both day 0 and day 1 significantly reduced the fungal bioburden of infected burns. UVC was found to be superior to a topical antifungal drug, nystatin cream. UVC was tested on normal mouse skin and no gross damage was observed 24 hours after 6.48 J/cm2. DNA lesions (cyclobutane pyrimidine dimers) were observed by immunofluorescence in normal mouse skin immediately after a 6.48 J/cm2 UVC exposure, but the lesions were extensively repaired at 24-hours after UVC exposure. PMID:21208209

Accelerating cine-MR Imaging in Mouse Hearts Using Compressed Sensing

PubMed Central

Wech, Tobias; Lemke, Angela; Medway, Debra; Stork, Lee-Anne; Lygate, Craig A; Neubauer, Stefan; Köstler, Herbert; Schneider, Jürgen E

2011-01-01

Purpose To combine global cardiac function imaging with compressed sensing (CS) in order to reduce scan time and to validate this technique in normal mouse hearts and in a murine model of chronic myocardial infarction. Materials and Methods To determine the maximally achievable acceleration factor, fully acquired cine data, obtained in sham and chronically infarcted (MI) mouse hearts were 2–4-fold undersampled retrospectively, followed by CS reconstruction and blinded image segmentation. Subsequently, dedicated CS sampling schemes were implemented at a preclinical 9.4 T magnetic resonance imaging (MRI) system, and 2- and 3-fold undersampled cine data were acquired in normal mouse hearts with high temporal and spatial resolution. Results The retrospective analysis demonstrated that an undersampling factor of three is feasible without impairing accuracy of cardiac functional parameters. Dedicated CS sampling schemes applied prospectively to normal mouse hearts yielded comparable left-ventricular functional parameters, and intra- and interobserver variability between fully and 3-fold undersampled data. Conclusion This study introduces and validates an alternative means to speed up experimental cine-MRI without the need for expensive hardware. J. Magn. Reson. Imaging 2011. © 2011 Wiley Periodicals, Inc. PMID:21932360

Utilizing nonlinear optical microscopy to investigate the development of early cancer in nude mice in vivo

NASA Astrophysics Data System (ADS)

Wang, Chun-Chin; Li, Feng-Chieh; Lin, Sung-Jan; Lo, Wen; Dong, Chen-Yuan

2007-07-01

In this investigation, we used in vivo nonlinear optical microscopy to image normal and carcinogen DMBA treated skin tissues of nude mice. We acquired two-photon autofluroescence and second harmonic generation (SHG) images of the skin tissue, and applied the ASI (Autofluorescence versus SHG Index) to the resulting image. This allows us to visualize and quantify the interaction between mouse skin cells and the surrounding connective tissue. We found that as the imaging depth increases, ASI has a different distribution in the normal and the treated skin tissues. Since the DMBA treated skin eventually became squamous cell carcinoma (SCC), our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy. We envision this approach to be effective in studying tumor biology and tumor treatment procedures.

Fertile offspring from sterile sex chromosome trisomic mice§

PubMed Central

Hirota, Takayuki; Ohta, Hiroshi; Powell, Benjamin E.; Mahadevaiah, Shantha K.; Ojarikre, Obah A.; Saitou, Mitinori; Turner, James M. A.

2017-01-01

Having the correct number of chromosomes is vital for normal development and health. Sex chromosome trisomy (SCT) affects 0.1% of the human population and is associated with infertility. We show that during reprogramming to induced pluripotent stem cells (iPSC), fibroblasts from sterile trisomic XXY and XYY mice lose the extra sex chromosome, by a phenomenon we term trisomy-biased chromosome loss (TCL). Resulting euploid XY iPSCs can be differentiated into the male germ cell lineage and functional sperm that can be used in intracytoplasmic sperm injection to produce chromosomally normal, fertile offspring. Sex chromosome loss is comparatively infrequent during mouse XX and XY iPSC generation. TCL also applies to other chromosomes, generating euploid iPSCs from cells of a Down syndrome mouse model. It can also create euploid iPSCs from human trisomic patient fibroblasts. The findings have relevance to overcoming infertility and other trisomic phenotypes. PMID:28818972

Will metformin postpone high-fat diet promotion of TRAMP mouse prostate cancer development and progression?

PubMed

Xu, Hua; Hu, Meng-Bo; Bai, Pei-de; Zhu, Wen-Hui; Ding, Qiang; Jiang, Hao-Wen

2014-12-01

We aimed to examine the effect of high-fat diet (HFD) on prostate cancer (PCa) development and progression and to investigate whether metformin would postpone PCa development and progression promoted by HFD. TRAMP mice were randomly divided into three groups: normal diet group, HFD group and metformin-HFD (Met-HFD) group. Mortality rate and tumor formation rate were examined. TRAMP mice were sacrificed and sampled on the 20th, 24(th), and 28th week, respectively. Serum levels of insulin and IGF-1 were tested by ELISA. Prostate tissue of TRAMP mice was used for HE staining. A total of 17 deaths of TRAMP mice were observed, including 3 (10 %) from the normal diet group, 10 (33.33 %) from the HFD group, and 4 (13.33 %) from Met-HFD group. The mortality rate of TRAMP mice from HFD group was significantly higher than that of normal diet group (P = 0.028), and metformin could moderately decrease the mortality rate by 60.01 % (P = 0.067). Tumor formation rates were not significantly different among the three groups. Levels of glucose, insulin, and IGF-1 tended to increase with TRAMP mice's age in HFD group. TRAMP mice from HFD group had higher serum insulin and IGF-1 levels. A moderate decrease in IGF-1 was also seen in Met-HFD group. HFD could promote TRAMP mouse PCa development and progression and metformin had moderate effect of reducing PCa mortality rate with a decrease in serum IGF-1 level.

Terahertz spectroscopy of brain tissue from a mouse model of Alzheimer's disease

NASA Astrophysics Data System (ADS)

Shi, Lingyan; Shumyatsky, Pavel; Rodríguez-Contreras, Adrián; Alfano, Robert

2016-01-01

The terahertz (THz) absorption and index of refraction of brain tissues from a mouse model of Alzheimer's disease (AD) and a control wild-type (normal) mouse were compared using THz time-domain spectroscopy (THz-TDS). Three dominating absorption peaks associated to torsional-vibrational modes were observed in AD tissue, at about 1.44, 1.8, and 2.114 THz, closer to the peaks of free tryptophan molecules than in normal tissue. A possible reason is that there is more free tryptophan in AD brain tissue, while in normal brain tissue more tryptophan is attached to other molecules. Our study suggests that THz-absorption modes may be used as an AD biomarker fingerprint in brain, and that THz-TDS is a promising technique for early diagnosis of AD.

Analysis of spatial heterogeneity in normal epithelium and preneoplastic alterations in mouse prostate tumor models

PubMed Central

Valkonen, Mira; Ruusuvuori, Pekka; Kartasalo, Kimmo; Nykter, Matti; Visakorpi, Tapio; Latonen, Leena

2017-01-01

Cancer involves histological changes in tissue, which is of primary importance in pathological diagnosis and research. Automated histological analysis requires ability to computationally separate pathological alterations from normal tissue with all its variables. On the other hand, understanding connections between genetic alterations and histological attributes requires development of enhanced analysis methods suitable also for small sample sizes. Here, we set out to develop computational methods for early detection and distinction of prostate cancer-related pathological alterations. We use analysis of features from HE stained histological images of normal mouse prostate epithelium, distinguishing the descriptors for variability between ventral, lateral, and dorsal lobes. In addition, we use two common prostate cancer models, Hi-Myc and Pten+/− mice, to build a feature-based machine learning model separating the early pathological lesions provoked by these genetic alterations. This work offers a set of computational methods for separation of early neoplastic lesions in the prostates of model mice, and provides proof-of-principle for linking specific tumor genotypes to quantitative histological characteristics. The results obtained show that separation between different spatial locations within the organ, as well as classification between histologies linked to different genetic backgrounds, can be performed with very high specificity and sensitivity. PMID:28317907

WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse

PubMed Central

Franco, Heather L.; Dai, Daisy; Lee, Kevin Y.; Rubel, Cory A.; Roop, Dennis; Boerboom, Derek; Jeong, Jae-Wook; Lydon, John P.; Bagchi, Indrani C.; Bagchi, Milan K.; DeMayo, Francesco J.

2011-01-01

WNT4, a member of the Wnt family of ligands, is critical for the development of the female reproductive tract. Analysis of Wnt4 expression in the adult uterus during pregnancy indicates that it may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo. To investigate the role of Wnt4 in adult uterine physiology, conditional ablation of Wnt4 using the PRcre mouse model was accomplished. Ablation of Wnt4 rendered female mice subfertile due to a defect in embryo implantation and subsequent defects in endometrial stromal cell survival, differentiation, and responsiveness to progesterone signaling. In addition to altered stromal cell function, the uteri of PRcre/+Wnt4f/f (Wnt4d/d) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands and the emergence of a p63-positive basal cell layer beneath the columnar luminal epithelial cells. The altered epithelial cell phenotype was further escalated by chronic estrogen treatment, which caused squamous cell metaplasia of the uterine epithelium in the Wnt4d/d mice. Thus, WNT4 is a critical regulator not only of proper postnatal uterine development, but also embryo implantation and decidualization.—Franco, H. L., Dai, D., Lee, K. Y., Rubel, C. S., Roop, D., Boerboom, D., Jeong, J.-W., Lydon, J.-P., Bagchi, I. C., Bagchi, M. K., DeMayo, F. J. WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse. PMID:21163860

Facilitation of endoglin-targeting cancer therapy by development/utilization of a novel genetically engineered mouse model expressing humanized endoglin (CD105).

PubMed

Toi, Hirofumi; Tsujie, Masanori; Haruta, Yuro; Fujita, Kanako; Duzen, Jill; Seon, Ben K

2015-01-15

Endoglin (ENG) is a TGF-β coreceptor and essential for vascular development and angiogenesis. A chimeric antihuman ENG (hENG) monoclonal antibody (mAb) c-SN6j (also known as TRC105) shows promising safety and clinical efficacy features in multiple clinical trials of patients with various advanced solid tumors. Here we developed a novel genetically engineered mouse model to optimize the ENG-targeting clinical trials. We designed a new targeting vector that contains exons 4-8 of hENG gene to generate novel genetically engineered mice (GEMs) expressing functional human/mouse chimeric (humanized) ENG with desired epitopes. Genotyping of the generated mice confirmed that we generated the desired GEMs. Immunohistochemical analysis demonstrated that humanized ENG protein of the GEMs expresses epitopes defined by 7 of our 8 anti-hENG mAbs tested. Surprisingly the homozygous GEMs develop normally and are healthy. Established breast and colon tumors as well as metastasis and tumor microvessels in the GEMs were effectively suppressed by systemic administration of anti-hENG mAbs. Additionally, test result indicates that synergistic potentiation of antitumor efficacy can be induced by simultaneous targeting of two distinct epitopes by anti-hENG mAbs. Sorafenib and capecitabine also showed antitumor efficacy in the GEMs. The presented novel GEMs are the first GEMs that express the targetable humanized ENG. Test results indicate utility of the GEMs for the clinically relevant studies. Additionally, we generated GEMs expressing a different humanized ENG containing exons 5-6 of hENG gene, and the homozygous GEMs develop normally and are healthy. © 2014 UICC.

Endothelin receptor antagonist macitentan or deletion of mouse mast cell protease 4 delays lesion development in atherosclerotic mice.

PubMed

Houde, Martin; Desbiens, Louisane; Schwertani, Adel; Pejler, Gunnar; Iglarz, Marc; D'Orléans-Juste, Pedro

2016-08-15

To determine the impact of mixed endothelin receptor antagonist and mouse mast cell protease-4 (mMCP-4) in the development of atherosclerosis in the mouse model. Apolipoprotein E (ApoE) KO mice were crossed with mMCP-4 KO mice to generate ApoE/mMCP-4 double KO mice. Atherosclerosis was induced with a normal- or high-fat diet for 12, 27 or 52weeks. Macitentan (30mg/kg/day), a dual ETA/ETB receptor antagonist, was given orally for 6weeks (27week protocol). At sacrifice, aortas and brachiocephalic arteries (BCAs) were collected. En face Sudan IV staining was performed on aortas and BCA sections were subjected to Masson's trichrome stain and α-smooth muscle actin labeling. Under normal diet, both macitentan treatment and the absence of mMCP-4 reduced the development of aortic atherosclerotic lesions in 27-week old ApoE KO mice, but mMCP-4 deletion failed to maintain this effect on 52-week old mice. Under high-fat diet (WD), macitentan, but not the absence of mMCP-4, reduced aortic lesion development in ApoE KO mice. On BCA lesions of 27-week old WD mice, macitentan treatment had a small impact while mMCP-4 deletion showed improved features of plaque stability. These results suggest that the inhibition of mMCP-4 reduces lesion spreading in the earlier phases of atherosclerosis development and can help stabilise the more advanced plaque. Macitentan treatment was more effective to prevent lesion spreading but did not improve plaque features to the same extent. Copyright © 2016 Elsevier Inc. All rights reserved.

Palatogenesis

PubMed Central

Levi, Benjamin; Brugman, Samantha; Wong, Victor W; Grova, Monica; Longaker, Michael T

2011-01-01

Cleft palate represents the second most common birth defect and carries substantial physiologic and social challenges for affected patients, as they often require multiple surgical interventions during their lifetime. A number of genes have been identified to be associated with the cleft palate phenotype, but etiology in the majority of cases remains elusive. In order to better understand cleft palate and both surgical and potential tissue engineering approaches for repair, we have performed an in-depth literature review into cleft palate development in humans and mice, as well as into molecular pathways underlying these pathologic developments. We summarize the multitude of pathways underlying cleft palate development, with the transforming growth factor β superfamily being the most commonly studied. Furthermore, while the majority of cleft palate studies are performed using a mouse model, studies focusing on tissue engineering have also focused heavily on mouse models. A paucity of human randomized controlled studies exists for cleft palate repair, and so far, tissue engineering approaches are limited. In this review, we discuss the development of the palate, explain the basic science behind normal and pathologic palate development in humans as well as mouse models and elaborate on how these studies may lead to future advances in palatal tissue engineering and cleft palate treatments. PMID:21964245

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.

High-fat, carbohydrate-free diet markedly aggravates obesity but prevents beta-cell loss and diabetes in the obese, diabetes-susceptible db/db strain.

PubMed

Mirhashemi, Farshad; Kluth, Oliver; Scherneck, Stephan; Vogel, Heike; Kluge, Reinhart; Schurmann, Annette; Joost, Hans-Georg; Neschen, Susanne

2008-01-01

We have previously reported that a high-fat, carbohydrate-free diet prevents diabetes and beta-cell destruction in the New Zealand Obese (NZO) mouse strain. Here we investigated the effect of diets with and without carbohydrates on obesity and development of beta-cell failure in a second mouse model of type 2 diabetes, the db/db mouse. When kept on a carbohydrate-containing standard (SD; with (w/w) 5.1, 58.3, and 17.6% fat, carbohydrates and protein, respectively) or high-fat diet (HFD; 14.6, 46.7 and 17.1%), db/db mice developed severe diabetes (blood glucose >20 mmol/l, weight loss, polydipsia and polyurea) associated with a selective loss of pancreatic beta-cells, reduced GLUT2 expression in the remaining beta-cells, and reduced plasma insulin levels. In contrast, db/db mice kept on a high-fat, carbohydrate-free diet (CFD; with 30.2 and 26.4% (w/w) fat or protein) did not develop diabetes and exhibited near-normal, hyperplastic islets in spite of a morbid obesity (fat content >60%) associated with hyperinsulinaemia. These data indicate that in genetically different mouse models of obesity-associated diabetes, obesity and dietary fat are not sufficient, and dietary carbohydrates are required, for beta-cell destruction.

SDF1 regulates leading process branching and speed of migrating interneurons

PubMed Central

Lysko, Daniel E.; Putt, Mary; Golden, Jeffrey A.

2011-01-01

Cell migration is required for normal embryonic development, yet how cells navigate complex paths while integrating multiple guidance cues remains poorly understood. During brain development, interneurons migrate from the ventral ganglionic eminence to the cerebral cortex within several migratory streams. They must exit these streams to invade the cortical plate. While SDF1-signaling is necessary for normal interneuron stream migration, how they switch from tangential stream migration to invade the cortical plate is unknown. Here we demonstrate that SDF1-signaling reduces interneuron branching frequency by reducing cAMP levels via a Gi-signaling pathway using an in vitro mouse explant system, resulting in the maintenance of stream migration. Blocking SDF1-signaling, or increasing branching frequency, results in stream exit and cortical plate invasion in mouse brain slices. These data support a novel model to understand how migrating interneurons switch from tangential migration to invade the cortical plate in which reducing SDF1-signaling increases leading process branching and slows the migration rate, permitting migrating interneurons to sense cortically directed guidance cues. PMID:21289183

Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis.

PubMed

Wu, Jianfeng; Huang, Zhe; Ren, Junming; Zhang, Zhirong; He, Peng; Li, Yangxin; Ma, Jianhui; Chen, Wanze; Zhang, Yingying; Zhou, Xiaojuan; Yang, Zhentao; Wu, Su-Qin; Chen, Lanfen; Han, Jiahuai

2013-08-01

Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1β (IL-1β), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis.

Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis

PubMed Central

Wu, Jianfeng; Huang, Zhe; Ren, Junming; Zhang, Zhirong; He, Peng; Li, Yangxin; Ma, Jianhui; Chen, Wanze; Zhang, Yingying; Zhou, Xiaojuan; Yang, Zhentao; Wu, Su-Qin; Chen, Lanfen; Han, Jiahuai

2013-01-01

Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1β (IL-1β), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis. PMID:23835476

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

PubMed

Imtiaz, Ayesha; Belyantseva, Inna A; Beirl, Alisha J; Fenollar-Ferrer, Cristina; Bashir, Rasheeda; Bukhari, Ihtisham; Bouzid, Amal; Shaukat, Uzma; Azaiez, Hela; Booth, Kevin T; Kahrizi, Kimia; Najmabadi, Hossein; Maqsood, Azra; Wilson, Elizabeth A; Fitzgerald, Tracy S; Tlili, Abdelaziz; Olszewski, Rafal; Lund, Merete; Chaudhry, Taimur; Rehman, Atteeq U; Starost, Matthew F; Waryah, Ali M; Hoa, Michael; Dong, Lijin; Morell, Robert J; Smith, Richard J H; Riazuddin, Sheikh; Masmoudi, Saber; Kindt, Katie S; Naz, Sadaf; Friedman, Thomas B

2018-03-01

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

EFFECTS OF VARIOUS IMMUNE RABBIT SERUMS ON THE CELLS OF SEVERAL TRANSPLANTED MOUSE LYMPHOMAS IN VITRO AND IN VIVO

PubMed Central

Mohos, Steven C.; Kidd, John G.

1957-01-01

Immune serums prepared in rabbits with antigens made from normal mouse organs and tissues that were presumably devoid of large numbers of lymphocytic cells (notably kidney, liver, brain, whole embryos, and erythrocytes) proved lethal for the cells of several transplanted mouse lymphomas in vitro in the presence of complement; but these immune serums, when given intraperitoneally in large amounts to susceptible mice that had been implanted subcutaneously with lymphoma cells of one or another of several types, failed entirely to inhibit growth of the lymphoma cells in vivo. In contrast, immune serums made with cells procured from transplanted mouse lymphomas as antigens, and those made with cells from normal mouse thymus or lymph nodes, acted even more powerfully upon the several types of lymphoma cells in vitro than did the immune serums prepared with normal mouse organs, and when given intraperitoneally to implanted mice they brought about death of the lymphoma cells in vivo, the effect being to a considerable extent specific and referable to an antibody that reacts with neoplastic and non-neoplastic lymphocytic cells of mice, as absorption experiments disclosed. In comparative tests, furthermore, the anti-lymphoma serums acted more powerfully upon the lymphoma cells in vivo than did such chemotherapeutic agents as amethopterin, azaguanine, ethionine, azaserine, and 6-mercaptopurine, given singly or in various combinations in maximal tolerated amounts, though their effects were not so powerful as those exerted by normal guinea pig serum on lymphoma cells of two types that are susceptible to its action in vivo. The significance of the findings was briefly discussed. PMID:13406182

Multiple oncogenic viruses are present in human breast tissues before development of virus associated breast cancer.

PubMed

Lawson, James S; Glenn, Wendy K

2017-01-01

Multiple oncogenic viruses including, mouse mammary tumor virus, bovine leukemia virus, human papilloma virus, and Epstein Barr virus, have been identified as separate infectious pathogens in human breast cancer. Here we demonstrate that these four viruses may be present in normal and benign breast tissues 1 to 11 years before the development of same virus breast cancer in the same patients. We combined the data we developed during investigations of the individual four oncogenic viruses and breast cancer. Patients who had benign breast biopsies 1-11 years prior to developing breast cancer were identified by pathology reports from a large Australian pathology service (Douglas Hanly Moir Pathology). Archival formalin fixed specimens from these patients were collected. The same archival specimens were used for (i) investigations of mouse mammary tumour virus (also known as human mammary tumour virus) conducted at the Icahn School of Medicine at Mount Sinai, New York and at the University of Pisa, Italy, (ii) bovine leukemia virus conducted at the University of California at Berkeley,(iii) human papilloma virus and Epstein Barr virus conducted at the University of New South Wales, Sydney, Australia. Seventeen normal breast tissues from cosmetic breast surgery conducted on Australian patients were used as controls. These patients were younger than those with benign and later breast cancer. Standard and in situ polymerase chain reaction (PCR) methods were used to identify the four viruses. The detailed methods are outlined in the separate publications.: mouse mammary tumor virus, human papilloma virus and Epstein Barr virus (Infect Agent Cancer 12:1, 2017, PLoS One 12:e0179367, 2017, Front Oncol 5:277, 2015, PLoS One 7:e48788, 2012). Epstein Barr virus and human papilloma virus were identified in the same breast cancer cells by in situ PCR. Mouse mammary tumour virus was identified in 6 (24%) of 25 benign breast specimens and in 9 (36%) of 25 breast cancer specimens which subsequently developed in the same patients. Bovine leukemia virus was identified in 18 (78%) of 23 benign breast specimens and in 20 (91%) of 22 subsequent breast cancers in the same patients. High risk human papilloma viruses were identified in 13 (72%) of 17 benign breast specimens and in 13 (76%) of 17 subsequent breast cancers in the same patients. Epstein Barr virus was not identified in any benign breast specimens but was identified in 3 (25%) of 12 subsequent breast cancers in the same patients. Mouse mammary tumour virus 3 (18%), bovine leukemia virus 6 (35%), high risk human papilloma virus 3 (18%) and Epstein Barr virus 5 (29%) were identified in 17 normal control breast specimens. These findings add to the evidence that multiple oncogenic viruses have potential roles in human breast cancer. This is an important observation because evidence of prior infection before the development of disease is a key criterion when assessing causation.

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination.

PubMed

Pereira, Glauber B; Meng, Fanxue; Kockara, Neriman T; Yang, Baoli; Wight, Patricia A

2013-02-01

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. Although removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is non-functional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. © 2012 International Society for Neurochemistry.

New insight into the role of the β3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout

PubMed Central

Ferguson, Carolyn; Hardy, Steven L; Werner, David F; Hileman, Stanley M; DeLorey, Timothy M; Homanics, Gregg E

2007-01-01

Background The β3 subunit of the γ-aminobutyric acid type A receptor (GABAA-R) has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of β3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated β3 gene was engineered. Results Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the β3 subunit was flanked by loxP sites (i.e., floxed). Crossing the floxed β3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of β3 was achieved by crossing floxed β3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal β3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of β3 was achieved using α CamKII-cre transgenic mice. Palate development was normal in forebrain selective β3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15–25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese. Conclusion Conditional inactivation of the β3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed β3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the β3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes. PMID:17927825

A Model of Cancer Stem Cells Derived from Mouse Induced Pluripotent Stem Cells

PubMed Central

Chen, Ling; Kasai, Tomonari; Li, Yueguang; Sugii, Yuh; Jin, Guoliang; Okada, Masashi; Vaidyanath, Arun; Mizutani, Akifumi; Satoh, Ayano; Kudoh, Takayuki; Hendrix, Mary J. C.; Salomon, David S.; Fu, Li; Seno, Masaharu

2012-01-01

Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. CSCs are considered derived from normal stem cells affected by the tumor microenvironment although the mechanism of development is not clear yet. In 2007, Yamanaka's group succeeded in generating Nanog mouse induced pluripotent stem (miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5′-untranslated region of the Nanog gene. Usually, iPS cells, just like embryonic stem cells, are considered to be induced into progenitor cells, which differentiate into various normal phenotypes depending on the normal niche. We hypothesized that CSCs could be derived from Nanog miPS cells in the conditioned culture medium of cancer cell lines, which is a mimic of carcinoma microenvironment. As a result, the Nanog miPS cells treated with the conditioned medium of mouse Lewis lung carcinoma acquired characteristics of CSCs, in that they formed spheroids expressing GFP in suspension culture, and had a high tumorigenicity in Balb/c nude mice exhibiting angiogenesis in vivo. In addition, these iPS-derived CSCs had a capacity of self-renewal and expressed the marker genes, Nanog, Rex1, Eras, Esg1 and Cripto, associated with stem cell properties and an undifferentiated state. Thus we concluded that a model of CSCs was originally developed from miPS cells and proposed the conditioned culture medium of cancer cell lines might perform as niche for producing CSCs. The model of CSCs and the procedure of their establishment will help study the genetic alterations and the secreted factors in the tumor microenvironment which convert miPS cells to CSCs. Furthermore, the identification of potentially bona fide markers of CSCs, which will help the development of novel anti-cancer therapies, might be possible though the CSC model. PMID:22511923

Biology and therapy of inherited retinal degenerative disease: insights from mouse models

PubMed Central

Veleri, Shobi; Lazar, Csilla H.; Chang, Bo; Sieving, Paul A.; Banin, Eyal; Swaroop, Anand

2015-01-01

Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Tremendous progress has been made over the last two decades in discovering genes and genetic defects that lead to retinal diseases. The primary focus has now shifted to uncovering disease mechanisms and designing treatment strategies, especially inspired by the successful application of gene therapy in some forms of congenital blindness in humans. Both spontaneous and laboratory-generated mouse mutants have been valuable for providing fundamental insights into normal retinal development and for deciphering disease pathology. Here, we provide a review of mouse models of human retinal degeneration, with a primary focus on diseases affecting photoreceptor function. We also describe models associated with retinal pigment epithelium dysfunction or synaptic abnormalities. Furthermore, we highlight the crucial role of mouse models in elucidating retinal and photoreceptor biology in health and disease, and in the assessment of novel therapeutic modalities, including gene- and stem-cell-based therapies, for retinal degenerative diseases. PMID:25650393

Histology and ultrastructure of transitional changes in skin morphology in the juvenile and adult four-striped mouse (Rhabdomys pumilio).

PubMed

Stewart, Eranée; Ajao, Moyosore Salihu; Ihunwo, Amadi Ogonda

2013-01-01

The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile and adult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer. Apart from these cells, the histology of the skin of the adult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin.

Nanoparticle targeted therapy against childhood acute lymphoblastic leukemia

NASA Astrophysics Data System (ADS)

Satake, Noriko; Lee, Joyce; Xiao, Kai; Luo, Juntao; Sarangi, Susmita; Chang, Astra; McLaughlin, Bridget; Zhou, Ping; Kenney, Elaina; Kraynov, Liliya; Arnott, Sarah; McGee, Jeannine; Nolta, Jan; Lam, Kit

2011-06-01

The goal of our project is to develop a unique ligand-conjugated nanoparticle (NP) therapy against childhood acute lymphoblastic leukemia (ALL). LLP2A, discovered by Dr. Kit Lam, is a high-affinity and high-specificity peptidomimetic ligand against an activated α4β1 integrin. Our study using 11 fresh primary ALL samples (10 precursor B ALL and 1 T ALL) showed that childhood ALL cells expressed activated α4β1 integrin and bound to LLP2A. Normal hematopoietic cells such as activated lymphocytes and monocytes expressed activated α4β1 integrin; however, normal hematopoietic stem cells showed low expression of α4β1 integrin. Therefore, we believe that LLP2A can be used as a targeted therapy for childhood ALL. The Lam lab has developed novel telodendrimer-based nanoparticles (NPs) which can carry drugs efficiently. We have also developed a human leukemia mouse model using immunodeficient NOD/SCID/IL2Rγ null mice engrafted with primary childhood ALL cells from our patients. LLP2A-conjugated NPs will be evaluated both in vitro and in vivo using primary leukemia cells and this mouse model. NPs will be loaded first with DiD near infra-red dye, and then with the chemotherapeutic agents daunorubicin or vincristine. Both drugs are mainstays of current chemotherapy for childhood ALL. Targeting properties of LLP2A-conjugated NPs will be evaluated by fluorescent microscopy, flow cytometry, MTS assay, and mouse survival after treatment. We expect that LLP2A-conjugated NPs will be preferentially delivered and endocytosed to leukemia cells as an effective targeted therapy.

Guilty as charged: all available evidence implicates complement's role in fetal demise.

PubMed

Girardi, Guillermina

2008-03-01

Appropriate complement inhibition is an absolute requirement for normal pregancy. Uncontrolled complement activation in the maternal-fetal interface leads to fetal death. Here we show that complement activation is a crucial and early mediator of pregnancy loss in two different mouse models of pregnancy loss. Using a mouse model of fetal loss and growth restriction (IUGR) induced by antiphospholipid antibodies (aPL), we examined the role of complement activation in fetal loss and IUGR. We found that C5a-C5aR interaction and neutrophils are key mediators of fetal injury. Treatment with heparin, the standard therapy for pregnant patients with aPL, prevents complement activation and protects mice from pregnancy complications induced by aPL, and anticoagulants that do not inhibit complement do not protect pregnancies. In an antibody-independent mouse model of spontaneous miscarriage and IUGR (CBA/JxDBA/2) we also identified C5a as an essential mediator. Complement activation caused dysregulation of the angiogenic factors required for normal placental development. In CBA/JxDBA/2 mice, we observed inflammatory infiltrates in placentas, functional deficiency of free vascular endothelial growth factor (VEGF), elevated levels of soluble VEGF receptor-1 (sVEGFR-1, also known as sFlt-1; a potent anti-angiogenic molecule), and defective placental development. Inhibition of complement activation blocked the increase in sVEGFR-1 and rescued pregnancies. Our studies in antibody-dependent and antibody-independent models of pregnancy complications identified complement activation as the key mediator of damage and will allow development of new interventions to prevent pregnancy loss and IUGR.

Mouse Model for Human Arginase Deficiency

PubMed Central

Iyer, Ramaswamy K.; Yoo, Paul K.; Kern, Rita M.; Rozengurt, Nora; Tsoa, Rosemarie; O'Brien, William E.; Yu, Hong; Grody, Wayne W.; Cederbaum, Stephen D.

2002-01-01

Deficiency of liver arginase (AI) causes hyperargininemia (OMIM 207800), a disorder characterized by progressive mental impairment, growth retardation, and spasticity and punctuated by sometimes fatal episodes of hyperammonemia. We constructed a knockout mouse strain carrying a nonfunctional AI gene by homologous recombination. Arginase AI knockout mice completely lacked liver arginase (AI) activity, exhibited severe symptoms of hyperammonemia, and died between postnatal days 10 and 14. During hyperammonemic crisis, plasma ammonia levels of these mice increased >10-fold compared to those for normal animals. Livers of AI-deficient animals showed hepatocyte abnormalities, including cell swelling and inclusions. Plasma amino acid analysis showed the mean arginine level in knockouts to be approximately fourfold greater than that for the wild type and threefold greater than that for heterozygotes; the mean proline level was approximately one-third and the ornithine level was one-half of the proline and ornithine levels, respectively, for wild-type or heterozygote mice—understandable biochemical consequences of arginase deficiency. Glutamic acid, citrulline, and histidine levels were about 1.5-fold higher than those seen in the phenotypically normal animals. Concentrations of the branched-chain amino acids valine, isoleucine, and leucine were 0.4 to 0.5 times the concentrations seen in phenotypically normal animals. In summary, the AI-deficient mouse duplicates several pathobiological aspects of the human condition and should prove to be a useful model for further study of the disease mechanism(s) and to explore treatment options, such as pharmaceutical administration of sodium phenylbutyrate and/or ornithine and development of gene therapy protocols. PMID:12052859

Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex.

PubMed

Schaefer, Alisa; Poluch, Sylvie; Juliano, Sharon

2008-04-01

Numerous functions related to neuronal migration are linked to the glycoprotein reelin. Reelin also elongates radial glia, which are disrupted in mutant reeler mice. Our lab developed a model of cortical dysplasia in ferrets that shares features with the reeler mouse, including impaired migration of neurons into the cerebral cortex and disrupted radial glia. Explants of normal ferret cortex in coculture with dysplastic ferret cortex restore the deficits in this model. To determine if reelin is integral to the repair, we used explants of P0 mouse cortex either of the wild type (WT) or heterozygous (het) for the reelin gene, as well as P0 reeler cortex (not containing reelin), in coculture with organotypic cultures of dysplastic ferret cortex. This arrangement revealed that all types of mouse cortical explants (WT, het, reeler) elongated radial glia in ferret cortical dysplasia, indicating that reelin is not required for proper radial glial morphology. Migration of cells into ferret neocortex, however, did not improve with explants of reeler cortex, but was almost normal after pairing with WT or het explants. We also placed an exogenous source of reelin in ferret cultures at the pial surface to reveal that migrating cells move toward the reelin source in dysplastic cortex; radial glia in these cultures were also improved toward normal. Our results demonstrate that the normotopic position of reelin is important for proper neuronal positioning, and that reelin is capable of elongating radial glial cells but is not the only radialization factor.

Abnormal bone formation induced by implantation of osteosarcoma-derived bone-inducing substance in the X-linked hypophosphatemic mouse

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

Yoshikawa, H.; Masuhara, K.; Takaoka, K.

1985-01-01

The X-linked hypophosphatemic mouse (Hyp) has been proposed as a model for the human familial hypophosphatemia (the most common form of vitamin D-resistant rickets). An osteosarcoma-derived bone-inducing substance was subcutaneously implanted into the Hyp mouse. The implant was consistently replaced by cartilage tissue at 2 weeks after implantation. The cartilage matrix seemed to be normal, according to the histological examination, and 35sulphur (TVS) uptake was also normal. Up to 4 weeks after implantation the cartilage matrix was completely replaced by unmineralized bone matrix and hematopoietic bone marrow. Osteoid tissue arising from the implantation of bone inducing substance in the Hypmore » mouse showed no radiologic or histologic sign of calcification. These findings suggest that the abnormalities of endochondral ossification in the Hyp mouse might be characterized by the failure of mineralization in cartilage and bone matrix. Analysis of the effects of bone-inducing substance on the Hyp mouse may help to give greater insight into the mechanism and treatment of human familial hypophosphatemia.« less

Glucose dysregulation and response to common anti-diabetic agents in the FATZO/Pco mouse

PubMed Central

Jackson, Charles Van; Zimmerman, Karen M.; Alsina-Fernandez, Jorge; Michael, M. Dodson; Emmerson, Paul J.; Coskun, Tamer

2017-01-01

The FATZO/Pco mouse is the result of a cross of the C57BL/6J and AKR/J strains. The crossing of these two strains and the selective inbreeding for obesity, insulin resistance and hyperglycemia has resulted in an inbred strain exhibiting obesity in the presumed presence of an intact leptin pathway. Routinely used rodent models for obesity and diabetes research have a monogenic defect in leptin signaling that initiates obesity. Given that obesity and its sequelae in humans are polygenic in nature and not associated with leptin signaling defects, the FATZO mouse may represent a more translatable rodent model for study of obesity and its associated metabolic disturbances. The FATZO mouse develops obesity spontaneously when fed a normal chow diet. Glucose intolerance with increased insulin levels are apparent in FATZO mice as young as 6 weeks of age. These progress to hyperglycemia/pre-diabetes and frank diabetes with decreasing insulin levels as they age. The disease in these mice is multi-faceted, similar to the metabolic syndrome apparent in obese individuals, and thus provides a long pre-diabetic state for determining the preventive value of new interventions. We have assessed the utility of this new model for the pre-clinical screening of agents to stop or slow progression of the metabolic syndrome to severe diabetes. Our assessment included: 1) characterization of the spontaneous development of disease, 2) comparison of metabolic disturbances of FATZO mice to control mice and 3) validation of the model with regard to the effectiveness of current and emerging anti-diabetic agents; rosiglitazone, metformin and semaglutide. Conclusion: Male FATZO mice spontaneously develop significant metabolic disease when compared to normal controls while maintaining hyperglycemia in the presence of high leptin levels and hyperinsulinemia. The disease condition responds to commonly used antidiabetic agents. PMID:28640857

Notch signaling dynamics in the adult healthy prostate and in prostatic tumor development.

PubMed

Pedrosa, Ana-Rita; Graça, José L; Carvalho, Sandra; Peleteiro, Maria C; Duarte, António; Trindade, Alexandre

2016-01-01

The Notch signaling pathway has been implicated in prostate development, maintenance and tumorigenesis by its key role in cell-fate determination, differentiation and proliferation. Therefore, we proposed to analyze Notch family members transcription and expression, including ligands (Dll1, 3, 4 and Jagged1 and 2), receptors (Notch1-4) and effectors (Hes1, 2, 5 and Hey1, 2, L), in both normal and tumor bearing mouse prostates to better understand the dynamics of Notch signaling in prostate tumorigenesis. Wild type mice and transgenic adenocarcinoma of the mouse prostate model (TRAMP) mice were sacrificed at 18, 24 or 30 weeks of age and the prostates collected and processed for either whole prostate or prostate cell specific populations mRNA analysis and for protein expression analysis by immunohistochemistry and immunofluorescence. We observed that Dll1 and Dll4 are expressed in the luminal compartment of the mouse healthy prostate, whereas Jagged2 expression is restricted to the basal and stromal compartment. Additionally, Notch2 and Notch4 are normally expressed in the prostate luminal compartment while Notch2 and Notch3 are also expressed in the stromal layer of the healthy prostate. As prostate tumor development takes place, there is up-regulation of Notch components. Particularly, the prostate tumor lesions have increased expression of Jagged1 and 2, of Notch3 and of Hey1. We have also detected the presence of activated Notch3 in prostatic tumors that co-express Jagged1 and ultimately the Hey1 effector. Taken together our results point out the Notch axis Jagged1-2/Notch3/Hey1 to be important for prostate tumor development and worthy of additional functional studies and validation in human clinical disease. © 2015 Wiley Periodicals, Inc.

Circadian Rhythm Regulates Development of Enamel in Mouse Mandibular First Molar

PubMed Central

Tao, Jiang; Zhai, Yue; Park, Hyun; Han, Junli; Dong, Jianhui; Xie, Ming; Gu, Ting; Lewi, Keidren; Ji, Fang; Jia, William

2016-01-01

Rhythmic incremental growth lines and the presence of melatonin receptors were discovered in tooth enamel, suggesting possible role of circadian rhythm. We therefore hypothesized that circadian rhythm may regulate enamel formation through melatonin receptors. To test this hypothesis, we examined expression of melatonin receptors (MTs) and amelogenin (AMELX), a maker of enamel formation, during tooth germ development in mouse. Using qRT-PCR and immunocytochemistry, we found that mRNA and protein levels of both MTs and AMELX in normal mandibular first molar tooth germs increased gradually after birth, peaked at 3 or 4 day postnatal, and then decreased. Expression of MTs and AMELX by immunocytochemistry was significantly delayed in neonatal mice raised in all-dark or all-light environment as well as the enamel development. Furthermore, development of tooth enamel was also delayed showing significant immature histology in those animals, especially for newborn mice raised in all daylight condition. Interestingly, disruption in circadian rhythm in pregnant mice also resulted in delayed enamel development in their babies. Treatment with melatonin receptor antagonist 4P-PDOT in pregnant mice caused underexpression of MTs and AMELX associated with long-lasting deficiency in baby enamel tissue. Electromicroscopic evidence demonstrated increased necrosis and poor enamel mineralization in ameloblasts. The above results suggest that circadian rhythm is important for normal enamel development at both pre- and postnatal stages. Melatonin receptors were partly responsible for the regulation. PMID:27494172

Circadian Rhythm Regulates Development of Enamel in Mouse Mandibular First Molar.

PubMed

Tao, Jiang; Zhai, Yue; Park, Hyun; Han, Junli; Dong, Jianhui; Xie, Ming; Gu, Ting; Lewi, Keidren; Ji, Fang; Jia, William

2016-01-01

Rhythmic incremental growth lines and the presence of melatonin receptors were discovered in tooth enamel, suggesting possible role of circadian rhythm. We therefore hypothesized that circadian rhythm may regulate enamel formation through melatonin receptors. To test this hypothesis, we examined expression of melatonin receptors (MTs) and amelogenin (AMELX), a maker of enamel formation, during tooth germ development in mouse. Using qRT-PCR and immunocytochemistry, we found that mRNA and protein levels of both MTs and AMELX in normal mandibular first molar tooth germs increased gradually after birth, peaked at 3 or 4 day postnatal, and then decreased. Expression of MTs and AMELX by immunocytochemistry was significantly delayed in neonatal mice raised in all-dark or all-light environment as well as the enamel development. Furthermore, development of tooth enamel was also delayed showing significant immature histology in those animals, especially for newborn mice raised in all daylight condition. Interestingly, disruption in circadian rhythm in pregnant mice also resulted in delayed enamel development in their babies. Treatment with melatonin receptor antagonist 4P-PDOT in pregnant mice caused underexpression of MTs and AMELX associated with long-lasting deficiency in baby enamel tissue. Electromicroscopic evidence demonstrated increased necrosis and poor enamel mineralization in ameloblasts. The above results suggest that circadian rhythm is important for normal enamel development at both pre- and postnatal stages. Melatonin receptors were partly responsible for the regulation.

Placental growth factor deficiency is associated with impaired cerebral vascular development in mice.

PubMed

Luna, Rayana Leal; Kay, Vanessa R; Rätsep, Matthew T; Khalaj, Kasra; Bidarimath, Mallikarjun; Peterson, Nichole; Carmeliet, Peter; Jin, Albert; Croy, B Anne

2016-02-01

Placental growth factor (PGF) is expressed in the developing mouse brain and contributes to vascularization and vessel patterning. PGF is dynamically expressed in fetal mouse brain, particularly forebrain, and is essential for normal cerebrovascular development. PGF rises in maternal plasma over normal human and mouse pregnancy but is low in many women with the acute onset hypertensive syndrome, pre-eclampsia (PE). Little is known about the expression of PGF in the fetus during PE. Pgf  (-/-) mice appear normal but recently cerebral vascular defects were documented in adult Pgf  (-/-) mice. Here, temporal-spatial expression of PGF is mapped in normal fetal mouse brains and cerebral vasculature development is compared between normal and congenic Pgf  (-/-) fetuses to assess the actions of PGF during cerebrovascular development. Pgf/PGF, Vegfa/VEGF, Vegf receptor (Vegfr)1 and Vegfr2 expression were examined in the brains of embryonic day (E)12.5, 14.5, 16.5 and 18.5 C57BL/6 (B6) mice using quantitative PCR and immunohistochemistry. The cerebral vasculature was compared between Pgf  (-/-) and B6 embryonic and adult brains using whole mount techniques. Vulnerability to cerebral ischemia was investigated using a left common carotid ligation assay. Pgf/PGF and Vegfr1 are highly expressed in E12.5-14.5 forebrain relative to VEGF and Vegfr2. Vegfa/VEGF is relatively more abundant in hindbrain (HB). PGF and VEGF expression were similar in midbrain. Delayed HB vascularization was seen at E10.5 and 11.5 in Pgf  (-/-) brains. At E14.5, Pgf  (-/-) circle of Willis showed unilateral hypoplasia and fewer collateral vessels, defects that persisted post-natally. Functionally, adult Pgf  (-/-) mice experienced cerebral ischemia after left common carotid arterial occlusion while B6 mice did not. Since Pgf  (-/-) mice were used, consequences of complete absence of maternal and fetal PGF were defined. Therefore, the effects of maternal versus fetal PGF deficiency on cerebrovascular development cannot be separated. However, as PGF was strongly expressed in the developing brain at all timepoints, we suggest that local PGF has a more important role than distant maternal or placental sources. Full PGF loss is not expected in PE pregnancies, predicting that the effects of PGF deficiency identified in this model will be more severe than any effects in PE-offspring. These studies provoke the question of whether PGF expression is decreased and cerebral vascular maldevelopment occurs in fetuses who experience a preeclamptic gestation. These individuals have already been reported to have elevated risk for stroke and cognitive impairments. N/A. This work was supported by awards from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and the Canadian Foundation for Innovation to B.A.C. and by training awards from the Universidade Federal de Pernambuco and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil to R.L.L.; Queen's University to V.R.K. and the Canadian Institutes of Health Research to M.T.R. The work of P.C. is supported by the Belgian Science Policy BELSPO-IUAP7/03, Structural funding by the Flemish Government-Methusalem funding, and the Flemish Science Fund-FWO grants. There were no competing interests. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Scanning Thin-Sheet Laser Imaging Microscopy Elucidates Details on Mouse Ear Development

PubMed Central

Kopecky, Benjamin; Johnson, Shane; Schmitz, Heather; Santi, Peter; Fritzsch, Bernd

2016-01-01

Background The mammalian inner ear is transformed from a flat placode into a three-dimensional (3D) structure with six sensory epithelia that allow for the perception of sound and both linear and angular acceleration. While hearing and balance problems are typically considered to be adult onset diseases, they may arise as a developmental perturbation to the developing ear. Future prevention of hearing or balance loss requires an understanding of how closely genetic mutations in model organisms reflect the human case, necessitating an objective multidimensional comparison of mouse ears with human ears that have comparable mutations in the same gene. Results Here, we present improved 3D analyses of normal murine ears during embryonic development using optical sections obtained through Thin-Sheet Laser Imaging Microscopy. We chronicle the transformation of an undifferentiated otic vesicle between mouse embryonic day 11.5 to a fully differentiated inner ear at postnatal day 15. Conclusions Our analysis of ear development provides new insights into ear development, enables unique perspectives into the complex development of the ear, and allows for the first full quantification of volumetric and linear aspects of ear growth. Our data provide the framework for future analysis of mutant phenotypes that are currently under-appreciated using only two dimensional renderings. PMID:22271591

Scanning thin-sheet laser imaging microscopy elucidates details on mouse ear development.

PubMed

Kopecky, Benjamin; Johnson, Shane; Schmitz, Heather; Santi, Peter; Fritzsch, Bernd

2012-03-01

The mammalian inner ear is transformed from a flat placode into a three-dimensional (3D) structure with six sensory epithelia that allow for the perception of sound and both linear and angular acceleration. While hearing and balance problems are typically considered to be adult onset diseases, they may arise as a developmental perturbation to the developing ear. Future prevention of hearing or balance loss requires an understanding of how closely genetic mutations in model organisms reflect the human case, necessitating an objective multidimensional comparison of mouse ears with human ears that have comparable mutations in the same gene. Here, we present improved 3D analyses of normal murine ears during embryonic development using optical sections obtained through Thin-Sheet Laser Imaging Microscopy. We chronicle the transformation of an undifferentiated otic vesicle between mouse embryonic day 11.5 to a fully differentiated inner ear at postnatal day 15. Our analysis of ear development provides new insights into ear development, enables unique perspectives into the complex development of the ear, and allows for the first full quantification of volumetric and linear aspects of ear growth. Our data provide the framework for future analysis of mutant phenotypes that are currently under-appreciated using only two dimensional renderings. Copyright © 2012 Wiley Periodicals, Inc.

The RNA-binding landscape of RBM10 and its role in alternative splicing regulation in models of mouse early development.

PubMed

Rodor, Julie; FitzPatrick, David R; Eyras, Eduardo; Cáceres, Javier F

2017-01-02

Mutations in the RNA-binding protein, RBM10, result in a human syndromic form of cleft palate, termed TARP syndrome. A role for RBM10 in alternative splicing regulation has been previously demonstrated in human cell lines. To uncover the cellular functions of RBM10 in a cell line that is relevant to the phenotype observed in TARP syndrome, we used iCLIP to identify its endogenous RNA targets in a mouse embryonic mandibular cell line. We observed that RBM10 binds to pre-mRNAs with significant enrichment in intronic regions, in agreement with a role for this protein in pre-mRNA splicing. In addition to protein-coding transcripts, RBM10 also binds to a variety of cellular RNAs, including non-coding RNAs, such as spliceosomal small nuclear RNAs, U2 and U12. RNA-seq was used to investigate changes in gene expression and alternative splicing in RBM10 KO mouse mandibular cells and also in mouse ES cells. We uncovered a role for RBM10 in the regulation of alternative splicing of common transcripts in both cell lines but also identified cell-type specific events. Importantly, those pre-mRNAs that display changes in alternative splicing also contain RBM10 iCLIP tags, suggesting a direct role of RBM10 in these events. Finally, we show that depletion of RBM10 in mouse ES cells leads to proliferation defects and to gross alterations in their differentiation potential. These results demonstrate a role for RBM10 in the regulation of alternative splicing in two cell models of mouse early development and suggests that mutations in RBM10 could lead to splicing changes that affect normal palate development and cause human disease.

Increased Expression of PcG Protein YY1 Negatively Regulates B Cell Development while Allowing Accumulation of Myeloid Cells and LT-HSC Cells

PubMed Central

Pan, Xuan; Jones, Morgan; Jiang, Jie; Zaprazna, Kristina; Yu, Duonan; Pear, Warren; Maillard, Ivan; Atchison, Michael L.

2012-01-01

Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs. PMID:22292011

Neuroanatomical phenotyping of the mouse brain with three-dimensional autofluorescence imaging

PubMed Central

Wong, Michael D.; Dazai, Jun; Altaf, Maliha; Mark Henkelman, R.; Lerch, Jason P.; Nieman, Brian J.

2012-01-01

The structural organization of the brain is important for normal brain function and is critical to understand in order to evaluate changes that occur during disease processes. Three-dimensional (3D) imaging of the mouse brain is necessary to appreciate the spatial context of structures within the brain. In addition, the small scale of many brain structures necessitates resolution at the ∼10 μm scale. 3D optical imaging techniques, such as optical projection tomography (OPT), have the ability to image intact large specimens (1 cm3) with ∼5 μm resolution. In this work we assessed the potential of autofluorescence optical imaging methods, and specifically OPT, for phenotyping the mouse brain. We found that both specimen size and fixation methods affected the quality of the OPT image. Based on these findings we developed a specimen preparation method to improve the images. Using this method we assessed the potential of optical imaging for phenotyping. Phenotypic differences between wild-type male and female mice were quantified using computer-automated methods. We found that optical imaging of the endogenous autofluorescence in the mouse brain allows for 3D characterization of neuroanatomy and detailed analysis of brain phenotypes. This will be a powerful tool for understanding mouse models of disease and development and is a technology that fits easily within the workflow of biology and neuroscience labs. PMID:22718750

Organ dose conversion coefficients based on a voxel mouse model and MCNP code for external photon irradiation.

PubMed

Zhang, Xiaomin; Xie, Xiangdong; Cheng, Jie; Ning, Jing; Yuan, Yong; Pan, Jie; Yang, Guoshan

2012-01-01

A set of conversion coefficients from kerma free-in-air to the organ absorbed dose for external photon beams from 10 keV to 10 MeV are presented based on a newly developed voxel mouse model, for the purpose of radiation effect evaluation. The voxel mouse model was developed from colour images of successive cryosections of a normal nude male mouse, in which 14 organs or tissues were segmented manually and filled with different colours, while each colour was tagged by a specific ID number for implementation of mouse model in Monte Carlo N-particle code (MCNP). Monte Carlo simulation with MCNP was carried out to obtain organ dose conversion coefficients for 22 external monoenergetic photon beams between 10 keV and 10 MeV under five different irradiation geometries conditions (left lateral, right lateral, dorsal-ventral, ventral-dorsal, and isotropic). Organ dose conversion coefficients were presented in tables and compared with the published data based on a rat model to investigate the effect of body size and weight on the organ dose. The calculated and comparison results show that the organ dose conversion coefficients varying the photon energy exhibits similar trend for most organs except for the bone and skin, and the organ dose is sensitive to body size and weight at a photon energy approximately <0.1 MeV.

A unified model of the excitability of mouse sensory and motor axons.

PubMed

Makker, Preet G S; Matamala, José Manuel; Park, Susanna B; Lees, Justin G; Kiernan, Matthew C; Burke, David; Moalem-Taylor, Gila; Howells, James

2018-06-19

Non-invasive nerve excitability techniques have provided valuable insight into the understanding of neurological disorders. The widespread use of mice in translational research on peripheral nerve disorders and by pharmaceutical companies during drug development requires valid and reliable models that can be compared to humans. This study established a novel experimental protocol that enables comparative assessment of the excitability properties of motor and sensory axons at the same site in mouse caudal nerve, compared the mouse data to data for motor and sensory axons in human median nerve at the wrist, and constructed a mathematical model of the excitability of mouse axons. In a separate study, ischaemia was employed as an experimental manoeuvre to test the translational utility of this preparation. The patterns of mouse sensory and motor excitability were qualitatively similar to human studies under normal and ischaemic conditions. The most conspicuous differences between mouse and human studies were observed in the recovery cycle and the response to hyperpolarization. Modelling showed that an increase in temperature in mouse axons could account for most of the differences in the recovery cycle. The modelling also suggested a larger hyperpolarization-activated conductance in mouse axons. The kinetics of this conductance appeared to be much slower raising the possibility that an additional or different hyperpolarization-activated cyclic-nucleotide gated (HCN) channel isoform underlies the accommodation to hyperpolarization in mouse axons. Given a possible difference in HCN isoforms, caution should be exercised in extrapolating from studies of mouse motor and sensory axons to human nerve disorders. This article is protected by copyright. All rights reserved.

CHANGES IN MOUSE CIRCULATING T-LYMPHOCYTE NUMBERS DURING A NORMAL CRYPTOSPORIDIUM MURIS INFECTION AND AFTER A SINGLE INJECTION OF METHYLPREDNISOLONE ACETATE

EPA Science Inventory

Cryptosporidium species are of public health significance in both developing nations and the industrialized nations of the world. Persons with immature or compromised immune systems are said to have increased risk or mortality once infected. The objective of this study was to c...

Generation of a mouse model with a reversible hypomorphic cytochrome P450 reductase gene: utility for tissue-specific rescue of the reductase expression, and insights from a resultant mouse model with global suppression of P450 reductase expression in extrahepatic tissues.

PubMed

Wei, Yuan; Zhou, Xin; Fang, Cheng; Li, Lei; Kluetzman, Kerri; Yang, Weizhu; Zhang, Qing-Yu; Ding, Xinxin

2010-07-01

A mouse model termed Cpr-low (CL) was recently generated, in which the expression of the cytochrome P450 reductase (Cpr) gene was globally down-regulated. The decreased CPR expression was accompanied by phenotypical changes, including reduced embryonic survival, decreases in circulating cholesterol, increases in hepatic P450 expression, and female infertility (accompanied by elevated serum testosterone and progesterone levels). In the present study, a complementary mouse model [named reversible-CL (r-CL)] was generated, in which the reduced CPR expression can be reversed in an organ-specific fashion. The neo cassette, which was inserted into the last Cpr intron in r-CL mice, can be deleted by Cre recombinase, thus returning the structure of the Cpr gene (and hence CPR expression) to normal in Cre-expressing cells. All previously identified phenotypes of the CL mice were preserved in the r-CL mice. As a first application of the r-CL model, we have generated an extrahepatic-CL (xh-CL) mouse for testing of the functions of CPR-dependent enzymes in all extrahepatic tissues. The xh-CL mice, generated by mating of r-CL mice with albumin-Cre mice, had normal CPR expression in hepatocytes but down-regulated CPR expression elsewhere. They were indistinguishable from wild-type mice in body and liver weights, circulating cholesterol levels, and hepatic microsomal P450 expression and activities; however, they still showed elevated serum testosterone and progesterone levels and sterility in females. Embryonic lethality was prevented in males, but apparently not in females, indicating a critical role for fetal hepatic CPR-dependent enzymes in embryonic development, at least in males.

Predicting Survival within the Lung Cancer Histopathological Hierarchy Using a Multi-Scale Genomic Model of Development

PubMed Central

Liu, Hongye; Kho, Alvin T; Kohane, Isaac S; Sun, Yao

2006-01-01

Background The histopathologic heterogeneity of lung cancer remains a significant confounding factor in its diagnosis and prognosis—spurring numerous recent efforts to find a molecular classification of the disease that has clinical relevance. Methods and Findings Molecular profiles of tumors from 186 patients representing four different lung cancer subtypes (and 17 normal lung tissue samples) were compared with a mouse lung development model using principal component analysis in both temporal and genomic domains. An algorithm for the classification of lung cancers using a multi-scale developmental framework was developed. Kaplan–Meier survival analysis was conducted for lung adenocarcinoma patient subgroups identified via their developmental association. We found multi-scale genomic similarities between four human lung cancer subtypes and the developing mouse lung that are prognostically meaningful. Significant association was observed between the localization of human lung cancer cases along the principal mouse lung development trajectory and the corresponding patient survival rate at three distinct levels of classical histopathologic resolution: among different lung cancer subtypes, among patients within the adenocarcinoma subtype, and within the stage I adenocarcinoma subclass. The earlier the genomic association between a human tumor profile and the mouse lung development sequence, the poorer the patient's prognosis. Furthermore, decomposing this principal lung development trajectory identified a gene set that was significantly enriched for pyrimidine metabolism and cell-adhesion functions specific to lung development and oncogenesis. Conclusions From a multi-scale disease modeling perspective, the molecular dynamics of murine lung development provide an effective framework that is not only data driven but also informed by the biology of development for elucidating the mechanisms of human lung cancer biology and its clinical outcome. PMID:16800721

Prenatal Correction of X-Linked Hypohidrotic Ectodermal Dysplasia.

PubMed

Schneider, Holm; Faschingbauer, Florian; Schuepbach-Mallepell, Sonia; Körber, Iris; Wohlfart, Sigrun; Dick, Angela; Wahlbuhl, Mandy; Kowalczyk-Quintas, Christine; Vigolo, Michele; Kirby, Neil; Tannert, Corinna; Rompel, Oliver; Rascher, Wolfgang; Beckmann, Matthias W; Schneider, Pascal

2018-04-26

Genetic deficiency of ectodysplasin A (EDA) causes X-linked hypohidrotic ectodermal dysplasia (XLHED), in which the development of sweat glands is irreversibly impaired, an condition that can lead to life-threatening hyperthermia. We observed normal development of mouse fetuses with Eda mutations after they had been exposed in utero to a recombinant protein that includes the receptor-binding domain of EDA. We administered this protein intraamniotically to two affected human twins at gestational weeks 26 and 31 and to a single affected human fetus at gestational week 26; the infants, born in week 33 (twins) and week 39 (singleton), were able to sweat normally, and XLHED-related illness had not developed by 14 to 22 months of age. (Funded by Edimer Pharmaceuticals and others.).

Norrin expression in endothelial cells in the developing mouse retina.

PubMed

Lee, Hanjae; Jo, Dong Hyun; Kim, Jin Hyoung; Kim, Jeong Hun

2013-06-01

Norrin, a protein that acts on Frizzled-4 receptor, participates in angiogenesis in a variety of contexts through the Wnt-signaling pathway. Specifically, Norrin is found to play a crucial role in retinal vascularization. Norrin's pivotal role in angiogenesis led us to investigate its expression and the primary source in the developing retina. In this study we demonstrate, for the first time, that Norrin protein is expressed along the retinal blood vessels. The expression of Norrin coincided with the pattern of vascular growth in the developing mouse retina, and its expression was identified from the endothelial cells of the retinal capillaries. Furthermore, Norrin was also expressed on endothelial cells of the developing human retina. Given that Norrin is crucial in the normal development and maintenance of ocular capillaries, our finding provides a hint of the involvement of Norrin in the self generative and protective mechanism of the endothelial cells in the developing retina. Copyright © 2012 Elsevier GmbH. All rights reserved.

Defining the cause of skewed X-chromosome inactivation in X-linked mental retardation by use of a mouse model.

PubMed

Muers, Mary R; Sharpe, Jacqueline A; Garrick, David; Sloane-Stanley, Jacqueline; Nolan, Patrick M; Hacker, Terry; Wood, William G; Higgs, Douglas R; Gibbons, Richard J

2007-06-01

Extreme skewing of X-chromosome inactivation (XCI) is rare in the normal female population but is observed frequently in carriers of some X-linked mutations. Recently, it has been shown that various forms of X-linked mental retardation (XLMR) have a strong association with skewed XCI in female carriers, but the mechanisms underlying this skewing are unknown. ATR-X syndrome, caused by mutations in a ubiquitously expressed, chromatin-associated protein, provides a clear example of XLMR in which phenotypically normal female carriers virtually all have highly skewed XCI biased against the X chromosome that harbors the mutant allele. Here, we have used a mouse model to understand the processes causing skewed XCI. In female mice heterozygous for a null Atrx allele, we found that XCI is balanced early in embryogenesis but becomes skewed over the course of development, because of selection favoring cells expressing the wild-type Atrx allele. Unexpectedly, selection does not appear to be the result of general cellular-viability defects in Atrx-deficient cells, since it is restricted to specific stages of development and is not ongoing throughout the life of the animal. Instead, there is evidence that selection results from independent tissue-specific effects. This illustrates an important mechanism by which skewed XCI may occur in carriers of XLMR and provides insight into the normal role of ATRX in regulating cell fate.

Bronchial airway gene expression signatures in mouse lung squamous cell carcinoma and their modulation by cancer chemopreventive agents

PubMed Central

Szabo, Eva; Miller, Mark Steven; Lubet, Ronald A.; You, Ming; Wang, Yian

2017-01-01

Due to exposure to environmental toxicants, a “field cancerization” effect occurs in the lung resulting in the development of a field of initiated but morphologically normal appearing cells in the damaged epithelium of bronchial airways with dysregulated gene expression patterns. Using a mouse model of lung squamous cell carcinoma (SCC), we performed transcriptome sequencing (RNA-Seq) to profile bronchial airway gene expression and found activation of the PI3K and Myc signaling networks in cytologically normal bronchial airway epithelial cells of mice with preneopastic lung SCC lesions, which was reversed by treatment with the PI3K Inhibitor XL-147 and pioglitazone, respectively. Activated MYC signaling was also present in premalignant and tumor tissues from human lung SCC patients. In addition, we identified a key microRNA, mmu-miR-449c-5p, whose suppression significantly up-regulated Myc expression in the normal bronchial airway epithelial cells of mice with early stage SCC lesions. We developed a novel bronchial genomic classifier in mice and validated it in humans. In the classifier, Ppbp (pro-platelet basic protein) was overexpressed 115 fold in the bronchial airways of mice with preneoplastic lung SCC lesions. This is the first report that demonstrates Ppbp as a novel biomarker in the bronchial airway for lung cancer diagnosis. PMID:27935865

Morphology of the lumbar transversospinal muscles examined in a mouse bearing a muscle fiber-specific nuclear marker.

PubMed

Cornwall, Jon; Deries, Marianne; Duxson, Marilyn

2010-12-01

Although the morphology of human lumbar transversospinal (TSP) muscles has been studied, little is known about the structure of these muscles in the mouse (Mus musculus). Such information is relevant given mice are often used as a "normal" phenotype for studies modeling human development. This study describes the gross morphology, muscle fiber arrangement, and innervation pattern of the mouse lumbar TSP muscles. A unique feature of the study is the use of a transgenic mouse line bearing a muscle-specific nuclear marker that allows clear delineation of muscle fiber and connective tissue boundaries. The lumbar TSP muscles of five mice were examined bilaterally; at each spinal level muscles attached to the caudal edge of the spinous process and passed caudally as a single complex unit. Fibers progressively terminated over the four vertebral segments caudad, with multiple points of muscle fiber attachment on each vertebra. Motor endplates, defined with acetylcholinesterase histochemistry, were consistently located half way along each muscle fiber, regardless of length, with all muscle fibers arranged in-parallel rather than in-series. These results provide information relevant to interpretation of developmental and functional studies involving this muscle group in the mouse and show mouse lumbar TSP muscles are different in form to descriptions of equivalent muscles in humans and horses.

Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

PubMed Central

Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

2015-01-01

Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

Anatomical Variation of the Tarsus in Common Inbred Mouse Strains.

PubMed

Richbourg, Heather A; Martin, Matthew J; Schachner, Emma R; McNulty, Margaret A

2017-03-01

Rodent models are used for a variety of orthopedic research applications; however, anatomy references include mostly artistic representations. Advanced imaging techniques, including micro-computed tomography (microCT), can provide more accurate representations of subtle anatomical characteristics. A recent microCT atlas of laboratory mouse (Mus musculus) anatomy depicts the central and tarsal bone III (T3) as a single bone, differing from previous references. Fusion of tarsal bones is generally characterized as pathological secondary to mutations associated with growth factors, and normal variation has not been documented in the mouse tarsus. Therefore, it is unclear if this fusion is a normal or a pathological characteristic. The aim of this study is to characterize the tarsus of the laboratory mouse and compare it to the rat and selected outgroup species (i.e., white-footed mouse) via microCT and histology to determine if the central and T3 are separate or fused into a single bone. Laboratory mice (C57/Bl6 [n = 17] and BalbC [n = 2]) and rats (n = 5) were scanned with microCT. A representative laboratory mouse from each strain was evaluated histologically via serial sagittal sections through the mid-tarsus. General pedal anatomy was similar between all species; however, the central and T3 bones were fused in all laboratory mice but not the rat or white-footed mouse. A band of hyaline cartilage was identified within the fused bone of the laboratory mice. We conclude that the fusion found is a normal characteristic in laboratory mice, but timing of the fusion remains ambiguous. Anat Rec, 300:450-459, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Spallanzani's mouse: a model of restoration and regeneration.

PubMed

Heber-Katz, E; Leferovich, J M; Bedelbaeva, K; Gourevitch, D

2004-01-01

The ability to regenerate is thought to be a lost phenotype in mammals, though there are certainly sporadic examples of mammalian regeneration. Our laboratory has identified a strain of mouse, the MRL mouse, which has a unique capacity to heal complex tissue in an epimorphic fashion, i.e., to restore a damaged limb or organ to its normal structure and function. Initial studies using through-and-through ear punches showed rapid full closure of the ear holes with cartilage growth, new hair follicles, and normal tissue architecture reminiscent of regeneration seen in amphibians as opposed to the scarring usually seen in mammals. Since the ear hole closure phenotype is a quantitative trait, this has been used to show-through extensive breeding and backcrossing--that the trait is heritable. Such analysis reveals that there is a complex genetic basis for this trait with multiple loci. One of the major phenotypes of the MRL mouse is a potent remodeling response with the absence or a reduced level of scarring. MRL healing is associated with the upregulation of the metalloproteinases MMP-2 and MMP-9 and the downregulation of their inhibitors TIMP-2 and TIMP-3, both present in inflammatory cells such as neutrophils and macrophages. This model has more recently been extended to the heart. In this case, a cryoinjury to the right ventricle leads to near complete scarless healing in the MRL mouse whereas scarring is seen in the control mouse. In the MRL heart, bromodeoxyuridine uptake by cardiomyocytes filling the wound site can be seen 60 days after injury. This does not occur in the control mouse. Function in the MRL heart, as measured by echocardiography, returns to normal.

Persistence of Cytosine Methylation of DNA following Fertilisation in the Mouse

PubMed Central

Li, Yan; O'Neill, Chris

2012-01-01

Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell-cycle following fertilization. An enzyme or mechanism capable of catalysing such rapid global demethylation has not been identified. The mouse is a widely used model for studying developmental epigenetics. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. We found when using conventional methods of immunolocalization that 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy by also performing tryptic digestion revealed a persistence of a methylated state. Analysis of methyl binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. The study shows that any 5meC remodelling that occurs during early development is not explained by a global active loss of 5meC staining during the cleavage stage of development and global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote. PMID:22292019

Effects of androgen on immunohistochemical localization of androgen receptor and Connexin 43 in mouse ovary.

PubMed

Yang, Mei; Li, Jianhua; An, Yulin; Zhang, Shuiwen

2015-10-01

Androgens have essential roles in the regulation of follicular development and female fertility. Androgen excess is the leading defect in polycystic ovary syndrome (PCOS) patients and involved in the ovarian dysfunction. The aim of this study was to elucidate the regarding regulatory role of androgen in the follicular development of female mouse. Immunohistochemical staining and Western blot analyses were performed to detect androgen receptor (AR) and Connexin 43 (Cx43) expression in ovaries from both control and testosterone-treated group mice. In this study, localizations of AR and Cx43 were dramatically altered in testosterone-treated mouse ovaries. In addition, AR expression was significantly increased, whereas Cx43 expression was markedly decreased after testosterone treatment. Alterations of AR and Cx43 expression by testosterone with concomitant reduction of MII oocytes. Overall, these results suggest the involvement of androgen in the regulation of AR and Cx43 localizations in mouse ovary. Alterations of AR and Cx43 expression by testosterone may affect normal folliculogenesis. Together these findings will enable us to begin understanding the important roles of AR and Cx43 actions in the regulation of follicular development, as well as providing insights into the role of AR and Cx43 actions in the androgen-associated reproductive diseases such as PCOS. Copyright © 2015 Elsevier Ltd. All rights reserved.

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

PubMed Central

Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

2012-01-01

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development. PMID:23227157

Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

PubMed

Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

2012-01-01

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

Genetic mouse embryo assay: improving performance and quality testing for assisted reproductive technology (ART) with a functional bioassay.

PubMed

Gilbert, Rebecca S; Nunez, Brandy; Sakurai, Kumi; Fielder, Thomas; Ni, Hsiao-Tzu

2016-03-24

Growing concerns about safety of ART on human gametes, embryos, clinical outcomes and long-term health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation. The one-cell mouse embryo assay (MEA) is the most widely used for development and quality testing of human ART products; however, concerns exist due to the insensitivity/variability of this bioassay which lacks standardization and involves subjective analysis by morphology alone rather than functional analysis of the developing embryos. We hypothesized that improvements to MEA by the use of functional molecular biomarkers could enhance sensitivity and improve detection of suboptimal materials/conditions. Fresh one-cell transgenic mouse embryos with green fluorescent protein (GFP) expression driven by Pou6f1 or Cdx2 control elements were harvested and cultured to blastocysts in varied test and control conditions to compare assessment by standard morphology alone versus the added dynamic expression of GFP for screening and selection of critical raw materials and detection of suboptimal culture conditions. Transgenic mouse embryos expressing functionally relevant biomarkers of normal early embryo development can be used to monitor the developmental impact of culture conditions. This novel approach provides a superior MEA that is more meaningful and sensitive for detection of embryotoxicity than morphological assessment alone.

Investigation of nuclear nano-morphology marker as a biomarker for cancer risk assessment using a mouse model

NASA Astrophysics Data System (ADS)

Bista, Rajan K.; Uttam, Shikhar; Hartman, Douglas J.; Qiu, Wei; Yu, Jian; Zhang, Lin; Brand, Randall E.; Liu, Yang

2012-06-01

The development of accurate and clinically applicable tools to assess cancer risk is essential to define candidates to undergo screening for early-stage cancers at a curable stage or provide a novel method to monitor chemoprevention treatments. With the use of our recently developed optical technology--spatial-domain low-coherence quantitative phase microscopy (SL-QPM), we have derived a novel optical biomarker characterized by structure-derived optical path length (OPL) properties from the cell nucleus on the standard histology and cytology specimens, which quantifies the nano-structural alterations within the cell nucleus at the nanoscale sensitivity, referred to as nano-morphology marker. The aim of this study is to evaluate the feasibility of the nuclear nano-morphology marker from histologically normal cells, extracted directly from the standard histology specimens, to detect early-stage carcinogenesis, assess cancer risk, and monitor the effect of chemopreventive treatment. We used a well-established mouse model of spontaneous carcinogenesis--ApcMin mice, which develop multiple intestinal adenomas (Min) due to a germline mutation in the adenomatous polyposis coli (Apc) gene. We found that the nuclear nano-morphology marker quantified by OPL detects the development of carcinogenesis from histologically normal intestinal epithelial cells, even at an early pre-adenomatous stage (six weeks). It also exhibits a good temporal correlation with the small intestine that parallels the development of carcinogenesis and cancer risk. To further assess its ability to monitor the efficacy of chemopreventive agents, we used an established chemopreventive agent, sulindac. The nuclear nano-morphology marker is reversed toward normal after a prolonged treatment. Therefore, our proof-of-concept study establishes the feasibility of the SL-QPM derived nuclear nano-morphology marker OPL as a promising, simple and clinically applicable biomarker for cancer risk assessment and evaluation of chemopreventive treatment.

Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities

PubMed Central

Lay, Angelina J.; Liang, Zhong; Rosen, Elliot D.; Castellino, Francis J.

2005-01-01

Anticoagulant protein C (PC) is important not only for maintenance of normal hemostasis, but also for regulating the host immune response during inflammation. Because mice with a designed total genetic deficiency in PC (PC–/– mice) die soon after birth, attempts to dissect PC function in various coagulation/inflammation-based pathologies through use of mice with less than 50% of normal PC levels have not been successful to date. In the current investigation, we have used a novel transgenic strategy to generate different mouse models expressing 1–18% of normal PC levels. In contrast to PC–/– mice, mice with only partial PC deficiency survived beyond birth and also developed thrombosis and inflammation. The onset and severity of these phenotypes vary significantly and are strongly dependent on plasma PC levels. Our findings additionally provide the first evidence that maternal PC is vital for sustaining pregnancy beyond 7.5 days postcoitum, likely by regulating the balance of coagulation and inflammation during trophoblast invasion. These low PC–expressing transgenic mouse lines provide novel animal models that can be used to elucidate the importance of PC in maintenance of the organism and in disease. PMID:15902301

Absence of Vsx1 expression in the normal and damaged mouse cornea

PubMed Central

Chow, Robert L.

2011-01-01

Purpose To examine the expression of visual system homeobox 1 (Vsx1) in the mouse cornea and its potential role in the corneal wound response pathway. Methods Expression of Vsx1 was examined by quantitative reverse-transcription PCR (qRT–PCR) in corneal tissue from developing and adult mice and from mice that had undergone alkali-burn corneal wounding. Immunolabeling and Vsx1 knock-in reporter gene expression in wild type and Vsx1 null-mice were used to confirm the qRT–PCR data. Results Using qRT–PCR, Vsx1 expression was not detected in either the postnatal or adult mouse cornea or in corneas following wounding. This qRT–PCR data was supported by the absence of specific Vsx1 immunolabeling and Vsx1 knock-in reporter expression in untreated and wounded corneas. Conclusions In mice, Vsx1 mRNA, protein or reporter gene expression is not detected in the normal or damaged cornea. These results make it uncertain what role VSX1/Vsx1 plays in corneal biology. Future experiments examining the pathogenicity of VSX1 mutations associated with corneal dystrophy are required to rule out species differences and possible non-cell autonomous roles for VSX1 in the cornea. PMID:21437200

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells

PubMed Central

Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino

2016-01-01

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

PubMed

Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino

2016-12-20

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography.

PubMed

Xue, Yanhong; Zhao, Wei; Du, Wen; Zhang, Xiang; Ji, Gang; Ying, Wang; Xu, Tao

2012-07-01

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.

Genome-wide analysis of alternative splicing in medulloblastoma identifies splicing patterns characteristic of normal cerebellar development

PubMed Central

Menghi, Francesca; Jacques, Thomas S.; Barenco, Martino; Schwalbe, Ed C.; Clifford, Steven C.; Hubank, Mike; Ham, Jonathan

2011-01-01

Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between paediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1262 genes identified as potentially generating tumour-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by RT-PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of Sonic Hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumour and normal fetal cerebellar samples shared significantly lower exon inclusion rates compared to normal adult cerebellum. We investigated whether tumour-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCPs) and found that Shh caused a decrease in the cassette exon inclusion rate of five out of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between post-natal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into post-mitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signalling pathways and a failure of cerebellar precursor cells to differentiate. PMID:21248070

The development of colon innervation in trisomy 16 mice and Hirschsprungs disease

PubMed Central

Li, Ji Cheng; Mi, Kai Hong; Zhou, Ji Lin; Busch, LC; Kuhnel, W

2001-01-01

AIM: To study the colon innervation of trisomy 16 mouse, an animal model for Down’s syndrome, and the expression of protein gene product 9.5 (PGP 9.5) in the stenosed segment of colon in Hirschsprungs disease (HD). METHODS: Trisomy 16 mouse breeding; cytogenetic analysis of trisomy 16 mice; and PGP 9.5 immunohistochemistry of colons of trisomy 16 mice and HD were carried out. RESULTS: Compared with their normal littermates, the nervous system of colon in trisomy 16 mice was abnormally developed. There existed developmental delay of muscular plexuses of colon, no submucosal plexus was found in the colon, and there was 5 mm aganglionic bowel aparting from the anus in trisomy 16 mice. The mesentery nerve fibers were as well developed as shown in their normal littermates. Abundant proliferation of PGP 9.5 positive nerve fibers was evealed in the stenosed segment of HD colon. CONCLUSION: Trisomy 16 mice could serve as an animal model for Hirschsprung’s disease for aganglionic bowel in the distal part of colon. Abundant proliferation of PGP 9.5 positive fibers resulted from extrinsic nerve compensation, since no ganglionic cells were observed in the stenosed segment of the colon in HD. HD has a genetic tendency. PMID:11819726

Analysis of experience-regulated transcriptome and imprintome during critical periods of mouse visual system development reveals spatiotemporal dynamics.

PubMed

Hsu, Chi-Lin; Chou, Chih-Hsuan; Huang, Shih-Chuan; Lin, Chia-Yi; Lin, Meng-Ying; Tung, Chun-Che; Lin, Chun-Yen; Lai, Ivan Pochou; Zou, Yan-Fang; Youngson, Neil A; Lin, Shau-Ping; Yang, Chang-Hao; Chen, Shih-Kuo; Gau, Susan Shur-Fen; Huang, Hsien-Sung

2018-03-15

Visual system development is light-experience dependent, which strongly implicates epigenetic mechanisms in light-regulated maturation. Among many epigenetic processes, genomic imprinting is an epigenetic mechanism through which monoallelic gene expression occurs in a parent-of-origin-specific manner. It is unknown if genomic imprinting contributes to visual system development. We profiled the transcriptome and imprintome during critical periods of mouse visual system development under normal- and dark-rearing conditions using B6/CAST F1 hybrid mice. We identified experience-regulated, isoform-specific and brain-region-specific imprinted genes. We also found imprinted microRNAs were predominantly clustered into the Dlk1-Dio3 imprinted locus with light experience affecting some imprinted miRNA expression. Our findings provide the first comprehensive analysis of light-experience regulation of the transcriptome and imprintome during critical periods of visual system development. Our results may contribute to therapeutic strategies for visual impairments and circadian rhythm disorders resulting from a dysfunctional imprintome.

Human endometrial cell coculture reduces the endocrine disruptor toxicity on mouse embryo development

PubMed Central

2012-01-01

Backgrounds Previous studies suggested that endocrine disruptors (ED) are toxic on preimplantation embryos and inhibit development of embryos in vitro culture. However, information about the toxicity of endocrine disruptors on preimplantation development of embryo in human reproductive environment is lacking. Methods Bisphenol A (BPA) and Aroclor 1254 (polychlorinated biphenyls) were used as endocrine disruptors in this study. Mouse 2-cell embryos were cultured in medium alone or vehicle or co-cultured with human endometrial epithelial layers in increasing ED concentrations. Results At 72 hours the percentage of normal blastocyst were decreased by ED in a dose-dependent manner while the co-culture system significantly enhanced the rate and reduced the toxicity of endocrine disruptors on the embryonic development in vitro. Conclusions In conclusion, although EDs have the toxic effect on embryo development, the co-culture with human endometrial cell reduced the preimplantation embryo from it thereby making human reproductive environment protective to preimplantation embryo from the toxicity of endocrine disruptors. PMID:22546201

New preservation method for mouse spermatozoa without freezing.

PubMed

Van Thuan, Nguyen; Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

2005-02-01

The objective of this study was to investigate the preservation of spermatozoa in a simple medium without freezing and to examine the effects of the preserved sperm on fertilization and development after injection into mature mouse oocytes. Mouse spermatozoa were collected from two caudae epididymides of mature B6D2F1 males and stored under various conditions: 1) in KSOMaa medium (potassium simplex optimized medium with amino acids) supplemented with 0, 1, or 4 mg/ml BSA and held at room temperature (RT, 27 degrees C); 2) in KSOMaa medium containing 4 mg/ml BSA (KSOM-BSA) and held at 4 degrees C, RT, or 37 degrees C (CO2 incubator); 3) in KSOM-BSA with osmolarity ranging from 271 to 2000 mOsmol, adjusted by addition of NaCl and held at 4 degrees C; and 4) a two-step preservation system consisting of storage in 800 mOsmol KSOM-BSA for 1 wk at RT followed by storage at -20 degrees C. Preservation of mouse spermatozoa at 4 degrees C in a medium with high osmolarity (700-1000 mOsmol) resulted in the highest frequency of live births after intracytoplasmic sperm injection (ICSI) into mature oocytes. The optimal conditions for preservation of mouse spermatozoa were 800 mOsmol KSOM containing 4 mg/ml BSA and a holding temperature of 4 degrees C. More than 40% of oocytes injected with sperm heads stored under these conditions for 2 mo developed to the morula/blastocyst stage in vitro and 39% of the embryos developed to term after transfer to recipient mice. Our results also indicate that mouse spermatozoa can be stored in 800 mOsmol KSOM-BSA medium at RT for 1 wk and then at -20 degrees C for up to 3 mo and retain their competence for ICSI. These new preservation methods permit extended conservation of viable spermatozoa that are capable of supporting normal embryonic development and the live birth of healthy offspring after ICSI.

The Ras/Raf signaling pathway is required for progression of mouse embryos through the two-cell stage.

PubMed Central

Yamauchi, N; Kiessling, A A; Cooper, G M

1994-01-01

We have used microinjection of antisense oligonucleotides, monoclonal antibody, and the dominant negative Ras N-17 mutant to interfere with Ras expression and function in mouse oocytes and early embryos. Microinjection of either ras antisense oligonucleotides or anti-Ras monoclonal antibody Y13-259 did not affect normal progression of oocytes through meiosis and arrest at metaphase II. However, microinjection of fertilized eggs with constructs expressing Ras N-17 inhibited subsequent development through the two-cell stage. The inhibitory effect of Ras N-17 was overcome by simultaneous injection of a plasmid expressing an active raf oncogene, indicating that it resulted from interference with the Ras/Raf signaling pathway. In contrast to the inhibition of two-cell embryo development resulting from microinjection of pronuclear stage eggs, microinjection of late two-cell embryos with Ras N-17 expression constructs did not affect subsequent cleavages and development to morulae and blastocysts. It thus appears that the Ras/Raf signaling pathway, presumably activated by autocrine growth factor stimulation, is specifically required at the two-cell stage, which is the time of transition between maternal and embryonic gene expression in mouse embryos. Images PMID:7935384

A reporter model to visualize imprinting stability at the Dlk1 locus during mouse development and in pluripotent cells.

PubMed

Swanzey, Emily; Stadtfeld, Matthias

2016-11-15

Genomic imprinting results in the monoallelic expression of genes that encode important regulators of growth and proliferation. Dysregulation of imprinted genes, such as those within the Dlk1-Dio3 locus, is associated with developmental syndromes and specific diseases. Our ability to interrogate causes of imprinting instability has been hindered by the absence of suitable model systems. Here, we describe a Dlk1 knock-in reporter mouse that enables single-cell visualization of allele-specific expression and prospective isolation of cells, simultaneously. We show that this 'imprinting reporter mouse' can be used to detect tissue-specific Dlk1 expression patterns in developing embryos. We also apply this system to pluripotent cell culture and demonstrate that it faithfully indicates DNA methylation changes induced upon cellular reprogramming. Finally, the reporter system reveals the role of elevated oxygen levels in eroding imprinted Dlk1 expression during prolonged culture and in vitro differentiation. The possibility to study allele-specific expression in different contexts makes our reporter system a useful tool to dissect the regulation of genomic imprinting in normal development and disease. © 2016. Published by The Company of Biologists Ltd.

Cocaine-and Amphetamine Regulated Transcript (CART) Peptide Is Expressed in Precursor Cells and Somatotropes of the Mouse Pituitary Gland

PubMed Central

Mortensen, Amanda H.

2016-01-01

Cocaine-and Amphetamine Regulated Transcript (CART) peptide is expressed in the brain, endocrine and neuroendocrine systems and secreted into the serum. It is thought to play a role in regulation of hypothalamic pituitary functions. Here we report a spatial and temporal analysis of Cart expression in the pituitaries of adult and developing normal and mutant mice with hypopituitarism. We found that Prop1 is not necessary for initiation of Cart expression in the fetal pituitary at e14.5, but it is required indirectly for maintenance of Cart expression in the postnatal anterior pituitary gland. Pou1f1 deficiency has no effect on Cart expression before or after birth. There is no 1:1 correspondence between CART and any particular cell type. In neonates, CART is detected primarily in non-proliferating, POU1F1-positive cells. CART is also found in some cells that express TSH and GH suggesting a correspondence with committed progenitors of the POU1F1 lineage. In summary, we have characterized the normal temporal and cell specific expression of CART in mouse development and demonstrate that postnatal CART expression in the pituitary gland requires PROP1. PMID:27685990

Atrial natriuretic peptide synthesis in atrial tumors of transgenic mice.

PubMed

Gardner, D G; Camargo, M J; Behringer, R R; Brinster, R L; Baxter, J D; Atlas, S A; Laragh, J H; Deschepper, C F

1992-04-01

Transgenic mice harboring a chimeric gene linking mouse protamine 1 5'-flanking sequence to the coding sequence of the simian virus 40 T-antigen develop spontaneous rhabdomyosarcomas of the right atria. The presence of the tumors is accompanied by dramatic elevations in plasma atrial natriuretic peptide (ANP) immunoreactivity (1,698 +/- 993 vs. 60 +/- 18 fmol/ml for controls) and hematocrit (56 +/- 8 vs. 51 +/- 2 for controls). The immunoreactive ANP (irANP) present in the tumors is similar in size to irANP found in normal mouse atria. ANP mRNA transcripts present in the tumors also appear to be very similar in overall size and 5'-termini to those produced in normal cardiac tissue. Microscopically, the tumors are composed of a disorganized array of densely packed abnormal-appearing cells. Immunocytochemistry and in situ hybridization analysis reveal considerable heterogeneity in ANP gene expression. ANP peptide and mRNA are detectable throughout the parenchyma of the tumors, but absolute levels of expression vary widely among different cells in the population. These tumors represent a potentially valuable model for the study of inappropriate ANP secretion and may provide a tissue source for the development of an ANP-producing atrial cell line.

Longitudinal in vivo muscle function analysis of the DMSXL mouse model of myotonic dystrophy type 1.

PubMed

Decostre, Valérie; Vignaud, Alban; Matot, Béatrice; Huguet, Aline; Ledoux, Isabelle; Bertil, Emilie; Gjata, Bernard; Carlier, Pierre G; Gourdon, Geneviève; Hogrel, Jean-Yves

2013-12-01

Myotonic dystrophy is the most common adult muscle dystrophy. In view of emerging therapies, which use animal models as a proof of principle, the development of reliable outcome measures for in vivo longitudinal study of mouse skeletal muscle function is becoming crucial. To satisfy this need, we have developed a device to measure ankle dorsi- and plantarflexion torque in rodents. We present an in vivo 8-month longitudinal study of the contractile properties of the skeletal muscles of the DMSXL mouse model of myotonic dystrophy type 1. Between 4 and 12 months of age, we observed a reduction in muscle strength in the ankle dorsi- and plantarflexors of DMSXL compared to control mice although the strength per muscle cross-section was normal. Mild steady myotonia but no abnormal muscle fatigue was also observed in the DMSXL mice. Magnetic resonance imaging and histological analysis performed at the end of the study showed respectively reduced muscle cross-section area and smaller muscle fibre diameter in DMSXL mice. In conclusion, our study demonstrates the feasibility of carrying out longitudinal in vivo studies of muscle function over several months in a mouse model of myotonic dystrophy confirming the feasibility of this method to test preclinical therapeutics. Copyright © 2013 Elsevier B.V. All rights reserved.

Skeletal Characterization of the Fgfr3 Mouse Model of Achondroplasia Using Micro-CT and MRI Volumetric Imaging.

PubMed

Shazeeb, Mohammed Salman; Cox, Megan K; Gupta, Anurag; Tang, Wen; Singh, Kuldeep; Pryce, Cynthia T; Fogle, Robert; Mu, Ying; Weber, William D; Bangari, Dinesh S; Ying, Xiaoyou; Sabbagh, Yves

2018-01-11

Achondroplasia, the most common form of dwarfism, affects more than a quarter million people worldwide and remains an unmet medical need. Achondroplasia is caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene which results in over-activation of the receptor, interfering with normal skeletal development leading to disproportional short stature. Multiple mouse models have been generated to study achondroplasia. The characterization of these preclinical models has been primarily done with 2D measurements. In this study, we explored the transgenic model expressing mouse Fgfr3 containing the achondroplasia mutation G380R under the Col2 promoter (Ach). Survival and growth rate of the Ach mice were reduced compared to wild-type (WT) littermates. Axial skeletal defects and abnormalities of the sternebrae and vertebrae were observed in the Ach mice. Further evaluation of the Ach mouse model was performed by developing 3D parameters from micro-computed tomography (micro-CT) and magnetic resonance imaging (MRI). The 3-week-old mice showed greater differences between the Ach and WT groups compared to the 6-week-old mice for all parameters. Deeper understanding of skeletal abnormalities of this model will help guide future studies for evaluating novel and effective therapeutic approaches for the treatment of achondroplasia.

Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells.

PubMed

Lv, Lei; Zhang, Tianwei; Yi, Qiyi; Huang, Yun; Wang, Zheng; Hou, Heli; Zhang, Huan; Zheng, Wei; Hao, Qiaomei; Guo, Zongyou; Cooke, Howard J; Shi, Qinghua

2012-08-01

Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.

Cloning and expression of sheep DNA methyltransferase 1 and its development-specific isoform.

PubMed

Taylor, Jane; Moore, Hannah; Beaujean, Nathalie; Gardner, John; Wilmut, Ian; Meehan, Richard; Young, Lorraine

2009-05-01

Unlike the mouse embryo, where loss of DNA methylation in the embryonic nucleus leaves cleavage stage embryos globally hypomethylated, sheep preimplantation embryos retain high levels of methylation until the blastocyst stage. We have cloned and sequenced sheep Dnmt1 and found it to be highly conserved with both the human and mouse homologues. Furthermore, we observed that the transcript normally expressed in adult somatic tissues is highly abundant in sheep oocytes. Throughout sheep preimplantation development the protein is retained in the cytoplasm whereas Dnmt1 transcript production declines after the embryonic genome activation at the 8-16 cell stage. Attempts to clone oocyte-specific 5' regions of Dnmt1, known to be present in the mouse and human gene, were unsuccessful. However, a novel ovine Dnmt1 exon, theoretically encoding 13 amino acids, was found to be expressed in sheep oocytes, preimplantation embryos and early fetal lineages, but not in the adult tissue. RNAi-mediated knockdown of this novel transcript resulted in embryonic developmental arrest at the late morula stage, suggesting an essential role for this isoform in sheep blastocyst formation. (c) 2008 Wiley-Liss, Inc.

An atlas of the prenatal mouse brain: gestational day 14.

PubMed

Schambra, U B; Silver, J; Lauder, J M

1991-11-01

A prenatal atlas of the mouse brain is presently unavailable and is needed for studies of normal and abnormal development, using techniques including immunocytochemistry and in situ hybridization. This atlas will be especially useful for researchers studying transgenic and mutant mice. This collection of photomicrographs and corresponding drawings of Gestational Day (GD) 14 mouse brain sections is an excerpt from a larger atlas encompassing GD 12-18. In composing this atlas, available published studies on the developing rodent brain were consulted to aid in the detailed labeling of embryonic brain structures. C57Bl/6J mice were mated for 1 h, and the presence of a copulation plug was designated as GD 0. GD 14 embryos were perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer and embedded in paraffin. Serial sections (10 microns thickness) were cut through whole heads in sagittal and horizontal planes. They were stained with hematoxylin and eosin and photographed. Magnifications were 43X and 31X for the horizontal and sagittal sections, respectively. Photographs were traced and line drawings prepared using an Adobe Illustrator on a Macintosh computer.

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.

Conditional N-WASP knockout in mouse brain implicates actin cytoskeleton regulation in hydrocephalus pathology.

PubMed

Jain, Neeraj; Lim, Lee Wei; Tan, Wei Ting; George, Bhawana; Makeyev, Eugene; Thanabalu, Thirumaran

2014-04-01

Cerebrospinal fluid (CSF) is produced by the choroid plexus and moved by multi-ciliated ependymal cells through the ventricular system of the vertebrate brain. Defects in the ependymal layer functionality are a common cause of hydrocephalus. N-WASP (Neural-Wiskott Aldrich Syndrome Protein) is a brain-enriched regulator of actin cytoskeleton and N-WASP knockout caused embryonic lethality in mice with neural tube and cardiac abnormalities. To shed light on the role of N-WASP in mouse brain development, we generated N-WASP conditional knockout mouse model N-WASP(fl/fl); Nestin-Cre (NKO-Nes). NKO-Nes mice were born with Mendelian ratios but exhibited reduced growth characteristics compared to their littermates containing functional N-WASP alleles. Importantly, all NKO-Nes mice developed cranial deformities due to excessive CSF accumulation and did not survive past weaning. Coronal brain sections of these animals revealed dilated lateral ventricles, defects in ciliogenesis, loss of ependymal layer integrity, reduced thickness of cerebral cortex and aqueductal stenosis. Immunostaining for N-cadherin suggests that ependymal integrity in NKO-Nes mice is lost as compared to normal morphology in the wild-type controls. Moreover, scanning electron microscopy and immunofluorescence analyses of coronal brain sections with anti-acetylated tubulin antibodies revealed the absence of cilia in ventricular walls of NKO-Nes mice indicative of ciliogenesis defects. N-WASP deficiency does not lead to altered expression of N-WASP regulatory proteins, Fyn and Cdc42, which have been previously implicated in hydrocephalus pathology. Taken together, our results suggest that N-WASP plays a critical role in normal brain development and implicate actin cytoskeleton regulation as a vulnerable axis frequently deregulated in hydrocephalus. Copyright © 2014 Elsevier Inc. All rights reserved.

Development, validation and characterization of a novel mouse model of Adynamic Bone Disease (ABD).

PubMed

Ng, Adeline H; Willett, Thomas L; Alman, Benjamin A; Grynpas, Marc D

2014-11-01

The etiology of Adynamic Bone Disease (ABD) is poorly understood but the hallmark of ABD is a lack of bone turnover. ABD occurs in renal osteodystrophy (ROD) and is suspected to occur in elderly patients on long-term anti-resorptive therapy. A major clinical concern of ABD is diminished bone quality and an increased fracture risk. To our knowledge, experimental animal models for ABD other than ROD-ABD have not been developed or studied. The objectives of this study were to develop a mouse model of ABD without the complications of renal ablation, and to characterize changes in bone quality in ABD relative to controls. To re-create the adynamic bone condition, 4-month old female Col2.3Δtk mice were treated with ganciclovir to specifically ablate osteoblasts, and pamidronate was used to inhibit osteoclastic resorption. Four groups of animals were used to characterize bone quality in ABD: Normal bone controls, No Formation controls, No Resorption controls, and an Adynamic group. After a 6-week treatment period, the animals were sacrificed and the bones were harvested for analyses. Bone quality assessments were conducted using established techniques including bone histology, quantitative backscattered electron imaging (qBEI), dual energy X-ray absorptiometry (DXA), microcomputed tomography (microCT), and biomechanical testing. Histomorphometry confirmed osteoblast-related hallmarks of ABD in our mouse model. Bone formation was near complete suppression in the No Formation and Adynamic specimens. Inhibition of bone resorption in the Adynamic group was confirmed by tartrate-resistant acid phosphatase (TRAP) stain. Normal bone mineral density and architecture were maintained in the Adynamic group, whereas the No Formation group showed a reduction in bone mineral content and trabecular thickness relative to the Adynamic group. As expected, the No Formation group had a more hypomineralized profile and the Adynamic group had a higher mean mineralization profile that is similar to suppressed bone turnover in human. This data confirms successful replication of the adynamic bone condition in a mouse without the complication of renal ablation. Our approach is the first model of ABD that uses pharmacological manipulation in a transgenic mouse to mimic the bone cellular dynamics observed in the human ABD condition. We plan to use our mouse model to investigate the adynamic bone condition in aging and to study changes to bone quality and fracture risk as a consequence of over-suppressed bone turnover. Copyright © 2014 Elsevier Inc. All rights reserved.

Autosomal dominant frontonasal dysplasia (atypical Greig syndrome): Lessons from the Xt mutant mouse

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

Cunningham, M.L.; Nunes, M.E.

1994-09-01

Greig syndrome is the autosomal dominant association of mild hypertelorism, variable polysyndactyly, and normal intelligence. Several families have been found to have translocations or deletions of 7p13 interrupting the normal expression of GLI3 (a zinc finger, DNA binding, transcription repressor). Recently, a mutation in the mouse homologue of GLI3 was found in the extra-toes mutant mouse (Xt). The phenotypic features of this mouse model include mild hypertelorism, postaxial polydactyly of the forelimbs, preaxial polydactyly of the hindlimbs, and variable tibial hemimelia. The homozygous mutant Xt/Xt have severe frontonasal dysplasia (FND), polysyndactyly of fore-and hindlimbs and invariable tibial hemimelia. We havemore » recently evaluated a child with severe (type D) frontonasal dysplasia, fifth finger camptodactyly, preaxial polydactyly of one foot, and ispilateral tibial hemimelia. His father was born with a bifid nose, broad columnella, broad feet, and a two centimeter leg length discrepancy. The paternal grandmother of the proband is phenotypically normal; however, her fraternal twin died at birth with severe facial anomalies. The paternal great-grandmother of the proband is phenotypically normal however her niece was born with moderate ocular hypertelorism. This pedigree is suggestive of an autosomal dominant form of frontonasal dysplasia with variable expressivity. The phenotypic features of our case more closely resemble the Xt mouse than the previously defined features of Greig syndrome in humans. This suggests that a mutation in GLI3 may be responsible for FND in this family. We are currently using polymorphic dinucleotide repeat markers flanking GLI3 in a attempt to demonstrate linkage in this pedigree. Demonstration of a GLI3 mutation in this family would broaden our view of the spectrum of phenotypes possible in Greig syndrome and could provide insight into genotype/phenotype correlation in FND.« less

Rapid and simple method for in vivo ex utero development of mouse embryo explants.

PubMed

Gonçalves, André B; Thorsteinsdóttir, Sólveig; Deries, Marianne

2016-01-01

The in utero development of mammals drastically reduces the accessibility of the mammalian embryo and therefore limits the range of experimental manipulation that can be done to study functions of genes or signaling pathways during embryo development. Over the past decades, tissue and organ-like culture methods have been developed with the intention of reproducing in vivo situations. Developing accessible and simple techniques to study and manipulate embryos is an everlasting challenge. Herein, we describe a reliable and quick technique to culture mid-gestation explanted mouse embryos on top of a floating membrane filter in a defined medium. Viability of the cultured tissues was assessed by apoptosis and proliferation analysis showing that cell proliferation is normal and there is only a slight increase in apoptosis after 12h of culture compared to embryos developing in utero. Moreover, differentiation and morphogenesis proceed normally as assessed by 3D imaging of the transformation of the myotome into deep back muscles. Not only does muscle cell differentiation occur as expected, but so do extracellular matrix organization and the characteristic splitting of the myotome into the three epaxial muscle groups. Our culture method allows for the culture and manipulation of mammalian embryo explants in a very efficient way, and it permits the manipulation of in vivo developmental events in a controlled environment. Explants grown under these ex utero conditions simulate real developmental events that occur in utero. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Kv7 channels are upregulated during striatal neuron development and promote maturation of human iPSC-derived neurons.

PubMed

Telezhkin, Vsevolod; Straccia, Marco; Yarova, Polina; Pardo, Monica; Yung, Sun; Vinh, Ngoc-Nga; Hancock, Jane M; Barriga, Gerardo Garcia-Diaz; Brown, David A; Rosser, Anne E; Brown, Jonathan T; Canals, Josep M; Randall, Andrew D; Allen, Nicholas D; Kemp, Paul J

2018-05-24

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.

Anatomy and Histology of the Human and Murine Prostate.

PubMed

Ittmann, Michael

2018-05-01

The human and murine prostate glands have similar functional roles in the generation of seminal fluid to assist in reproduction. There are significant differences in the anatomy and histology of murine and human prostate and knowledge of the normal anatomy and histology of the murine prostate is essential to interpreting changes in genetically engineered mouse models. In this review, the normal anatomy and histology of both human and mouse prostate will be described. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

PubMed

Liu, Bigang; Gong, Shuai; Li, Qiuhui; Chen, Xin; Moore, John; Suraneni, Mahipal V; Badeaux, Mark D; Jeter, Collene R; Shen, Jianjun; Mehmood, Rashid; Fan, Qingxia; Tang, Dean G

2017-08-08

This project was undertaken to address a critical cancer biology question: Is overexpression of the pluripotency molecule Nanog sufficient to initiate tumor development in a somatic tissue? Nanog1 is critical for the self-renewal and pluripotency of ES cells, and its retrotransposed homolog, NanogP8 is preferentially expressed in somatic cancer cells. Our work has shown that shRNA-mediated knockdown of NanogP8 in prostate, breast, and colon cancer cells inhibits tumor regeneration whereas inducible overexpression of NanogP8 promotes cancer stem cell phenotypes and properties. To address the key unanswered question whether tissue-specific overexpression of NanogP8 is sufficient to promote tumor development in vivo , we generated a NanogP8 transgenic mouse model, in which the ARR 2 PB promoter was used to drive NanogP8 cDNA. Surprisingly, the ARR 2 PB-NanogP8 transgenic mice were viable, developed normally, and did not form spontaneous tumors in >2 years. Also, both wild type and ARR 2 PB-NanogP8 transgenic mice responded similarly to castration and regeneration and castrated ARR 2 PB-NanogP8 transgenic mice also did not develop tumors. By crossing the ARR 2 PB-NanogP8 transgenic mice with ARR 2 PB-Myc (i.e., Hi-Myc) mice, we found that the double transgenic (i.e., ARR 2 PB-NanogP8; Hi-Myc) mice showed similar tumor incidence and histology to the Hi-Myc mice. Interestingly, however, we observed white dots in the ventral lobes of the double transgenic prostates, which were characterized as overgrown ductules/buds featured by crowded atypical Nanog-expressing luminal cells. Taken together, our present work demonstrates that transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

RNA-seq based transcriptomic map reveals new insights into mouse salivary gland development and maturation.

PubMed

Gluck, Christian; Min, Sangwon; Oyelakin, Akinsola; Smalley, Kirsten; Sinha, Satrajit; Romano, Rose-Anne

2016-11-16

Mouse models have served a valuable role in deciphering various facets of Salivary Gland (SG) biology, from normal developmental programs to diseased states. To facilitate such studies, gene expression profiling maps have been generated for various stages of SG organogenesis. However these prior studies fall short of capturing the transcriptional complexity due to the limited scope of gene-centric microarray-based technology. Compared to microarray, RNA-sequencing (RNA-seq) offers unbiased detection of novel transcripts, broader dynamic range and high specificity and sensitivity for detection of genes, transcripts, and differential gene expression. Although RNA-seq data, particularly under the auspices of the ENCODE project, have covered a large number of biological specimens, studies on the SG have been lacking. To better appreciate the wide spectrum of gene expression profiles, we isolated RNA from mouse submandibular salivary glands at different embryonic and adult stages. In parallel, we processed RNA-seq data for 24 organs and tissues obtained from the mouse ENCODE consortium and calculated the average gene expression values. To identify molecular players and pathways likely to be relevant for SG biology, we performed functional gene enrichment analysis, network construction and hierarchal clustering of the RNA-seq datasets obtained from different stages of SG development and maturation, and other mouse organs and tissues. Our bioinformatics-based data analysis not only reaffirmed known modulators of SG morphogenesis but revealed novel transcription factors and signaling pathways unique to mouse SG biology and function. Finally we demonstrated that the unique SG gene signature obtained from our mouse studies is also well conserved and can demarcate features of the human SG transcriptome that is different from other tissues. Our RNA-seq based Atlas has revealed a high-resolution cartographic view of the dynamic transcriptomic landscape of the mouse SG at various stages. These RNA-seq datasets will complement pre-existing microarray based datasets, including the Salivary Gland Molecular Anatomy Project by offering a broader systems-biology based perspective rather than the classical gene-centric view. Ultimately such resources will be valuable in providing a useful toolkit to better understand how the diverse cell population of the SG are organized and controlled during development and differentiation.

Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain.

PubMed

Ye, Xin; Smallwood, Philip; Nathans, Jeremy

2011-01-01

The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here, we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (Ndp(AP)). In the CNS, Ndp(AP) expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of Ndp(AP) expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, Ndp(AP) expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea. Copyright © 2010 Elsevier B.V. All rights reserved.

Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain

PubMed Central

Ye, Xin; Smallwood, Philip; Nathans, Jeremy

2011-01-01

The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (NdpAP). In the CNS, NdpAP expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of NdpAP expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, NdpAP expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea. PMID:21055480

Both nuclear and cytoplasmic components are defective in oocytes of the B6.Y(TIR) sex-reversed female mouse.

PubMed

Amleh, A; Smith, L; Chen, H; Taketo, T

2000-03-15

In the mammalian gonadal primordium, activation of the Sry gene on the Y chromosome initiates a cascade of genetic events leading to testicular organization whereas its absence results in ovarian differentiation. An exception occurs when the Y chromosome of Mus musculus domesticus from Tirano, Italy (Y(TIR)), is placed on the C57BL/6J (B6) genetic background. The B6.Y(TIR) progeny develop only ovaries or ovotestes despite Sry transcription in fetal life. Consequently, the XY offspring with bilateral ovaries develop into apparently normal females, but their eggs fail to develop after fertilization. Our previous studies have shown that the primary cause of infertility can be attributed to oocytes rather than their surrounding somatic cells in the XY ovary. This study attempted to identify the defects in oocytes from the B6.Y(TIR) female mouse. We examined the developmental potential of embryos from XY and XX females after exchanging their nuclear components by microsurgery following in vitro maturation and fertilization. The results suggest that both nuclear and cytoplasmic components are defective in oocytes from XY females. In the XY fetal ovary, most germ cells entered meiosis and their autosomes appeared to synapse normally while the X and Y chromosomes remained unpaired during meiotic prophase. This lack of X-Y pairing probably caused aneuploidy in some secondary oocytes following in vitro maturation. However, normal numbers of chromosomes in the rest of the secondary oocytes indicate that aneuploidy alone can not explain the nuclear defect in oocytes. Copyright 2000 Academic Press.

Hair and skin sterols in normal mice and those with deficient dehydrosterol reductase (DHCR7), the enzyme associated with Smith-Lemli-Opitz syndrome.

PubMed

Serra, Montserrat; Matabosch, Xavier; Ying, Lee; Watson, Gordon; Shackleton, Cedric

2010-11-01

Our recent studies have focused on cholesterol synthesis in mouse models for 7-dehydrosterolreductase (DHCR7) deficiency, also known as Smith-Lemli-Opitz syndrome. Investigations of such mutants have relied on tissue and blood levels of the cholesterol precursor 7-dehydrocholesterol (7DHC) and its 8-dehydro isomer. In this investigation by gas chromatography/mass spectrometry (GC/MS) we have identified and quantified cholesterol and its precursors (7DHC, desmosterol, lathosterol, lanosterol and cholest-7,24-dien-3β-ol) in mouse hair. The components were characterized and their concentrations were compared to those found in mouse skin and serum. Hair appeared unique in that desmosterol was a major sterol component, almost matching in concentration cholesterol itself. In DHCR7 deficient mice, dehydrodesmosterol (DHD) was the dominant hair Δ(7) sterol. Mutant mouse hair had much higher concentrations of 7-dehydrosterols relative to cholesterol than did serum or tissue at all ages studied. The 7DHC/C ratio in hair was typically about sevenfold the value in serum or skin and the DHD/D ratio was 100× that of the serum 7DHC/C ratio. Mutant mice compensate for their DHCR7 deficiency with maturity, and the tissue and blood 7DHC/C become close to normal. That hair retains high relative concentrations of the dehydro precursors suggests that the apparent up-regulation of Dhcr7 seen in liver is slower to develop at the site of hair cholesterol synthesis. Copyright © 2010 Elsevier Ltd. All rights reserved.

Characterization of Cancer Stem-Like Cells Derived from Mouse Induced Pluripotent Stem Cells Transformed by Tumor-Derived Extracellular Vesicles

PubMed Central

Yan, Ting; Mizutani, Akifumi; Chen, Ling; Takaki, Mai; Hiramoto, Yuki; Matsuda, Shuichi; Shigehiro, Tsukasa; Kasai, Tomonari; Kudoh, Takayuki; Murakami, Hiroshi; Masuda, Junko; Hendrix, Mary J. C.; Strizzi, Luigi; Salomon, David S.; Fu, Li; Seno, Masaharu

2014-01-01

Several studies have shown that cancer niche can perform an active role in the regulation of tumor cell maintenance and progression through extracellular vesicles-based intercellular communication. However, it has not been reported whether this vesicle-mediated communication affects the malignant transformation of normal stem cells/progenitors. We have previously reported that the conditioned medium derived from the mouse Lewis Lung Carcinoma (LLC) cell line can convert mouse induced pluripotent stem cells (miPSCs) into cancer stem cells (CSCs), indicating that normal stem cells when placed in an aberrant microenvironment can give rise to functionally active CSCs. Here, we focused on the contribution of tumor-derived extracellular vesicles (tEVs) that are secreted from LLC cells to induce the transformation of miPSCs into CSCs. We isolated tEVs from the conditioned medium of LLC cells, and then the differentiating miPSCs were exposed to tEVs for 4 weeks. The resultant tEV treated cells (miPS-LLCev) expressed Nanog and Oct3/4 proteins comparable to miPSCs. The frequency of sphere formation of the miPS-LLCev cells in suspension culture indicated that the self-renewal capacity of the miPS-LLCev cells was significant. When the miPS-LLCev cells were subcutaneously transplanted into Balb/c nude mice, malignant liposarcomas with extensive angiogenesis developed. miPS-LLCevPT and miPS-LLCevDT, the cells established from primary site and disseminated liposarcomas, respectively, showed their capacities to self-renew and differentiate into adipocytes and endothelial cells. Moreover, we confirmed the secondary liposarcoma development when these cells were transplanted. Taken together, these results indicate that miPS-LLCev cells possess CSC properties. Thus, our current study provides the first evidence that tEVs have the potential to induce CSC properties in normal tissue stem cells/progenitors. PMID:25057308

Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia.

PubMed

Mižíková, Ivana; Ruiz-Camp, Jordi; Steenbock, Heiko; Madurga, Alicia; Vadász, István; Herold, Susanne; Mayer, Konstantin; Seeger, Werner; Brinckmann, Jürgen; Morty, Rory E

2015-06-01

Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking of collagen and elastin, which imparts stability and functionality to the ECM. During aberrant late lung development in bronchopulmonary dysplasia (BPD) patients and animal models of BPD, alveolarization is blocked, and the function of ECM cross-linking enzymes is deregulated, suggesting that perturbed ECM cross-linking may impact alveolarization. In a hyperoxia (85% O2)-based mouse model of BPD, blunted alveolarization was accompanied by alterations to lung collagen and elastin levels and cross-linking. Total collagen levels were increased (by 63%). The abundance of dihydroxylysinonorleucine collagen cross-links and the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio were increased by 11 and 18%, respectively, suggestive of a profibrotic state. In contrast, insoluble elastin levels and the abundance of the elastin cross-links desmosine and isodesmosine in insoluble elastin were decreased by 35, 30, and 21%, respectively. The lung collagen-to-elastin ratio was threefold increased. Treatment of hyperoxia-exposed newborn mice with the lysyl oxidase inhibitor β-aminopropionitrile partially restored normal collagen levels, normalized the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio, partially normalized desmosine and isodesmosine cross-links in insoluble elastin, and partially restored elastin foci structure in the developing septa. However, β-aminopropionitrile administration concomitant with hyperoxia exposure did not improve alveolarization, evident from unchanged alveolar surface area and alveoli number, and worsened septal thickening (increased by 12%). These data demonstrate that collagen and elastin cross-linking are perturbed during the arrested alveolarization of developing mouse lungs exposed to hyperoxia. Copyright © 2015 the American Physiological Society.

In vitro suppression of spontaneous erythrocyte autoimmune responses with lymphocytes activated with concanavalin A

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

Ramshaw, I.A.; Woodsworth, M.; Eidinger, D.

1979-01-01

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to BrMRBC. Mitogenic concentrations of Con A prevented themore » development of both the PFC and T/sub DTH/ responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and T/sub DTH/ response against BrMRBC.« less

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution.

PubMed

Denoyer, Delphine; Pearson, Helen B; Clatworthy, Sharnel A S; Smith, Zoe M; Francis, Paul S; Llanos, Roxana M; Volitakis, Irene; Phillips, Wayne A; Meggyesy, Peter M; Masaldan, Shashank; Cater, Michael A

2016-06-14

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.

Noninvasive bioluminescence imaging of normal and spontaneously transformed prostate tissue in mice.

PubMed

Lyons, Scott K; Lim, Ed; Clermont, Anne O; Dusich, Joan; Zhu, Lingyun; Campbell, Kenneth D; Coffee, Richard J; Grass, David S; Hunter, John; Purchio, Tony; Jenkins, Darlene

2006-05-01

Several transgenic mouse models of prostate cancer have been developed recently that are able to recapitulate many key biological features of the human condition. It would, therefore, be desirable to employ these models to test the efficacy of new therapeutics before clinical trial; however, the variable onset and non-visible nature of prostate tumor development limit their use for such applications. We now report the generation of a transgenic reporter mouse that should obviate these limitations by enabling noninvasive in vivo bioluminescence imaging of normal and spontaneously transformed prostate tissue in the mouse. We used an 11-kb fragment of the human prostate-specific antigen (PSA) promoter to achieve specific and robust expression of firefly luciferase in the prostate glands of transgenic mice. Ex vivo bioluminescence imaging and in situ hybridization analysis confirmed that luciferase expression was restricted to the epithelium in all four lobes of the prostate. We also show that PSA-Luc mice exhibit decreased but readily detectable levels of in vivo bioluminescence over extended time periods following androgen ablation. These results suggest that this reporter should enable in vivo imaging of both androgen-dependent and androgen-independent prostate tumor models. As proof-of-principle, we show that we could noninvasively image SV40 T antigen-induced prostate tumorigenesis in mice with PSA-Luc. Furthermore, we show that our noninvasive imaging strategy can be successfully used to image tumor response to androgen ablation in transgenic mice and, as a result, that we can rapidly identify individual animals capable of sustaining tumor growth in the absence of androgen.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution

PubMed Central

Denoyer, Delphine; Pearson, Helen B.; Clatworthy, Sharnel A.S.; Smith, Zoe M.; Francis, Paul S.; Llanos, Roxana M.; Volitakis, Irene; Phillips, Wayne A.; Meggyesy, Peter M.; Masaldan, Shashank; Cater, Michael A.

2016-01-01

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed. PMID:27175597

Tumour and normal tissue radiobiology in mouse models: how close are mice to mini-humans?

PubMed

Koontz, Bridget F; Verhaegen, Frank; De Ruysscher, Dirk

2017-01-01

Animal modelling is essential to the study of radiobiology and the advancement of clinical radiation oncology by providing preclinical data. Mouse models in particular have been highly utilized in the study of both tumour and normal tissue radiobiology because of their cost effectiveness and versatility. Technology has significantly advanced in preclinical radiation techniques to allow highly conformal image-guided irradiation of small animals in an effort to mimic human treatment capabilities. However, the biological and physical limitations of animal modelling should be recognized and considered when interpreting preclinical radiotherapy (RT) studies. Murine tumour and normal tissue radioresponse has been shown to vary from human cellular and molecular pathways. Small animal irradiation techniques utilize different anatomical boundaries and may have different physical properties than human RT. This review addresses the difference between the human condition and mouse models and discusses possible strategies for future refinement of murine models of cancer and radiation for the benefit of both basic radiobiology and clinical translation.

Tumour and normal tissue radiobiology in mouse models: how close are mice to mini-humans?

PubMed Central

Verhaegen, Frank; De Ruysscher, Dirk

2017-01-01

Animal modelling is essential to the study of radiobiology and the advancement of clinical radiation oncology by providing preclinical data. Mouse models in particular have been highly utilized in the study of both tumour and normal tissue radiobiology because of their cost effectiveness and versatility. Technology has significantly advanced in preclinical radiation techniques to allow highly conformal image-guided irradiation of small animals in an effort to mimic human treatment capabilities. However, the biological and physical limitations of animal modelling should be recognized and considered when interpreting preclinical radiotherapy (RT) studies. Murine tumour and normal tissue radioresponse has been shown to vary from human cellular and molecular pathways. Small animal irradiation techniques utilize different anatomical boundaries and may have different physical properties than human RT. This review addresses the difference between the human condition and mouse models and discusses possible strategies for future refinement of murine models of cancer and radiation for the benefit of both basic radiobiology and clinical translation. PMID:27612010

Monitoring p53 by MDM2 and MDMX is required for endocrine pancreas development and function in a spatio-temporal manner.

PubMed

Zhang, Yiwei; Zeng, Shelya X; Hao, Qian; Lu, Hua

2017-03-01

Although p53 is not essential for normal embryonic development, it plays a pivotal role in many biological and pathological processes, including cell fate determination-dependent and independent events and diseases. The expression and activity of p53 largely depend on its two biological inhibitors, MDM2 and MDMX, which have been shown to form a complex in order to tightly control p53 to an undetectable level during early stages of embryonic development. However, more delicate studies using conditional gene-modification mouse models show that MDM2 and MDMX may function separately or synergistically on p53 regulation during later stages of embryonic development and adulthood in a cell and tissue-specific manner. Here, we report the role of the MDM2/MDMX-p53 pathway in pancreatic islet morphogenesis and functional maintenance, using mouse lines with specific deletion of MDM2 or MDMX in pancreatic endocrine progenitor cells. Interestingly, deletion of MDM2 results in defects of embryonic endocrine pancreas development, followed by neonatal hyperglycemia and lethality, by inducing pancreatic progenitor cell apoptosis and inhibiting cell proliferation. However, unlike MDM2-knockout animals, mice lacking MDMX in endocrine progenitor cells develop normally. But, surprisingly, the survival rate of adult MDMX-knockout mice drastically declines compared to control mice, as blockage of neonatal development of endocrine pancreas by inhibition of cell proliferation and subsequent islet dysfunction and hyperglycemia eventually lead to type 1 diabetes-like disease with advanced diabetic nephropathy. As expected, both MDM2 and MDMX deletion-caused pancreatic defects are completely rescued by loss of p53, verifying the crucial role of the MDM2 and/or MDMX in regulating p53 in a spatio-temporal manner during the development, functional maintenance, and related disease progress of endocrine pancreas. Also, our study suggests a possible mouse model of advanced diabetic nephropathy, which is complementary to other established diabetic models and perhaps useful for the development of anti-diabetes therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

High-resolution detection of 13C multiplets from the conscious mouse brain by ex vivo NMR spectroscopy

PubMed Central

Marin-Valencia, Isaac; Good, Levi B.; Ma, Qian; Jeffrey, F. Mark; Malloy, Craig R.; Pascual, Juan M.

2011-01-01

Glucose readily supplies the brain with the majority of carbon needed to sustain neurotransmitter production and utilization., The rate of brain glucose metabolism can be computed using 13C nuclear magnetic resonance (NMR) spectroscopy by detecting changes in 13C contents of products generated by cerebral metabolism. As previously observed, scalar coupling between adjacent 13C carbons (multiplets) can provide additional information to 13C contents for the computation of metabolic rates. Most NMR studies have been conducted in large animals (often under anesthesia) because the mass of the target organ is a limiting factor for NMR. Yet, despite the challengingly small size of the mouse brain, NMR studies are highly desirable because the mouse constitutes a common animal model for human neurological disorders. We have developed a method for the ex vivo resolution of NMR multiplets arising from the brain of an awake mouse after the infusion of [1,6-13C2]glucose. NMR spectra obtained by this method display favorable signal-to-noise ratios. With this protocol, the 13C multiplets of glutamate, glutamine, GABA and aspartate achieved steady state after 150 min. The method enables the accurate resolution of multiplets over time in the awake mouse brain. We anticipate that this method can be broadly applicable to compute brain fluxes in normal and transgenic mouse models of neurological disorders. PMID:21946227

Enteric oxalate elimination is induced and oxalate is normalized in a mouse model of primary hyperoxaluria following intestinal colonization with Oxalobacter

PubMed Central

Gjymishka, Altin; Salido, Eduardo C.; Allison, Milton J.; Freel, Robert W.

2011-01-01

Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691–698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals. PMID:21163900

Subchronic Arsenic Exposure Induces Anxiety-Like Behaviors in Normal Mice and Enhances Depression-Like Behaviors in the Chemically Induced Mouse Model of Depression

PubMed Central

Chang, Chia-Yu; Guo, How-Ran; Tsai, Wan-Chen; Yang, Kai-Lin; Lin, Li-Chuan

2015-01-01

Accumulating evidence implicates that subchronic arsenic exposure causes cerebral neurodegeneration leading to behavioral disturbances relevant to psychiatric disorders. However, there is still little information regarding the influence of subchronic exposure to arsenic-contaminated drinking water on mood disorders and its underlying mechanisms in the cerebral prefrontal cortex. The aim of this study is to assess the effects of subchronic arsenic exposure (10 mg/LAs2O3 in drinking water) on the anxiety- and depression-like behaviors in normal mice and in the chemically induced mouse model of depression by reserpine pretreatment. Our findings demonstrated that 4 weeks of arsenic exposure enhance anxiety-like behaviors on elevated plus maze (EPM) and open field test (OFT) in normal mice, and 8 weeks of arsenic exposure augment depression-like behaviors on tail suspension test (TST) and forced swimming test (FST) in the reserpine pretreated mice. In summary, in this present study, we demonstrated that subchronic arsenic exposure induces only the anxiety-like behaviors in normal mice and enhances the depression-like behaviors in the reserpine induced mouse model of depression, in which the cerebral prefrontal cortex BDNF-TrkB signaling pathway is involved. We also found that eight weeks of subchronic arsenic exposure are needed to enhance the depression-like behaviors in the mouse model of depression. These findings imply that arsenic could be an enhancer of depressive symptoms for those patients who already had the attribute of depression. PMID:26114099

Estrogen Responsiveness of the TFIID Subunit TAF4B in the Normal Mouse Ovary and in Ovarian Tumors1

PubMed Central

Wardell, Jennifer R.; Hodgkinson, Kendra M.; Binder, April K.; Seymour, Kimberly A.; Korach, Kenneth S.; Vanderhyden, Barbara C.; Freiman, Richard N.

2013-01-01

ABSTRACT Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation. PMID:24068106

Estrogen responsiveness of the TFIID subunit TAF4B in the normal mouse ovary and in ovarian tumors.

PubMed

Wardell, Jennifer R; Hodgkinson, Kendra M; Binder, April K; Seymour, Kimberly A; Korach, Kenneth S; Vanderhyden, Barbara C; Freiman, Richard N

2013-11-01

Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation.

Subchronic Arsenic Exposure Induces Anxiety-Like Behaviors in Normal Mice and Enhances Depression-Like Behaviors in the Chemically Induced Mouse Model of Depression.

PubMed

Chang, Chia-Yu; Guo, How-Ran; Tsai, Wan-Chen; Yang, Kai-Lin; Lin, Li-Chuan; Cheng, Tain-Junn; Chuu, Jiunn-Jye

2015-01-01

Accumulating evidence implicates that subchronic arsenic exposure causes cerebral neurodegeneration leading to behavioral disturbances relevant to psychiatric disorders. However, there is still little information regarding the influence of subchronic exposure to arsenic-contaminated drinking water on mood disorders and its underlying mechanisms in the cerebral prefrontal cortex. The aim of this study is to assess the effects of subchronic arsenic exposure (10 mg/LAs2O3 in drinking water) on the anxiety- and depression-like behaviors in normal mice and in the chemically induced mouse model of depression by reserpine pretreatment. Our findings demonstrated that 4 weeks of arsenic exposure enhance anxiety-like behaviors on elevated plus maze (EPM) and open field test (OFT) in normal mice, and 8 weeks of arsenic exposure augment depression-like behaviors on tail suspension test (TST) and forced swimming test (FST) in the reserpine pretreated mice. In summary, in this present study, we demonstrated that subchronic arsenic exposure induces only the anxiety-like behaviors in normal mice and enhances the depression-like behaviors in the reserpine induced mouse model of depression, in which the cerebral prefrontal cortex BDNF-TrkB signaling pathway is involved. We also found that eight weeks of subchronic arsenic exposure are needed to enhance the depression-like behaviors in the mouse model of depression. These findings imply that arsenic could be an enhancer of depressive symptoms for those patients who already had the attribute of depression.

Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

PubMed

Bol, S A; van Steeg, H; van Oostrom, C T; Tates, A D; Vrieling, H; de Groot, A J; Mullenders, L H; van Zeeland, A A; Jansen, J G

1999-05-01

The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.

Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

PubMed Central

Kobayashi, Dione T.; Olson, Rory J.; Sly, Laurel; Swanson, Chad J.; Chung, Brett; Naryshkin, Nikolai; Narasimhan, Jana; Bhattacharyya, Anuradha; Mullenix, Michael; Chen, Karen S.

2011-01-01

Objectives Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA. Methods A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed. Results The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA. Conclusions This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts. PMID:21904622

Follicular growth and atresia in the mouse

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

Oakberg, E. F.

1978-01-01

Follicles were classified on the basis of number of layers of follicle cells, presence and degree of development of the zona pellucida, and presence of an antrum. Formation of an antrum in follicles with less than 7-8 cell layers and/or presence of necrotic cells was considered indicative of degeneration. When classified in this manner, the data suggest that follicles and their contained oocytes are committed to either normal development or atresia by the time a third layer of granulosa cells is formed.

Identification of Pro-Differentiation p53 Target Genes and Evaluation of Expression in Normal and Malignant Mammary Gland

DTIC Science & Technology

2009-04-01

progenitor cells ’ ability to survive under non-attachment culture conditions, which is composed of primary mammoshpere and secondary mammosphere. Both... cell lineages in early mouse development depend on SOX2 function. Genes Dev, 17: 126-140, 2003 8. Chambers, I., Colby, D., Robertson, M., Nichols, J...Rivett, D., Jones, K., and Dalton, S. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development , 132: 885-896

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

NASA Technical Reports Server (NTRS)

Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

1996-01-01

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

Coordinated maturational regulation of PHEX and renal phosphate transport inhibitory activity: evidence for the pathophysiological role of PHEX in X-linked hypophosphatemia.

PubMed

Nesbitt, T; Fujiwara, I; Thomas, R; Xiao, Z S; Quarles, L D; Drezner, M K

1999-12-01

The mechanism by which inactivating mutations of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) cause X-linked hypophosphatemia remains unknown. However, recent reports suggest errant PHEX activity in osteoblasts may fail to inactivate a phosphaturic factor produced by these cells. To test this possibility, we examined coordinated maturational expression of PHEX and production of phosphate transport inhibitory activity in osteoblasts from normal and hyp-mice. We assessed the inhibitory activity in conditioned medium by examining the effects on opossum kidney cell phosphate transport and osteoblast PHEX expression by reverse transcriptase-polymerase chain reaction during a 17-day maturational period. Inhibitory activity increased as a function of osteoblast maturational stage, with no activity after 3 days and persistent activity by 6 days of culture. More significantly, equal phosphate transport inhibitory activity in conditioned medium from normal and hyp-mouse osteoblasts (control 1.90 +/- 0.12, normal 1.48 +/- 0.10, hyp 1.45 +/- 0.04 nmol/mg of protein/minute) was observed at 6 days. However, by 10 days hyp-mouse osteoblasts exhibited greater inhibitory activity than controls, and by 17 days the difference in phosphate transport inhibition maximized (control 2.08 +/- 0.09, normal 1.88 +/- 0.06, hyp 1.58 +/- 0.06 nmol/mg of protein/minute). Concurrently, we observed absent PHEX expression in normal osteoblasts after 3 days, limited production at 6 days, and significant production by day 10 of culture, while hyp-mouse osteoblasts exhibited limited PHEX activity secondary to an inactivating mutation. The data suggest that the presence of inactivating PHEX mutations results in the enhanced renal phosphate transport inhibitory activity exhibited by hyp-mouse osteoblasts.

Effects of deer velvet extract from Formosan sika deer on the embryonic development and anti-oxidative enzymes mRNA expression in mouse embryos.

PubMed

Cheng, Shih-Lin; Lai, Yi-Ling; Lee, Ming-Che; Shen, Perng-Chih; Liu, Shyh-Shyan; Liu, Bing-Tsan

2014-07-03

The deer velvet or its extracts has been widely used in clinic. It has been used in promoting reproductive performances and treating of oxidation and aging process. The aim of this study is to investigate the effects of velvet extract from Formosan sika deer (Formosan sika deer; Cervus nippon taiouanus, FSD) velvet on mouse embryonic development and anti-oxidant ability in vitro. Mouse 4-cells embryos were divided into 16 groups for 72 h in vitro incubation. The embryonic development stages and morphology were evaluated every 12h in experimental period. The quantitative real time PCR was used to measure the CuZn-SOD, GPx and CAT mRNA expression of the blastocysts. The 4-cells embryos of hydrogen peroxide (HP) groups did not continue developing after oxidant stress challenged. The blastocyst developmental rate (90.0-90.4%, P>0.05) and normal morphological rate (84.4-85.1%, P>0.05) of the 1% and 2% DV extract groups were similar to those in the control group (90.7% and 88.8%, respectively). The embryos challenged by HP (5, 10 and 25 μM) and subsequently incubated in mHTF medium with 1% and 2% of deer velvet (DV) extracts were able to continue development; the blastocyst developmental rate of these groups were similar to that in the control group. The relative mRNA expression of the focused anti-oxidative enzymes in the mouse embryos did not significantly differ among the designed DV treatment groups (P>0.05). The FSD velvet extract in adequate concentration could promote anti-oxidative enzymes mRNA expression followed the challenge of hydrogen peroxide, relieve the mouse embryo under oxidative stress, and maintain the blastocyst developmental ability in vitro. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Activation of Wnt signalling promotes development of dysplasia in Barrett's oesophagus.

PubMed

Moyes, Lisa H; McEwan, Hamish; Radulescu, Sorina; Pawlikowski, Jeff; Lamm, Catherine G; Nixon, Colin; Sansom, Owen J; Going, James J; Fullarton, Grant M; Adams, Peter D

2012-09-01

Barrett's oesophagus is a precursor of oesophageal adenocarcinoma, via intestinal metaplasia and dysplasia. Risk of cancer increases substantially with dysplasia, particularly high-grade dysplasia. Thus, there is a clinical need to identify and treat patients with early-stage disease (metaplasia and low-grade dysplasia) that are at high risk of cancer. Activated Wnt signalling is critical for normal intestinal development and homeostasis, but less so for oesophageal development. Therefore, we asked whether abnormally increased Wnt signalling contributes to the development of Barrett's oesophagus (intestinal metaplasia) and/or dysplasia. Forty patients with Barrett's metaplasia, dysplasia or adenocarcinoma underwent endoscopy and biopsy. Mice with tamoxifen- and β-naphthoflavone-induced expression of activated β-catenin were used to up-regulate Wnt signalling in mouse oesophagus. Immunohistochemistry of β-catenin, Ki67, a panel of Wnt target genes, and markers of intestinal metaplasia was performed on human and mouse tissues. In human tissues, expression of nuclear activated β-catenin was found in dysplasia, particularly high grade. Barrett's metaplasia did not show high levels of activated β-catenin. Up-regulation of Ki67 and Wnt target genes was also mostly associated with high-grade dysplasia. Aberrant activation of Wnt signalling in mouse oesophagus caused marked tissue disorganization with features of dysplasia, but only selected molecular indicators of metaplasia. Based on these results in human tissues and a mouse model, we conclude that abnormal activation of Wnt signalling likely plays only a minor role in initiation of Barrett's metaplasia but a more critical role in progression to dysplasia. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes.

PubMed

Khosla, S; Dean, W; Brown, D; Reik, W; Feil, R

2001-03-01

Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 + FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 + FCS fetuses. No alterations were detected for the imprinted gene MEST: Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development.

Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia

PubMed Central

Roberts, Edward W.; Deonarine, Andrew; Jones, James O.; Denton, Alice E.; Feig, Christine; Lyons, Scott K.; Espeli, Marion; Kraman, Matthew; McKenna, Brendan; Wells, Richard J.B.; Zhao, Qi; Caballero, Otavia L.; Larder, Rachel; Coll, Anthony P.; O’Rahilly, Stephen; Brindle, Kevin M.; Teichmann, Sarah A.; Tuveson, David A.

2013-01-01

Fibroblast activation protein-α (FAP) identifies stromal cells of mesenchymal origin in human cancers and chronic inflammatory lesions. In mouse models of cancer, they have been shown to be immune suppressive, but studies of their occurrence and function in normal tissues have been limited. With a transgenic mouse line permitting the bioluminescent imaging of FAP+ cells, we find that they reside in most tissues of the adult mouse. FAP+ cells from three sites, skeletal muscle, adipose tissue, and pancreas, have highly similar transcriptomes, suggesting a shared lineage. FAP+ cells of skeletal muscle are the major local source of follistatin, and in bone marrow they express Cxcl12 and KitL. Experimental ablation of these cells causes loss of muscle mass and a reduction of B-lymphopoiesis and erythropoiesis, revealing their essential functions in maintaining normal muscle mass and hematopoiesis, respectively. Remarkably, these cells are altered at these sites in transplantable and spontaneous mouse models of cancer-induced cachexia and anemia. Thus, the FAP+ stromal cell may have roles in two adverse consequences of cancer: their acquisition by tumors may cause failure of immunosurveillance, and their alteration in normal tissues contributes to the paraneoplastic syndromes of cachexia and anemia. PMID:23712428

Gab3-deficient mice exhibit normal development and hematopoiesis and are immunocompetent.

PubMed

Seiffert, Martina; Custodio, Joseph M; Wolf, Ingrid; Harkey, Michael; Liu, Yan; Blattman, Joseph N; Greenberg, Philip D; Rohrschneider, Larry R

2003-04-01

Gab proteins are intracellular scaffolding and docking molecules involved in signaling pathways mediated by various growth factor, cytokine, or antigen receptors. Gab3 has been shown to act downstream of the macrophage colony-stimulating factor receptor, c-Fms, and to be important for macrophage differentiation. To analyze the physiological role of Gab3, we used homologous recombination to generate mice deficient in Gab3. Gab3(-/-) mice develop normally, are visually indistinguishable from their wild-type littermates, and are healthy and fertile. To obtain a detailed expression pattern of Gab3, we generated Gab3-specific monoclonal antibodies. Immunoblotting revealed a predominant expression of Gab3 in lymphocytes and bone marrow-derived macrophages. However, detailed analysis demonstrated that hematopoiesis in mice lacking Gab3 is not impaired and that macrophages develop in normal numbers and exhibit normal function. The lack of Gab3 expression during macrophage differentiation is not compensated for by increased levels of Gab1 or Gab2 mRNA. Furthermore, Gab3-deficient mice have no major immune deficiency in T- and B-lymphocyte responses to protein antigens or during viral infection. In addition, allergic responses in Gab3-deficient mice appeared to be normal. Together, these data demonstrate that loss of Gab3 does not result in detectable defects in normal mouse development, hematopoiesis, or immune system function.

Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia

PubMed Central

Huang, Peng; Schier, Alexander F.

2009-01-01

Summary Cilia have been implicated in Hedgehog (Hh) and Wnt signaling in mouse but not in Drosophila. To determine whether the role of cilia is conserved in zebrafish, we generated maternal-zygotic (MZ) oval (ovl; ift88) mutants that lack all cilia. MZovl mutants display normal canonical and non-canonical Wnt signaling but show defects in Hh signaling. As in mouse, zebrafish cilia are required to mediate the activities of Hh, Ptc, Smo and PKA. However, in contrast to mouse Ift88 mutants, which show a dramatic reduction in Hh signaling, zebrafish MZovl mutants display dampened, but expanded, Hh pathway activity. This activity is largely due to gli1, the expression of which is fully dependent on Hh signaling in mouse but not in zebrafish. These results reveal a conserved requirement for cilia in transducing the activity of upstream regulators of Hh signaling but distinct phenotypic effects due to differential regulation and differing roles of transcriptional mediators. PMID:19700616

Perioperative haemostatic management of haemophilic mice using normal mouse plasma.

PubMed

Tatsumi, K; Ohashi, K; Kanegae, K; Shim, I K; Okano, T

2013-11-01

Intense haemostatic interventions are required to avoid bleeding complications when surgical procedures are performed on haemophilia patients. The objective of this study was to establish an appropriate protocol for perioperative haemostatic management of haemophilic mice. We assessed the prophylactic haemostatic effects of normal mouse plasma (NMP) on haemophilia B (HB) mice for both a skin flap procedure and a laparotomy. When 500 μL of NMP was administered to the mice, plasma factor IX (FIX:C) levels peaked at 15.1% immediately after intravenous (IV) administration, at 6.1% 2 h after intraperitoneal (IP) administration and at 2.7% 6 h after subcutaneous administration. Administering 500 μL of NMP via IP or IV 30 min in advance enabled the skin flap procedure to be performed safely without any complications. After the laparotomy procedure, several mice in the IP administration group exhibited lethal bleeding, but all mice survived in the IV administration group. Anti-mouse FIX inhibitors did not develop, even after repetitive administrations of NMP. However, human FIX concentrates, especially plasma-derived concentrates, elicited the anti-human FIX inhibitors. The results show that administering 500 μL of NMP via IV or IP 30 min in advance enables surgical procedures to be safely performed on HB mice, and that IV administration is more desirable than IP if the procedure requires opening of the abdominal wall. © 2013 John Wiley & Sons Ltd.

Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia

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

Vandenberg, P.; Khillan, J.S.; Prockop, D.J.

A minigene version of the human gene for type II procollagen (COL2AI) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro{alpha} chains that associate with normal pro{alpha} chains and thereby cause degradation of the shortened and normal pro{alpha} chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing themore » minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro{alpha}1(II) chains that were disulfide-linked to normal mouse pro{alpha}1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.« less

B lymphocytes not required for progression from insulitis to diabetes in non-obese diabetic mice.

PubMed

Charlton, B; Zhang, M D; Slattery, R M

2001-12-01

Previous studies have implicated B lymphocytes in the pathogenesis of diabetes in the non-obese diabetic (NOD) mouse. While it is clear that B lymphocytes are necessary, it has not been clear at which stage of disease they play a role; early, late or both. To clarify when B lymphocytes are needed, T lymphocytes were transferred from 5-week-old NOD female mice to age-matched NOD/severe combined immunodeficiency (SCID) recipient mice. NOD/SCID mice, which lack functionally mature T and B lymphocytes, do not normally develop insulitis or insulin-dependent diabetes melitus (IDDM). The NOD/SCID mice that received purified T lymphocytes from 5-week-old NOD mice subsequently developed insulitis and diabetes even though they did not have detectable B lymphocytes. This suggests that while B lymphocytes may be essential for an initial priming event they are not requisite for disease progression in the NOD mouse.

The histone demethylase Jarid1b ensures faithful mouse development by protecting developmental genes from aberrant H3K4me3.

PubMed

Albert, Mareike; Schmitz, Sandra U; Kooistra, Susanne M; Malatesta, Martina; Morales Torres, Cristina; Rekling, Jens C; Johansen, Jens V; Abarrategui, Iratxe; Helin, Kristian

2013-04-01

Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications.

The Histone Demethylase Jarid1b Ensures Faithful Mouse Development by Protecting Developmental Genes from Aberrant H3K4me3

PubMed Central

Kooistra, Susanne M.; Malatesta, Martina; Morales Torres, Cristina; Rekling, Jens C.; Johansen, Jens V.; Abarrategui, Iratxe; Helin, Kristian

2013-01-01

Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications. PMID:23637629

Knockout of RNA Binding Protein MSI2 Impairs Follicle Development in the Mouse Ovary: Characterization of MSI1 and MSI2 during Folliculogenesis

PubMed Central

Sutherland, Jessie M.; Sobinoff, Alexander P.; Gunter, Kara M.; Fraser, Barbara A.; Pye, Victoria; Bernstein, Ilana R.; Boon, Evan; Siddall, Nicole A.; De Andres, Luisa I.; Hime, Gary R.; Holt, Janet E.; Graf, Thomas; McLaughlin, Eileen A.

2015-01-01

Characterizing the mechanisms underlying follicle development in the ovary is crucial to understanding female fertility and is an area of increasing research interest. The RNA binding protein Musashi is essential for post-transcriptional regulation of oocyte maturation in Xenopus and is expressed during ovarian development in Drosophila. In mammals Musashi is important for spermatogenesis and male fertility, but its role in the ovary has yet to be characterized. In this study we determined the expression of mammalian Musashi proteins MSI1 and MSI2 during mouse folliculogenesis, and through the use of a MSI2-specific knockout mouse model we identified that MSI2 is essential for normal follicle development. Time-course characterization of MSI1 and MSI2 revealed distinct differences in steady-state mRNA levels and protein expression/localization at important developmental time-points during folliculogenesis. Using a gene-trap mouse model that inactivates Msi2, we observed a significant decrease in ovarian mass, and change in follicle-stage composition due to developmental blocking of antral stage follicles and pre-antral follicle loss through atresia. We also confirmed that hormonally stimulated Msi2-deficient mice produce significantly fewer MII oocytes (60.9% less than controls, p < 0.05). Furthermore, the majority of these oocytes are of poor viability (62.2% non-viable/apoptotic, p < 0.05), which causes a reduction in female fertility evidenced by decreased litter size in Msi2-deficient animals (33.1% reduction to controls, p < 0.05). Our findings indicate that MSI1 and MSI2 display distinct expression profiles during mammalian folliculogenesis and that MSI2 is required for pre-antral follicle development. PMID:26131972

Piperlongumine inhibits LMP1/MYC-dependent mouse B-lymphoma cells

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

Han, Seong-Su; Tompkins, Van S.; Son, Dong-Ju

2013-07-12

Highlights: •Mouse model of human Burkitt lymphoma revealed cancer inhibition by PL. •Treatment with PL led to apoptosis of malignant but not normal B cells. •PL inhibited LMP1–NF-κB–Myc-dependent target genes including p21-encoding Cdkn1a. •PL holds promise for new interventions approaches to hematologic malignancies. -- Abstract: Piperlongumine (PL), isolated from the fruit of Long pepper, Piper longum, is a cancer-inhibiting compound that selectively kills tumor cells while sparing their normal counterparts. Here we evaluated the efficacy with which PL suppresses malignant B cells derived from a newly developed, double-transgenic mouse model of human endemic Burkitt lymphoma (BL), designated mCD40-LMP1/iMyc{sup Eμ}. PLmore » inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplastic but not normal B cells. Treatment with PL resulted in downregulation of EBV-encoded LMP1, cellular Myc, constitutive NF-κB activity, and a host of LMP1-Myc-NF-κB-regulated target genes including Aurka, Bcat1, Bub1b, Ccnb1, Chek1, Fancd2, Tfrc and Xrcc6. Of note, p21{sup Cip1}-encoding Cdkn1a was suppressed independent of changes in Trp53 mRNA levels and p53 DNA-binding activity. Considering the central role of the LMP1–NF-κB–Myc axis in B-lineage neoplasia, these findings further our understanding of the mechanisms by which PL inhibits B-lymphoma and provide a preclinical rationale for the inclusion of PL in new interventions in blood cancers.« less

Early Mitochondrial Adaptations in Skeletal Muscle to Diet-Induced Obesity Are Strain Dependent and Determine Oxidative Stress and Energy Expenditure But Not Insulin Sensitivity

PubMed Central

Sena, Sandra; Sloan, Crystal; Tebbi, Ali; Han, Yong Hwan; O'Neill, Brian T.; Cooksey, Robert C.; Jones, Deborah; Holland, William L.; McClain, Donald A.; Abel, E. Dale

2012-01-01

This study sought to elucidate the relationship between skeletal muscle mitochondrial dysfunction, oxidative stress, and insulin resistance in two mouse models with differential susceptibility to diet-induced obesity. We examined the time course of mitochondrial dysfunction and insulin resistance in obesity-prone C57B and obesity-resistant FVB mouse strains in response to high-fat feeding. After 5 wk, impaired insulin-mediated glucose uptake in skeletal muscle developed in both strains in the absence of any impairment in proximal insulin signaling. Impaired mitochondrial oxidative capacity preceded the development of insulin resistant glucose uptake in C57B mice in concert with increased oxidative stress in skeletal muscle. By contrast, mitochondrial uncoupling in FVB mice, which prevented oxidative stress and increased energy expenditure, did not prevent insulin resistant glucose uptake in skeletal muscle. Preventing oxidative stress in C57B mice treated systemically with an antioxidant normalized skeletal muscle mitochondrial function but failed to normalize glucose tolerance and insulin sensitivity. Furthermore, high fat-fed uncoupling protein 3 knockout mice developed increased oxidative stress that did not worsen glucose tolerance. In the evolution of diet-induced obesity and insulin resistance, initial but divergent strain-dependent mitochondrial adaptations modulate oxidative stress and energy expenditure without influencing the onset of impaired insulin-mediated glucose uptake. PMID:22510273

Targeted disruption of glutathione peroxidase 4 (GPx4) in mouse skin epithelial cells impairs postnatal hair follicle morphogenesis that is partially rescued through inhibition of COX-2

PubMed Central

Sengupta, Aniruddha; Lichti, Ulrike F.; Carlson, Bradley A.; Cataisson, Christophe; Ryscavage, Andrew O.; Mikulec, Carol; Conrad, Marcus; Fischer, Susan M.; Hatfield, Dolph L.; Yuspa, Stuart H.

2013-01-01

Selenoproteins are essential molecules for the mammalian antioxidant network. We previously demonstrated that targeted loss of all selenoproteins in mouse epidermis disrupted skin and hair development and caused premature death. In the current study we targeted specific selenoproteins for epidermal deletion to determine whether similar phenotypes developed. Keratinocyte-specific knockout mice lacking either the glutathione peroxidase 4 (GPx4) or thioredoxin reductase 1 (TR1) gene were generated by cre-lox technology using K14-cre. TR1 knockout mice had a normal phenotype in resting skin while GPx4 loss in epidermis caused epidermal hyperplasia, dermal inflammatory infiltrate, dysmorphic hair follicles and alopecia in perinatal mice. Unlike epidermal ablation of all selenoproteins, mice ablated for GPx4 recovered after 5 weeks and had a normal lifespan. GPx1 and TR1 were upregulated in the skin and keratinocytes of GPx4 knockout mice. GPx4 deletion reduces keratinocyte adhesion in culture and increases lipid peroxidation and COX-2 levels in cultured keratinocytes and whole skin. Feeding a COX-2 inhibitor to nursing mothers partially prevents development of the abnormal skin phenotype in knockout pups. These data link the activity of cutaneous GPx4 to the regulation of COX-2 and hair follicle morphogenesis and provide insight into the function of individual selenoprotein activity in maintaining cutaneous homeostasis. PMID:23364477

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells

PubMed Central

van den Brink, Susanne C.; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A.; Martinez Arias, Alfonso

2014-01-01

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call ‘gastruloids’. PMID:25371360

Altered behavior and neural activity in conspecific cagemates co-housed with mouse models of brain disorders.

PubMed

Yang, Hyunwoo; Jung, Seungmoon; Seo, Jinsoo; Khalid, Arshi; Yoo, Jung-Seok; Park, Jihyun; Kim, Soyun; Moon, Jangsup; Lee, Soon-Tae; Jung, Keun-Hwa; Chu, Kon; Lee, Sang Kun; Jeon, Daejong

2016-09-01

The psychosocial environment is one of the major contributors of social stress. Family members or caregivers who consistently communicate with individuals with brain disorders are considered at risk for physical and mental health deterioration, possibly leading to mental disorders. However, the underlying neural mechanisms of this phenomenon remain poorly understood. To address this, we developed a social stress paradigm in which a mouse model of epilepsy or depression was housed long-term (>4weeks) with normal conspecifics. We characterized the behavioral phenotypes and electrophysiologically investigated the neural activity of conspecific cagemate mice. The cagemates exhibited deficits in behavioral tasks assessing anxiety, locomotion, learning/memory, and depression-like behavior. Furthermore, they showed severe social impairment in social behavioral tasks involving social interaction or aggression. Strikingly, behavioral dysfunction remained in the cagemates 4weeks following co-housing cessation with the mouse models. In an electrophysiological study, the cagemates showed an increased number of spikes in medial prefrontal cortex (mPFC) neurons. Our results demonstrate that conspecifics co-housed with mouse models of brain disorders develop chronic behavioral dysfunctions, and suggest a possible association between abnormal mPFC neural activity and their behavioral pathogenesis. These findings contribute to the understanding of the psychosocial and psychiatric symptoms frequently present in families or caregivers of patients with brain disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.

PubMed

van den Brink, Susanne C; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A; Martinez Arias, Alfonso

2014-11-01

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'. © 2014. Published by The Company of Biologists Ltd.

Agglutination of Mouse Erythrocytes by Eperythrozoon coccoides

PubMed Central

Iralu, Vichazelhu; Ganong, Kevin D.

1983-01-01

Erythrocytes from blood of mice infected with Eperythrozoon coccoides for 3 or 4 days agglutinated spontaneously. Washed E. coccoides particles agglutinated washed erythrocytes of uninfected mice. E. coccoides-mediated agglutination of normal mouse erythrocytes would be an excellent system for studies of bacterial adhesion. Images PMID:6832825

Tumor vessel normalization after aerobic exercise enhances chemotherapeutic efficacy.

PubMed

Schadler, Keri L; Thomas, Nicholas J; Galie, Peter A; Bhang, Dong Ha; Roby, Kerry C; Addai, Prince; Till, Jacob E; Sturgeon, Kathleen; Zaslavsky, Alexander; Chen, Christopher S; Ryeom, Sandra

2016-10-04

Targeted therapies aimed at tumor vasculature are utilized in combination with chemotherapy to improve drug delivery and efficacy after tumor vascular normalization. Tumor vessels are highly disorganized with disrupted blood flow impeding drug delivery to cancer cells. Although pharmacologic anti-angiogenic therapy can remodel and normalize tumor vessels, there is a limited window of efficacy and these drugs are associated with severe side effects necessitating alternatives for vascular normalization. Recently, moderate aerobic exercise has been shown to induce vascular normalization in mouse models. Here, we provide a mechanistic explanation for the tumor vascular normalization induced by exercise. Shear stress, the mechanical stimuli exerted on endothelial cells by blood flow, modulates vascular integrity. Increasing vascular shear stress through aerobic exercise can alter and remodel blood vessels in normal tissues. Our data in mouse models indicate that activation of calcineurin-NFAT-TSP1 signaling in endothelial cells plays a critical role in exercise-induced shear stress mediated tumor vessel remodeling. We show that moderate aerobic exercise with chemotherapy caused a significantly greater decrease in tumor growth than chemotherapy alone through improved chemotherapy delivery after tumor vascular normalization. Our work suggests that the vascular normalizing effects of aerobic exercise can be an effective chemotherapy adjuvant.

Broad AOX expression in a genetically tractable mouse model does not disturb normal physiology

PubMed Central

Szibor, Marten; Dhandapani, Praveen K.; Dufour, Eric; Holmström, Kira M.; Zhuang, Yuan; Salwig, Isabelle; Wittig, Ilka; Heidler, Juliana; Gizatullina, Zemfira; Fuchs, Helmut; Gailus-Durner, Valérie; de Angelis, Martin Hrabě; Nandania, Jatin; Velagapudi, Vidya; Wietelmann, Astrid; Rustin, Pierre; Gellerich, Frank N.; Braun, Thomas

2017-01-01

ABSTRACT Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo. PMID:28067626

Production of MPS VII mouse (Gustm(hE540A·mE536A)Sly) doubly tolerant to human and mouse β-glucuronidase

PubMed Central

Tomatsu, Shunji; Orii, Koji O.; Vogler, Carole; Grubb, Jeffrey H.; Snella, Elizabeth M.; Gutierrez, Monica; Dieter, Tatiana; Holden, Christopher C.; Sukegawa, Kazuko; Orii, Tadao; Kondo, Naomi; Sly, William S.

2006-01-01

Mucopolysaccharidosis VII (MPS VII, Sly syndrome) is an autosomal recessive lysosomal storage disease caused by β-glucuronidase (GUS) deficiency. A naturally occurring mouse model of that disease has been very useful for studying experimental approaches to therapy. However, immune responses can complicate evaluation of the long-term benefits of enzyme replacement or gene therapy delivered to adult MPS VII mice. To make this model useful for studying the long-term effectiveness and side effects of experimental therapies delivered to adult mice, we developed a new MPS VII mouse model, which is tolerant to both human and murine GUS. To achieve this, we used homologous recombination to introduce simultaneously a human cDNA transgene expressing inactive human GUS into intron 9 of the murine Gus gene and a targeted active site mutation (E536A) into the adjacent exon 10. When the heterozygote products of germline transmission were bred to homozygosity, the homozygous mice expressed no GUS enzyme activity but expressed inactive human GUS protein highly and were tolerant to immune challenge with human enzyme. Expression of the mutant murine Gus gene was reduced to about 10% of normal levels, but the inactive murine GUS enzyme also conferred tolerance to murine GUS. This MPS VII mouse model should be useful to evaluate therapeutic responses in adult mice receiving repetitive doses of enzyme or mice receiving gene therapy as adults. Heterozygotes expressed only 9.5–26% of wild-type levels of murine GUS instead of the expected 50%, indicating a dominant-negative effect of the mutant enzyme monomers on the activity of GUS tetramers in different tissues. Corrective gene therapy in this model should provide high enough levels of expression of normal GUS monomers to overcome the dominant negative effect of mutant monomers on newly synthesized GUS tetramers in most tissues. PMID:12700165

Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis

PubMed Central

2014-01-01

Background While microRNA (miRNA) expression is known to be altered in a variety of human malignancies contributing to cancer development and progression, the potential role of miRNA dysregulation in malignant mast cell disease has not been previously explored. The purpose of this study was to investigate the potential contribution of miRNA dysregulation to the biology of canine mast cell tumors (MCTs), a well-established spontaneous model of malignant mast cell disease. Methods We evaluated the miRNA expression profiles from biologically low-grade and biologically high-grade primary canine MCTs using real-time PCR-based TaqMan Low Density miRNA Arrays and performed real-time PCR to evaluate miR-9 expression in primary canine MCTs, malignant mast cell lines, and normal bone marrow-derived mast cells (BMMCs). Mouse mast cell lines and BMMCs were transduced with empty or pre-miR-9 expressing lentiviral constructs and cell proliferation, caspase 3/7 activity, and invasion were assessed. Transcriptional profiling of cells overexpressing miR-9 was performed using Affymetrix GeneChip Mouse Gene 2.0 ST arrays and real-time PCR was performed to validate changes in mRNA expression. Results Our data demonstrate that unique miRNA expression profiles correlate with the biological behavior of primary canine MCTs and that miR-9 expression is increased in biologically high grade canine MCTs and malignant cell lines compared to biologically low grade tumors and normal canine BMMCs. In transformed mouse malignant mast cell lines expressing either wild-type (C57) or activating (P815) KIT mutations and mouse BMMCs, miR-9 overexpression significantly enhanced invasion but had no effect on cell proliferation or apoptosis. Transcriptional profiling of normal mouse BMMCs and P815 cells possessing enforced miR-9 expression demonstrated dysregulation of several genes, including upregulation of CMA1, a protease involved in activation of matrix metalloproteases and extracellular matrix remodeling. Conclusions Our findings demonstrate that unique miRNA expression profiles correlate with the biological behavior of canine MCTs. Furthermore, dysregulation of miR-9 is associated with MCT metastasis potentially through the induction of an invasive phenotype, identifying a potentially novel pathway for therapeutic intervention. PMID:24517413

What underlies the diversity of brain tumors?

PubMed Central

Swartling, Fredrik J.; Hede, Sanna-Maria; Weiss, William A.

2012-01-01

Glioma and medulloblastoma represent the most commonly occurring malignant brain tumors in adults and in children respectively. Recent genomic and transcriptional approaches present a complex group of diseases, and delineate a number of molecular subgroups within tumors that share a common histopathology. Differences in cells of origin, regional niches, developmental timing and genetic events all contribute to this heterogeneity. In an attempt to recapitulate the diversity of brain tumors, an increasing array of genetically engineered mouse models (GEMMs) has been developed. These models often utilize promoters and genetic drivers from normal brain development, and can provide insight into specific cells from which these tumors originate. GEMMs show promise in both developmental biology and developmental therapeutics. This review describes numerous murine brain tumor models in the context of normal brain development, and the potential for these animals to impact brain tumor research. PMID:23085857

Kindler syndrome protein Kindlin-1 is mainly expressed in adult tissues originating from ectoderm/endoderm.

PubMed

Zhan, Jun; Yang, Mei; Zhang, Jing; Guo, YongQing; Liu, Wei; Zhang, HongQuan

2015-05-01

Mutations of integrin-interacting protein Kindlin-1 cause Kindler syndrome and deregulation of Kindlin-1 is implicated in human cancers. The Kindlin-1-related diseases are confined in limited tissue types. However, Kindlin-1 tissue distribution and the dogma that governs Kindlin-1 expression in normal human body are elusive. This study examined Kindlin-1 expression in normal human adult organs, human and mouse embryonic organs by immunohistochemical analyses. We identified a general principle that the level of Kindlin-1 expression in tissues is tightly correlated with the corresponding germ layers from which these tissues originate. We compared the expression of Kindlin-1 with Kindlin-2 and found that Kindlin-1 is highly expressed in epithelial tissues derived from ectoderm and endoderm, whereas Kindlin-2 is mainly expressed in mesoderm-derived tissues. Likewise, Kindlin-1 was also found highly expressed in endoderm/ectoderm-derived tissues in human and mouse embryos. Our findings indicate that Kindlin-1 may play an importance role in the development of endoderm/ectoderm related tissues.

An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth

PubMed Central

Saha, Nayanendu; Eissman, Moritz F.; Xu, Kai; Llerena, Carmen; Kusebauch, Ulrike; Ding, Bi-Sen; Cao, Zhongwei; Rafii, Shahin; Ernst, Matthias; Scott, Andrew M.; Nikolov, Dimitar B.; Lackmann, Martin

2016-01-01

The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance. PMID:27503072

An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth.

PubMed

Atapattu, Lakmali; Saha, Nayanendu; Chheang, Chanly; Eissman, Moritz F; Xu, Kai; Vail, Mary E; Hii, Linda; Llerena, Carmen; Liu, Zhanqi; Horvay, Katja; Abud, Helen E; Kusebauch, Ulrike; Moritz, Robert L; Ding, Bi-Sen; Cao, Zhongwei; Rafii, Shahin; Ernst, Matthias; Scott, Andrew M; Nikolov, Dimitar B; Lackmann, Martin; Janes, Peter W

2016-08-22

The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance. © 2016 Atapattu et al.

Cross-species identification of genomic drivers of squamous cell carcinoma development across preneoplastic intermediates

PubMed Central

Chitsazzadeh, Vida; Coarfa, Cristian; Drummond, Jennifer A.; Nguyen, Tri; Joseph, Aaron; Chilukuri, Suneel; Charpiot, Elizabeth; Adelmann, Charles H.; Ching, Grace; Nguyen, Tran N.; Nicholas, Courtney; Thomas, Valencia D.; Migden, Michael; MacFarlane, Deborah; Thompson, Erika; Shen, Jianjun; Takata, Yoko; McNiece, Kayla; Polansky, Maxim A.; Abbas, Hussein A.; Rajapakshe, Kimal; Gower, Adam; Spira, Avrum; Covington, Kyle R.; Xiao, Weimin; Gunaratne, Preethi; Pickering, Curtis; Frederick, Mitchell; Myers, Jeffrey N.; Shen, Li; Yao, Hui; Su, Xiaoping; Rapini, Ronald P.; Wheeler, David A.; Hawk, Ernest T.; Flores, Elsa R.; Tsai, Kenneth Y.

2016-01-01

Cutaneous squamous cell carcinoma (cuSCC) comprises 15–20% of all skin cancers, accounting for over 700,000 cases in USA annually. Most cuSCC arise in association with a distinct precancerous lesion, the actinic keratosis (AK). To identify potential targets for molecularly targeted chemoprevention, here we perform integrated cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar ultraviolet radiation-driven Hairless mouse model. We identify the major transcriptional drivers of this progression sequence, showing that the key genomic changes in cuSCC development occur in the normal skin to AK transition. Our data validate the use of this ultraviolet radiation-driven mouse cuSCC model for cross-species analysis and demonstrate that cuSCC bears deep molecular similarities to multiple carcinogen-driven SCCs from diverse sites, suggesting that cuSCC may serve as an effective, accessible model for multiple SCC types and that common treatment and prevention strategies may be feasible. PMID:27574101

Molecular architecture underlying fluid absorption by the developing inner ear

PubMed Central

Honda, Keiji; Kim, Sung Huhn; Kelly, Michael C; Burns, Joseph C; Constance, Laura; Li, Xiangming; Zhou, Fei; Hoa, Michael; Kelley, Matthew W; Morell, Robert J

2017-01-01

Mutations of SLC26A4 are a common cause of hearing loss associated with enlargement of the endolymphatic sac (EES). Slc26a4 expression in the developing mouse endolymphatic sac is required for acquisition of normal inner ear structure and function. Here, we show that the mouse endolymphatic sac absorbs fluid in an SLC26A4-dependent fashion. Fluid absorption was sensitive to ouabain and gadolinium but insensitive to benzamil, bafilomycin and S3226. Single-cell RNA-seq analysis of pre- and postnatal endolymphatic sacs demonstrates two types of differentiated cells. Early ribosome-rich cells (RRCs) have a transcriptomic signature suggesting expression and secretion of extracellular proteins, while mature RRCs express genes implicated in innate immunity. The transcriptomic signature of mitochondria-rich cells (MRCs) indicates that they mediate vectorial ion transport. We propose a molecular mechanism for resorption of NaCl by MRCs during development, and conclude that disruption of this mechanism is the root cause of hearing loss associated with EES. PMID:28994389

Metabotropic glutamate receptor 5 responses dictate differentiation of neural progenitors to NMDA-responsive cells in fragile X syndrome.

PubMed

Achuta, Venkat Swaroop; Grym, Heli; Putkonen, Noora; Louhivuori, Verna; Kärkkäinen, Virve; Koistinaho, Jari; Roybon, Laurent; Castrén, Maija L

2017-04-01

Disrupted metabotropic glutamate receptor 5 (mGluR5) signaling is implicated in many neuropsychiatric disorders, including autism spectrum disorder, found in fragile X syndrome (FXS). Here we report that intracellular calcium responses to the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) are augmented, and calcium-dependent mGluR5-mediated mechanisms alter the differentiation of neural progenitors in neurospheres derived from human induced pluripotent FXS stem cells and the brains of mouse model of FXS. Treatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) prevents an abnormal clustering of DHPG-responsive cells that are responsive to activation of ionotropic receptors in mouse FXS neurospheres. MPEP also corrects morphological defects of differentiated cells and enhanced migration of neuron-like cells in mouse FXS neurospheres. Unlike in mouse neurospheres, MPEP increases the differentiation of DHPG-responsive radial glial cells as well as the subpopulation of cells responsive to both DHPG and activation of ionotropic receptors in human neurospheres. However, MPEP normalizes the FXS-specific increase in the differentiation of cells responsive only to N-methyl-d-aspartate (NMDA) present in human neurospheres. Exposure to MPEP prevents the accumulation of intermediate basal progenitors in embryonic FXS mouse brain suggesting that rescue effects of GluR5 antagonist are progenitor type-dependent and species-specific differences of basal progenitors may modify effects of MPEP on the cortical development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419-437, 2017. © 2016 Wiley Periodicals, Inc.

Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

PubMed

Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2015-03-01

High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain

PubMed Central

Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

2013-01-01

An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain. PMID:23440889

Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain.

PubMed

Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

2013-01-01

An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain.

Injury, inflammation and the emergence of human specific genes

DTIC Science & Technology

2016-07-12

genes in circulating and resident human immune cells can be studied in mice after the transplantation and engraft- ment of human hemato- lymphoid immune...Martinek J, Strowig T, Gearty SV, Teichmann LL, et al. Development and function of human innate immune cells in a humanized mouse model. Nat Bio...normal wound repair and regeneration, we hypothesize that the preponderance of human-specific genes expressed in human inflammatory cells is commensurate

In vivo photoacoustic imaging of mouse embryos

NASA Astrophysics Data System (ADS)

Laufer, Jan; Norris, Francesca; Cleary, Jon; Zhang, Edward; Treeby, Bradley; Cox, Ben; Johnson, Peter; Scambler, Pete; Lythgoe, Mark; Beard, Paul

2012-06-01

The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.

Thalidomide Ameliorates Inflammation and Vascular Injury but Aggravates Tubular Damage in the Irradiated Mouse Kidney

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

Scharpfenecker, Marion, E-mail: m.scharpfenecker@nki.nl; Floot, Ben; Russell, Nicola S.

Purpose: The late side effects of kidney irradiation include vascular damage and fibrosis, which are promoted by an irradiation-induced inflammatory response. We therefore treated kidney-irradiated mice with the anti-inflammatory and angiogenesis-modulating drug thalidomide in an attempt to prevent the development of late normal tissue damage and radiation nephropathy in the mouse kidney. Methods and Materials: Kidneys of C57Bl/6 mice were irradiated with a single dose of 14 Gy. Starting from week 16 after irradiation, the mice were fed with thalidomide-containing chow (100 mg/kg body weight/day). Gene expression and kidney histology were analyzed at 40 weeks and blood samples at 10, 20, 30, andmore » 40 weeks after irradiation. Results: Thalidomide improved the vascular structure and vessel perfusion after irradiation, associated with a normalization of pericyte coverage. The drug also reduced infiltration of inflammatory cells but could not suppress the development of fibrosis. Irradiation-induced changes in hematocrit and blood urea nitrogen levels were not rescued by thalidomide. Moreover, thalidomide worsened tubular damage after irradiation and also negatively affected basal tubular function. Conclusions: Thalidomide improved the inflammatory and vascular side effects of kidney irradiation but could not reverse tubular toxicity, which probably prevented preservation of kidney function.« less

Diversification of intrinsic motoneuron electrical properties during normal development and botulinum toxin-induced muscle paralysis in early postnatal mice.

PubMed

Nakanishi, S T; Whelan, P J

2010-05-01

During early postnatal development, between birth and postnatal days 8-11, mice start to achieve weight-bearing locomotion. In association with the progression of weight-bearing locomotion there are presumed developmental changes in the intrinsic electrical properties of spinal -motoneurons. However, these developmental changes in the properties of -motoneuron properties have not been systematically explored in mice. Here, data are presented documenting the developmental changes of selected intrinsic motoneuron electrical properties, including statistically significant changes in action potential half-width, intrinsic excitability and diversity (quantified as coefficient of variation) of rheobase current, afterhyperpolarization half-decay time, and input resistance. In various adult mammalian preparations, the maintenance of intrinsic motoneuron electrical properties is dependent on activity and/or transmission-sensitive motoneuron-muscle interactions. In this study, we show that botulinum toxin-induced muscle paralysis led to statistically significant changes in the normal development of intrinsic motoneuron electrical properties in the postnatal mouse. This suggests that muscle activity during early neonatal life contributes to the development of normal motoneuron electrical properties.

Enhancement of thermal response of normal and malignant tissues by Corynebacterium parvum. [Mice

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

Urano, M.; Yamashita, T.; Suit, H.D.

1984-06-01

Further studies were carried out on the combined effects of Corynebacterium parvum and hyperthermia on animal tissues and cultured Chinese hamster ovary cells. Experimental animals were C3Hf/Sed mice derived from a defined flora mouse colony. Tumors were eighth-generation isotransplants of a spontaneous fibrosarcoma, FSa-II. Hyperthermia was given by immersing the mouse foot or culture flasks in the constant temperature water bath. Present experiments include thermal enhancement of C. parvum at different temperatures, effect of the agent on the kinetics of thermal resistance, and the mechanism of the thermal enhancement. The thermal enhancement by C. parvum was independent of temperature inmore » a range between 42.5 and 46.5 degrees, and it increased with decreasing temperature. The analysis of the Arrhenius plot suggested a comparable activation energy for combined treatments and for heat alone between 42.5 and 46.5 degrees. The thermal resistance developed very rapidly in both normal and tumor tissues. Systemic administration of C. parvum failed to modify the kinetics of thermal resistance. Several experiments were attempted in order to disclose the mechanism. A single injection of C. parvum-induced macrophages failed to enhance thermal response of the mouse foot, while 3 daily injections of the macrophages enhanced the response, indicating that the enhancement by C. parvum is at least partly attributed to the C. parvum-induced macrophages. Whole-body irradiation of 6 Gy and/or administration of anti-mouse T-cell serum and histamine failed to inhibit the C. parvum enhancement of thermal response. No thermal enhancement was observed for Chinese hamster ovary cells treated at 43.0 degrees in vitro with C. parvum or thiomersalate, a preservative supplemented in C. parvum, although cytotoxic effect was shown at a high concentration of thiomersalate.« less

Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues

PubMed Central

Müller, Brigitte; Ellinwood, N. M.; Lorenz, Birgit; Stieger, Knut

2018-01-01

Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB) repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30) in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months) retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL), suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted. PMID:29765300

A Rat Model for Human Ductal Carcinoma In Situ

DTIC Science & Technology

2006-09-01

complexes in early stages of mouse mammary preneoplasia. Cell Growth Differ 12:285-95. 14. Gudmundur Thordarson , Adrian V. Lee, Meghan McCarty...S.M., Thordarson , G., Collins, G., Talamantes, F., and Nandi, S. (1995) In vivo growth stimulation of collagen gel embedded normal human and mouse

The Molecular Basis of Muscular Dystrophy in the mdx Mouse: A Point Mutation

NASA Astrophysics Data System (ADS)

Sicinski, Piotr; Geng, Yan; Ryder-Cook, Allan S.; Barnard, Eric A.; Darlison, Mark G.; Barnard, Pene J.

1989-06-01

The mdx mouse is an X-linked myopathic mutant, an animal model for human Duchenne muscular dystrophy. In both mouse and man the mutations lie within the dystrophin gene, but the phenotypic differences of the disease in the two species confer much interest on the molecular basis of the mdx mutation. The complementary DNA for mouse dystrophin has been cloned, and the sequence has been used in the polymerase chain reaction to amplify normal and mdx dystrophin transcripts in the area of the mdx mutation. Sequence analysis of the amplification products showed that the mdx mouse has a single base substitution within an exon, which causes premature termination of the polypeptide chain.

Selective binding of lectins to normal and neoplastic urothelium in rat and mouse bladder carcinogenesis models.

PubMed

Zupančič, Daša; Kreft, Mateja Erdani; Romih, Rok

2014-01-01

Bladder cancer adjuvant intravesical therapy could be optimized by more selective targeting of neoplastic tissue via specific binding of lectins to plasma membrane carbohydrates. Our aim was to establish rat and mouse models of bladder carcinogenesis to investigate in vivo and ex vivo binding of selected lectins to the luminal surface of normal and neoplastic urothelium. Male rats and mice were treated with 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water and used for ex vivo and in vivo lectin binding experiments. Urinary bladder samples were also used for paraffin embedding, scanning electron microscopy and immunofluorescence labelling of uroplakins. During carcinogenesis, the structure of the urinary bladder luminal surface changed from microridges to microvilli and ropy ridges and the expression of urothelial-specific glycoproteins uroplakins was decreased. Ex vivo and in vivo lectin binding experiments gave comparable results. Jacalin (lectin from Artocarpus integrifolia) exhibited the highest selectivity for neoplastic compared to normal urothelium of rats and mice. The binding of lectin from Amaranthus caudatus decreased in rat model and increased in mouse carcinogenesis model, indicating interspecies variations of plasma membrane glycosylation. Lectin from Datura stramonium showed higher affinity for neoplastic urothelium compared to the normal in rat and mouse model. The BBN-induced animal models of bladder carcinogenesis offer a promising approach for lectin binding experiments and further lectin-mediated targeted drug delivery research. Moreover, in vivo lectin binding experiments are comparable to ex vivo experiments, which should be considered when planning and optimizing future research.

p120 catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas

PubMed Central

Hendley, Audrey M.; Provost, Elayne; Bailey, Jennifer M.; Wang, Yue J.; Cleveland, Megan H.; Blake, Danielle; Bittman, Ross W.; Roeser, Jeffrey C.; Maitra, Anirban; Reynolds, Albert B.; Leach, Steven D.

2015-01-01

The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120f/f pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development. PMID:25523391

NOGGIN IS REQUIRED FOR NORMAL LOBE PATTERNING AND DUCTAL BUDDING IN THE MOUSE PROSTATE

PubMed Central

Cook, Crist; Vezina, Chad M.; Hicks, Sarah M.; Shaw, Aubie; Yu, Min; Peterson, Richard E.; Bushman, Wade

2008-01-01

Mesenchymal expression of the BMP antagonist NOGGIN during prostate development plays a critical role in pre-natal ventral prostate development and opposes BMP4-mediated inhibition of cell proliferation during postnatal ductal development. Morphologic examination of newborn Noggin-/- male fetuses revealed genitourinary anomalies including cryptorchidism, incomplete separation of the hindgut from the urogenital sinus (UGS), absence of the ventral mesenchymal pad and a complete loss of ventral prostate (VP) budding. Examination of lobe-specific marker expression in the E14 Noggin-/- UGS rescued by transplantation under the renal capsule of a male nude mouse confirmed a complete loss of VP determination. More modest effects were observed in the other lobes, including decreased number of ductal buds in the dorsal and lateral prostates of newborn Noggin-/- males. BMP4 and BMP7 have been shown to inhibit ductal budding and outgrowth by negatively regulating epithelial cell proliferation. We show here that NOGGIN can neutralize budding inhibition by BMP4 and rescues branching morphogenesis of BMP4-exposed UGS in organ culture and show that the effects of BMP4 and NOGGIN activities converge on P63+ epithelial cells located at nascent duct tips. Together, these studies show that the BMP-NOGGIN axis regulates patterning of the ventral prostate, regulates ductal budding, and controls proliferation of P63+ epithelial cells in the nascent ducts of developing mouse prostate. PMID:18028901

Development and characterization of a mouse with profound biotinidase deficiency: a biotin-responsive neurocutaneous disorder.

PubMed

Pindolia, Kirit; Jordan, Megan; Guo, Caiying; Matthews, Nell; Mock, Donald M; Strovel, Erin; Blitzer, Miriam; Wolf, Barry

2011-02-01

Biotinidase deficiency is the primary enzymatic defect in biotin-responsive, late-onset multiple carboxylase deficiency. Untreated children with profound biotinidase deficiency usually exhibit neurological symptoms including lethargy, hypotonia, seizures, developmental delay, sensorineural hearing loss and optic atrophy; and cutaneous symptoms including skin rash, conjunctivitis and alopecia. Although the clinical features of the disorder markedly improve or are prevented with biotin supplementation, some symptoms, once they occur, such as developmental delay, hearing loss and optic atrophy, are usually irreversible. To prevent development of symptoms, the disorder is screened for in the newborn period in essentially all states and in many countries. In order to better understand many aspects of the pathophysiology of the disorder, we have developed a transgenic biotinidase-deficient mouse. The mouse has a null mutation that results in no detectable serum biotinidase activity or cross-reacting material to antibody prepared against biotinidase. When fed a biotin-deficient diet these mice develop neurological and cutaneous symptoms, carboxylase deficiency, mild hyperammonemia, and exhibit increased urinary excretion of 3-hydroxyisovaleric acid and biotin and biotin metabolites. The clinical features are reversed with biotin supplementation. This biotinidase-deficient animal can be used to study systematically many aspects of the disorder and the role of biotinidase, biotin and biocytin in normal and in enzyme-deficient states. Copyright © 2010 Elsevier Inc. All rights reserved.

Atmosphere stabilization and element recycle in an experimental mouse-algal system

NASA Technical Reports Server (NTRS)

Smernoff, David T.

1986-01-01

Life support systems based on bioregeneration rely on the control and manipulation of organisms. Experiments conducted with a gas-closed mouse-algal system designed to investigate principles of photosynthetic gas exchange focus primarily on observing gas exchange phenomena under varying algal environmental conditions and secondarily on studying element cycling through compartments of the experimental system. Inherent instabilities exit between the uptake and release of carbon dioxide CO2 and oxygen O2 by the mouse and algae. Variations in light intensity and cell density alter the photosynthetic rate of the algae and enable maintenance of physiologic concentrations of CO2 and O2. Different nitrogen sources (urea and nitrate) result in different algal assimilatory quotients (AQ). Combinations of photosynthetic rate and AQ ratio manipulations have been examined for their potential in stabilizing atmospheric gas concentrations in the gas-closed algal-mouse system. Elemental mass balances through the experimental systems compartments are being studied with the concurrent development of a mathematical simulation model. Element cycling experiments include quantification of elemental flows through system compartments and wet oxidation of system waste materials for use as an algal nutrient source. Oxidized waste products demonstrate inhibitory properties although dilution has been shown to allow normal growth.

Age dependent regulation of bone-mass and renal function by the MEPE ASARM-motif

PubMed Central

Zelenchuk, Lesya V; Hedge, Anne-Marie; Rowe, Peter S N

2015-01-01

Context Mice with null mutations in Matrix Extracellular Phosphoglycoprotein (MEPE) have increased bone mass, increased trabecular density and abnormal cancellous bone (MN-mice). These defects worsen with age and MEPE over expression induces opposite effects. Also, Genome Wide Association studies show MEPE plays a major role in bone mass. We hypothesized the conserved C-terminal MEPE ASARM-motif is chiefly responsible for regulating bone mass and trabecular structure. Design To test our theory we over expressed C-terminal ASARM-peptide in MN-mice using the Col1α1 promoter (MNAt-mice). We then compared the bone and renal phenotypes of the MNAt-mouse with the MN-mouse and the X-linked hypophosphatemic rickets mouse (HYP). The HYP mouse over expresses ASARM-peptides and is defective for the PHEX gene. Results The MN-mouse developed increased bone mass, bone strength and trabecular abnormalities that worsened markedly with age. Defects in bone formation were chiefly responsible with suppressed sclerostin and increased active β-catenin. Increased uric acid levels also suggested abnormalities in purine-metabolism and a reduced fractional excretion of uric acid signaled additional renal transport changes. The MN mouse developed a worsening hyperphosphatemia and reduced FGF23 with age. An increase in the fractional excretion of phosphate (FEP) despite the hyperphosphatemia confirms an imbalance in kidney-intestinal phosphate regulation. Also, the MN mice showed an increased creatinine clearance suggesting hyperfiltration. A reversal of the MN bone-renal phenotype changes occurred with the MNAt mice including the apparent hyperfiltration. The MNAt mice also developed localized hypomineralization, hypophosphatemia and increased FGF23. Conclusions The C-terminal ASARM-motif plays a major role in regulating bone–mass and cancellous structure as mice age. In healthy mice, the processing and release of free ASARM-peptide is chiefly responsible for preserving normal bone and renal function. Free ASARM-peptide also effects renal mineral phosphate handling by influencing FGF23 expression. These findings have implications for understanding age-dependent osteoporosis, unraveling drug-targets and developing treatments. PMID:26051469

Sexual dimorphism and the effects of the X-linked Tfm locus on hexobarbitone metabolism and action in mice.

PubMed

King, D K; Shapiro, B H

1981-09-01

1 Normal males of the testicular feminized strain of mice (Tfm) had longer hexobarbitone-induced sleeping times than females, and hepatic hexobarbitone hydroxylase activity different in that the Km was higher and the Vmax lower in the male. 2 Castration and androgen replacement studies indicated that testicular androgens were responsible for the sexual differences in drug metabolism found in this mouse strain. 3 Hepatic hexobarbitone metabolism and action were feminized in the intact, androgen-insensitive, genetically male Tfm mouse. Furthermore, hexobarbitone hydroxylase activities were less responsive to large doses of testosterone in Tfm mice than in normal males. 4 The Tfm mouse with a deficiency in androgen receptors responded to the enzyme-inductive effects of phenobarbitone and softwood bedding, indicating that these inducers do not act through the androgen receptors.

Mediation of mouse natural cytotoxic activity by tumour necrosis factor

NASA Astrophysics Data System (ADS)

Ortaldo, John R.; Mason, Llewellyn H.; Mathieson, Bonnie J.; Liang, Shu-Mei; Flick, David A.; Herberman, Ronald B.

1986-06-01

Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors1. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.

Comparative thermometric coagulation studies of plasmas from normal outbred Swiss Webster mice and persons.

PubMed

Tsang, V C; Wyatt, C R; Damian, R T

1979-06-01

The functional capabilities of a thermometric clot-timer have been demonstrated in a comparative study of human and mouse plasma coagulation. The influence of some variables on coagulation times of mouse and human plasmas were examined in activated partial thromboplastin time, one-stage prothrombin time, and Russell's viper venom time assays. Mouse plasma coagulation times were generally shorter and more reproducible than those of human plasma. Optimal assay conditions are also described.

Structure and Function of the Splice Variants of TMPRSS2-ERG, a Prevalent Genomic Alteration in Prostate Cancer

DTIC Science & Technology

2011-09-01

the ETS family of transcription factors showing diverse expression patterns in human tissues (Turner and Watson, 2008). ERG, similar to other...and adult mouse tissues . Most striking of these observations was highly selective and abundant expression of erg protein in endothelial cells of...mouse tissues . We for the first time clarified that endogenous ERG was not expressed in normal mouse prostate epithelium (Mohamed et al., 2010

Effect of human alpha 2HS glycoprotein on mouse macrophage function.

PubMed Central

Lewis, J G; André, C M

1980-01-01

alpha 2HS glycoprotein was isolated from normal adult serum. The ability of alpha 2HS glycoprotein to promote the endocytosis of radiolabelled DNA and radiolabelled latex particles by mouse macrophages was investigated. The results using both radiolabelled latex particles and radiolabelled DNA show that alpha 2HS glycoprotein enhances the ability of mouse macrophages to take up these radiolabelled substrates as compared to control cells. Images Figure 1 Figure 2 PMID:7439929

Performance evaluation of the Abbott CELL-DYN Emerald for use as a bench-top analyzer in a research setting.

PubMed

Khoo, T-L; Xiros, N; Guan, F; Orellana, D; Holst, J; Joshua, D E; Rasko, J E J

2013-08-01

The CELL-DYN Emerald is a compact bench-top hematology analyzer that can be used for a three-part white cell differential analysis. To determine its utility for analysis of human and mouse samples, we evaluated this machine against the larger CELL-DYN Sapphire and Sysmex XT2000iV hematology analyzers. 120 human (normal and abnormal) and 30 mouse (normal and abnormal) samples were analyzed on both the CELL-DYN Emerald and CELL-DYN Sapphire or Sysmex XT2000iV analyzers. For mouse samples, the CELL-DYN Emerald analyzer required manual recalibration based on the histogram populations. Analysis of the CELL-DYN Emerald showed excellent precision, within accepted ranges (white cell count CV% = 2.09%; hemoglobin CV% = 1.68%; platelets CV% = 4.13%). Linearity was excellent (R² ≥ 0.99), carryover was minimal (<1%), and overall interinstrument agreement was acceptable for both human and mouse samples. Comparison between the CELL-DYN Emerald and Sapphire analyzers for human samples or Sysmex XT2000iV analyzer for mouse samples showed excellent correlation for all parameters. The CELL-DYN Emerald was generally comparable to the larger reference analyzer for both human and mouse samples. It would be suitable for use in satellite research laboratories or as a backup system in larger laboratories. © 2012 John Wiley & Sons Ltd.

Effects of Acanthopanax senticosus on Brain Injury Induced by Simulated Spatial Radiation in Mouse Model Based on Pharmacokinetics and Comparative Proteomics

PubMed Central

Cheng, Cuilin; Baranenko, Denis; Wang, Jiaping; Li, Yongzhi; Lu, Weihong

2018-01-01

The active compounds in Acanthopanax senticosus (AS) have different pharmacokinetic characteristics in mouse models. Cmax and AUC of Acanthopanax senticosus polysaccharides (ASPS) were significantly reduced in radiation-injured mice, suggesting that the blood flow of mouse was blocked or slowed, due to the pathological state of ischemia and hypoxia, which are caused by radiation. In contrast, the ability of various metabolizing enzymes to inactivate, capacity of biofilm transport decrease, and lessening of renal blood flow accounts for radiation, resulting in the accumulation of syringin and eleutheroside E in the irradiated mouse. Therefore, there were higher pharmacokinetic parameters—AUC, MRT, and t1/2 of the two compounds in radiation-injured mouse, when compared with normal mouse. In order to investigate the intrinsic mechanism of AS on radiation injury, AS extract’s protective effects on brain, the main part of mouse that suffered from radiation, were explored. The function of AS extract in repressing expression changes of radiation response proteins in prefrontal cortex (PFC) of mouse brain included tubulin protein family (α-, β-tubulin subunits), dihydropyrimidinase-related protein 2 (CRMP2), γ-actin, 14-3-3 protein family (14-3-3ζ, ε), heat shock protein 90β (HSP90β), and enolase 2. The results demonstrated the AS extract had positive effects on nerve cells’ structure, adhesion, locomotion, fission, and phagocytosis, through regulating various action pathways, such as Hippo, phagosome, PI3K/Akt (phosphatidylinositol 3 kinase/protein kinase B), Neurotrophin, Rap1 (Ras-related protein RAP-1A), gap junction glycolysis/gluconeogenesis, and HIF-1 (Hypoxia-inducible factor 1) signaling pathways to maintain normal mouse neurological activity. All of the results indicated that AS may be a promising alternative medicine for the treatment of radiation injury in mouse brain. It would be tested that whether the bioactive ingredients of AS could be effective through the blood–brain barrier in the future. PMID:29342911

Urea impairs β cell glycolysis and insulin secretion in chronic kidney disease

PubMed Central

Koppe, Laetitia; Nyam, Elsa; Vivot, Kevin; Manning Fox, Jocelyn E.; Dai, Xiao-Qing; Nguyen, Bich N.; Attané, Camille; Moullé, Valentine S.; MacDonald, Patrick E.; Ghislain, Julien

2016-01-01

Disorders of glucose homeostasis are common in chronic kidney disease (CKD) and are associated with increased mortality, but the mechanisms of impaired insulin secretion in this disease remain unclear. Here, we tested the hypothesis that defective insulin secretion in CKD is caused by a direct effect of urea on pancreatic β cells. In a murine model in which CKD is induced by 5/6 nephrectomy (CKD mice), we observed defects in glucose-stimulated insulin secretion in vivo and in isolated islets. Similarly, insulin secretion was impaired in normal mouse and human islets that were cultured with disease-relevant concentrations of urea and in islets from normal mice treated orally with urea for 3 weeks. In CKD mouse islets as well as urea-exposed normal islets, we observed an increase in oxidative stress and protein O-GlcNAcylation. Protein O-GlcNAcylation was also observed in pancreatic sections from CKD patients. Impairment of insulin secretion in both CKD mouse and urea-exposed islets was associated with reduced glucose utilization and activity of phosphofructokinase 1 (PFK-1), which could be reversed by inhibiting O-GlcNAcylation. Inhibition of O-GlcNAcylation also restored insulin secretion in both mouse models. These results suggest that insulin secretory defects associated with CKD arise from elevated circulating levels of urea that increase islet protein O-GlcNAcylation and impair glycolysis. PMID:27525435

Comparison of animal discs used in disc research to human lumbar disc: torsion mechanics and collagen content.

PubMed

Showalter, Brent L; Beckstein, Jesse C; Martin, John T; Beattie, Elizabeth E; Espinoza Orías, Alejandro A; Schaer, Thomas P; Vresilovic, Edward J; Elliott, Dawn M

2012-07-01

Experimental measurement and normalization of in vitro disc torsion mechanics and collagen content for several animal species used in intervertebral disc research and comparing these with the human disc. To aid in the selection of appropriate animal models for disc research by measuring torsional mechanical properties and collagen content. There is lack of data and variability in testing protocols for comparing animal and human disc torsion mechanics and collagen content. Intervertebral disc torsion mechanics were measured and normalized by disc height and polar moment of inertia for 11 disc types in 8 mammalian species: the calf, pig, baboon, goat, sheep, rabbit, rat, and mouse lumbar discs, and cow, rat, and mouse caudal discs. Collagen content was measured and normalized by dry weight for the same discs except the rat and the mouse. Collagen fiber stretch in torsion was calculated using an analytical model. Measured torsion parameters varied by several orders of magnitude across the different species. After geometric normalization, only the sheep and pig discs were statistically different from human discs. Fiber stretch was found to be highly dependent on the assumed initial fiber angle. The collagen content of the discs was similar, especially in the outer annulus where only the calf and goat discs were statistically different from human. Disc collagen content did not correlate with torsion mechanics. Disc torsion mechanics are comparable with human lumbar discs in 9 of 11 disc types after normalization by geometry. The normalized torsion mechanics and collagen content of the multiple animal discs presented are useful for selecting and interpreting results for animal disc models. Structural organization of the fiber angle may explain the differences that were noted between species after geometric normalization.

Comparison of Animal Discs Used in Disc Research to Human Lumbar Disc: Torsion Mechanics and Collagen Content

PubMed Central

Showalter, Brent L.; Beckstein, Jesse C.; Martin, John T.; Beattie, Elizabeth E.; Orías, Alejandro A. Espinoza; Schaer, Thomas P.; Vresilovic, Edward J.; Elliott, Dawn M.

2012-01-01

Study Design Experimental measurement and normalization of in vitro disc torsion mechanics and collagen content for several animal species used in intervertebral disc research and comparing these to the human disc. Objective To aid in the selection of appropriate animal models for disc research by measuring torsional mechanical properties and collagen content. Summary of Background Data There is lack of data and variability in testing protocols for comparing animal and human disc torsion mechanics and collagen content. Methods Intervertebral disc torsion mechanics were measured and normalized by disc height and polar moment of inertia for 11 disc types in 8 mammalian species: the calf, pig, baboon, goat, sheep, rabbit, rat, and mouse lumbar, and cow, rat, and mouse caudal. Collagen content was measured and normalized by dry weight for the same discs except the rat and mouse. Collagen fiber stretch in torsion was calculated using an analytical model. Results Measured torsion parameters varied by several orders of magnitude across the different species. After geometric normalization, only the sheep and pig discs were statistically different from human. Fiber stretch was found to be highly dependent on the assumed initial fiber angle. The collagen content of the discs was similar, especially in the outer annulus where only the calf and goat discs were statistically different from human. Disc collagen content did not correlate with torsion mechanics. Conclusion Disc torsion mechanics are comparable to human lumbar discs in 9 of 11 disc types after normalization by geometry. The normalized torsion mechanics and collagen content of the multiple animal discs presented is useful for selecting and interpreting results for animal models of the disc. Structural composition of the disc, such as initial fiber angle, may explain the differences that were noted between species after geometric normalization. PMID:22333953

Enamelin Is Critical for Ameloblast Integrity and Enamel Ultrastructure Formation

PubMed Central

Hu, Jan C.-C.; Hu, Yuanyuan; Lu, Yuhe; Smith, Charles E.; Lertlam, Rangsiyakorn; Wright, John Timothy; Suggs, Cynthia; McKee, Marc D.; Beniash, Elia; Kabir, M. Enamul; Simmer, James P.

2014-01-01

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam−/− mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam−/− mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using β-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam−/− background did not fully recover enamel formation while a medium expresser in the Enam+/− background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation. PMID:24603688

Polymorphic Expression of a Human Superficial Bladder Tumor Antigen Defined by Mouse Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Fradet, Yves; Islam, Nazrul; Boucher, Lucie; Parent-Vaugeois, Carmen; Tardif, Marc

1987-10-01

Three mouse monoclonal antibodies (mAbs), which define a highly restricted antigen, were obtained by simultaneous immunizations with superficial papillary bladder tumor cells and mouse polyclonal serum against normal urothelium. The antigen was detected by the avidin/biotin/peroxidase method in 30/44 superficial bladder tumors (68%) but in only 4/27 infiltrating urothelial cancers (with much less intensity). No normal adult or fetal tissues tested expressed the antigen, including normal urothelium from 40 individuals, 13 of whom had a bladder tumor positive for the antigen. Only 1 of 45 nonbladder tumors showed some reactivity with one of the three mAbs. Serological tests on a large panel of human cancer cell lines and normal cultured cells were negative. The antigen is highly stable and well preserved on paraffin-embedded tissues. Electrophoretic transfer blot experiments with fresh tumor extracts showed that all three mAbs react with a determinant on a component of 300,000 Mr (pI 9.5) and 62,000 Mr (pI 6.5). The antigen shows polymorphic expression at the cellular level on tissue sections and also at a molecular level on immunoblots where the two bands are differentially detected on extracts of a series of tumors but are not visualized on normal urothelium extracts. The characteristics of this antigenic system suggest that it may provide some insights about the biology of bladder cancer. Specific detection of the antigen on 70% of superficial bladder tumors with normal cytology may be useful for their diagnosis and follow-up.

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility

PubMed Central

Núnez-Ollé, Marc; Jung, Carole; Terré, Berta; Balsiger, Norman A.; Plata, Cristina; Roset, Ramon; Pardo-Pastor, Carlos; Garrido, Marta; Rojas, Santiago; Alameda, Francesc; Lloreta, Josep; Martín-Caballero, Juan; Flores, Juana M.; Stracker, Travis H.; Valverde, Miguel A.; Muñoz, Francisco J.; Gil-Gómez, Gabriel

2017-01-01

Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients. PMID:29245899

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility.

PubMed

Núnez-Ollé, Marc; Jung, Carole; Terré, Berta; Balsiger, Norman A; Plata, Cristina; Roset, Ramon; Pardo-Pastor, Carlos; Garrido, Marta; Rojas, Santiago; Alameda, Francesc; Lloreta, Josep; Martín-Caballero, Juan; Flores, Juana M; Stracker, Travis H; Valverde, Miguel A; Muñoz, Francisco J; Gil-Gómez, Gabriel

2017-11-21

Cyclin O (encoded by CCNO ) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo , we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno -/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno +/- mice also developed hydrocephalus and affected Ccno -/- and Ccno +/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno -/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.

Armed Forces Institute of Regenerative Medicine

DTIC Science & Technology

2009-01-01

constructs healed faster than controls and were able to self -organize into skin that appeared almost identical to normal mouse skin. Research...mouse model using the device. They also determined that printed constructs healed faster than controls and were able to self - organize into skin...iiiAFIRM Annual Report 2009 IV Scarless Wound Healing IV-1 Background

Molecular Solutions to Low Vision Resulting from Battlefield Injuries

DTIC Science & Technology

2006-05-01

promote optic nerve regeneration; (5) dry eye bydetermining how to minimize dry eye after LASIK refractive surgery by developing new tests to predict...Nerve Loss in LASIK Surgery, Invest. Ophthalmol. Vis. Sci. 2006 47: E-Abstract 4600. CONCLUSIONS We conclude that in the normal mouse conjunctiva, the...Mimicking Nerve Loss in LASIK Surgery, Invest. Ophthalmol. Vis. Sci. 2006 47: E-Abstract 4600. Services Email this article to a friend Similar articles in

Development and Function of the Mouse Vestibular System in the Absence of Gravity Perception

NASA Technical Reports Server (NTRS)

Wolgemuth, Debra J.

2005-01-01

The hypothesis that was tested in this research was that the absence of gravity perception, such as would occur in space, would affect the development and function of the vestibular and central nervous systems. Further, we postulated that these effects would be more significant at specific stages of post-natal development of the animal. We also proposed the use of molecular genetic approaches that would provide important information as to the hierarchy of gene function during the development and subsequent function of the vestibular system. The tilted (tlt) mutant mouse has been characterized as lacking the ability to provide sensory input to the gravity receptors. The tlt/tlt mutant mice were a particularly attractive model for the study of vestibular function since the primary defect was limited to the receptor part of the vestibular system, and there were no detectable abnormal phenotypes in other organ systems. The goal of the proposed studies was to assess immediate and delayed effects of the lack of gravity perception on the vestibular system. Particular attention was paid to characterizing primarily affected periods of vestibular morphogenesis, and to identifying downstream genetic pathways that are altered in the CNS of the tlt/tlt mutant mouse. The specific aims were: (1) to characterize the postnatal morphogenesis of the CNS in the tlt mutant mouse, using detailed morphometric analysis of isolated vestibular ganglia and brain tissue at different stages of postnatal development and assessment of apoptotic cell death; (2) to examine the expression of selected genes implicated by mutational analysis to be important in vestibular development or function by in situ hybridization or immunohistochemistry in the mutant mice; and (3) to identify other genes involved in vestibular development and function, using differential cloning strategies to isolate genes whose expression is changed in the mutant versus normal vestibular system.

Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer.

PubMed

Whiteaker, Jeffrey R; Zhang, Heidi; Zhao, Lei; Wang, Pei; Kelly-Spratt, Karen S; Ivey, Richard G; Piening, Brian D; Feng, Li-Chia; Kasarda, Erik; Gurley, Kay E; Eng, Jimmy K; Chodosh, Lewis A; Kemp, Christopher J; McIntosh, Martin W; Paulovich, Amanda G

2007-10-01

Despite their potential to impact diagnosis and treatment of cancer, few protein biomarkers are in clinical use. Biomarker discovery is plagued with difficulties ranging from technological (inability to globally interrogate proteomes) to biological (genetic and environmental differences among patients and their tumors). We urgently need paradigms for biomarker discovery. To minimize biological variation and facilitate testing of proteomic approaches, we employed a mouse model of breast cancer. Specifically, we performed LC-MS/MS of tumor and normal mammary tissue from a conditional HER2/Neu-driven mouse model of breast cancer, identifying 6758 peptides representing >700 proteins. We developed a novel statistical approach (SASPECT) for prioritizing proteins differentially represented in LC-MS/MS datasets and identified proteins over- or under-represented in tumors. Using a combination of antibody-based approaches and multiple reaction monitoring-mass spectrometry (MRM-MS), we confirmed the overproduction of multiple proteins at the tissue level, identified fibulin-2 as a plasma biomarker, and extensively characterized osteopontin as a plasma biomarker capable of early disease detection in the mouse. Our results show that a staged pipeline employing shotgun-based comparative proteomics for biomarker discovery and multiple reaction monitoring for confirmation of biomarker candidates is capable of finding novel tissue and plasma biomarkers in a mouse model of breast cancer. Furthermore, the approach can be extended to find biomarkers relevant to human disease.

Surface Antigens Common to Mouse Cleavage Embryos and Primitive Teratocarcinoma Cells in Culture

PubMed Central

Artzt, Karen; Dubois, Philippe; Bennett, Dorothea; Condamine, Hubert; Babinet, Charles; Jacob, François

1973-01-01

Syngeneic antisera have been produced in mouse strain 129/Sv-CP males against the primitive cells of teratocarcinoma. These sera react specifically with the primitive cells and are negative on various types of differentiated teratoma cells derived from the same original tumor. They are negative on all other mouse cells tested, with the exception of male germ cells and cleavage-stage embryos. Thus, teratoma cells possess cell-surface antigens in common with normal cleavage-stage embryos. Images PMID:4355379

Evaluation of diffusion kurtosis imaging in ex vivo hypomyelinated mouse brains.

PubMed

Kelm, Nathaniel D; West, Kathryn L; Carson, Robert P; Gochberg, Daniel F; Ess, Kevin C; Does, Mark D

2016-01-01

Diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), and DKI-derived white matter tract integrity metrics (WMTI) were experimentally evaluated ex vivo through comparisons to histological measurements and established magnetic resonance imaging (MRI) measures of myelin in two knockout mouse models with varying degrees of hypomyelination. DKI metrics of mean and radial kurtosis were found to be better indicators of myelin content than conventional DTI metrics. The biophysical WMTI model based on the DKI framework reported on axon water fraction with good accuracy in cases with near normal axon density, but did not provide additional specificity to myelination. Overall, DKI provided additional information regarding white matter microstructure compared with DTI, making it an attractive method for future assessments of white matter development and pathology. Copyright © 2015 Elsevier Inc. All rights reserved.

Epidermolysis Bullosa Acquisita: Autoimmunity to Anchoring Fibril Collagen

PubMed Central

Chen, Mei; Kim, Gene H.; Prakash, Lori; Woodley, David T.

2012-01-01

Epidermolysis bullosa acquisita (EBA) is a rare and acquired autoimmune subepidermal bullous disease of the skin and mucosa. EBA includes various distinct clinical manifestations resembling Bullous Pemphigus, Brunsting-Perry pemphigoid, or cicatricial pemphigoid. These patients have autoantibodies against type VII collagen, an integral component of anchoring fibrils, which are responsible for attaching the dermis to the epidermis. Destruction or perturbation of the normally functioning anchoring fibrils clinically results in skin fragility, blisters, erosions, scars, milia and nail loss, all features reminiscent of genetic dystrophic epidermolysis bullosa. These anti-type VII collagen antibodies are “pathogenic” because when injected into a mouse, the mouse develops an EBA-like blistering disease. Currently treatment is often unsatisfactory, however some success has been achieved with colchichine, dapsone, photopheresis, plasmaphresis, infliximab, rituximab and IVIG. PMID:21955050

Selective Neurodegeneration, Without Neurofibrillary Tangles, in a Mouse Model of Niemann-Pick C Disease

PubMed Central

German, Dwight C.; Quintero, E. Matthew; Liang, Chang-Lin; Ng, Benton; Punia, Surender; Xie, Chonglun; Dietschy, John M.

2012-01-01

The BALB/c mouse model of Niemann-Pick type C (NPC) disease exhibits neuropathological similarities to the human condition. There is an age-related cerebral atrophy, demyelination of the corpus callosum, and degeneration of cerebellar Purkinje cells in the NPC mouse. In human NPC, many cortical and subcortical neurons contain neurofibrillary tangles, which are thought by some investigators to play an important role in the neurodegenerative process. The purpose of the present study was to determine whether neurodegeneration occurs in the NPC mouse, in brain regions other than the cerebellum and whether the degeneration is related to the presence of neurofibrillary tangles. Using light microscopic methods with immunohistochemistry, electron microscopy, and cell counting methods, 11-week-old NPC+/+ and NPC−/− animals were examined. In the NPC−/− mice, there were 96% fewer Purkinje cells, 28% fewer neurons in the prefrontal cortex, 20% fewer neurons in the thalamus, and 63% fewer glial cells in the corpus callosum. On the other hand, previous studies indicate normal numbers of neurons and glial cells in these same neuroanatomical regions in young NPC−/− mice. There were normal numbers of cholinergic neurons in sections assessed in the striatum and basal forebrain in the 11-week-old animals and no evidence of neurofibrillary tangles within cells. The present data indicate that both neurons and glial cells die in the NPC mouse but that all cells are not equally vulnerable. There was no evidence for neurofibrillary tangles in the NPC mouse, and therefore the degenerative process in the mouse is unrelated to the neurofibrillary tangle. PMID:11298365

New animal models to study the role of tyrosinase in normal retinal development.

PubMed

Lavado, Alfonso; Montoliu, Lluis

2006-01-01

Albino animals display a hypopigmented phenotype associated with several visual abnormalities, including rod photoreceptor cell deficits, abnormal patterns of connections between the eye and the brain and a general underdevelopment of central retina. Oculocutaneous albinism type I, a common form of albinism, is caused by mutations in the tyrosinase gene. In mice, the albino phenotype can be corrected by functional tyrosinase transgenes. Tyrosinase transgenic animals not only show normal pigmentation but the correction of all visual abnormalities associated with albinism, confirming a role of tyrosinase, a key enzyme in melanin biosynthesis, in normal retinal development. Here, we will discuss recent work carried out with new tyrosinase transgenic mouse models, to further analyse the role of tyrosinase in retinal development. We will first report a transgenic model with inducible tyrosinase expression that has been used to address the regulated activation of this gene and its associated effects on the development of the visual system. Second, we will comment on an interesting yeast artificial chromosome (YAC)-tyrosinase transgene, lacking important regulatory elements, that has highlighted the significance of local interactions between the retinal pigment epithelium (RPE) and developing neural retina.

Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms

PubMed Central

Antony, N.; McDougall, A. R.; Mantamadiotis, T.; Cole, T. J.; Bird, A. D.

2016-01-01

During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1−/− mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1−/− mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1−/− mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development. PMID:27150575

Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

PubMed

Igetei, Joseph E; Liddell, Susan; El-Faham, Marwa; Doenhoff, Michael J

2016-04-01

A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine β-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or β-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.

THE EFFECT OF ANTISERUM, ALONE AND WITH HYDROCORTISONE, ON FOETAL MOUSE BONES IN CULTURE

PubMed Central

Fell, Honor B.; Weiss, L.

1965-01-01

1. The effects of normal rabbit serum and of rabbit antiserum to whole foetal mouse tissues, on the isolated limb bones of late foetal mice were studied in organ culture, and the influence of hydrocortisone on these effects was investigated. 2. Unheated normal serum caused slight loss of metachromatic material from the cartilage matrix, and some resorption of both cartilage and bone. 3. In unheated antiserum to foetal mouse tissues, the terminal cartilage was smaller and less metachromatic than in paired controls in normal serum, while osteoclasis was so intense that in many explants the bone had almost disappeared. The amount of necrosis varied with different batches of antiserum. 4. The changes produced by normal serum and antiserum could be largely prevented by heating the sera to 57°C for 45 minutes. 5. The effects could also be inhibited by the addition of hydrocortisone to the unheated sera; as little as 0.1 µg hydrocortisone per ml of medium had a well marked protective action. 6. It is suggested that (a) unheated antiserum causes a release of lysosomal enzymes with consequent breakdown of intercellular material, (b) this release is due to an indirect action on the lysosome via an increased permeability of the cell membrane, (c) hydrocortisone does not affect the antigen-antibody reaction, but inhibits the autolytic changes that normally follow this reaction, possibly by stabilising both the lysosomal and cell membranes. PMID:14276776

Pharmacological reversal of synaptic plasticity deficits in the mouse model of fragile X syndrome by group II mGluR antagonist or lithium treatment.

PubMed

Choi, Catherine H; Schoenfeld, Brian P; Bell, Aaron J; Hinchey, Paul; Kollaros, Maria; Gertner, Michael J; Woo, Newton H; Tranfaglia, Michael R; Bear, Mark F; Zukin, R Suzanne; McDonald, Thomas V; Jongens, Thomas A; McBride, Sean M J

2011-03-22

Fragile X syndrome is the leading single gene cause of intellectual disabilities. Treatment of a Drosophila model of Fragile X syndrome with metabotropic glutamate receptor (mGluR) antagonists or lithium rescues social and cognitive impairments. A hallmark feature of the Fragile X mouse model is enhanced mGluR-dependent long-term depression (LTD) at Schaffer collateral to CA1 pyramidal synapses of the hippocampus. Here we examine the effects of chronic treatment of Fragile X mice in vivo with lithium or a group II mGluR antagonist on mGluR-LTD at CA1 synapses. We find that long-term lithium treatment initiated during development (5-6 weeks of age) and continued throughout the lifetime of the Fragile X mice until 9-11 months of age restores normal mGluR-LTD. Additionally, chronic short-term treatment beginning in adult Fragile X mice (8 weeks of age) with either lithium or an mGluR antagonist is also able to restore normal mGluR-LTD. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of Fragile X syndrome is an important advance, in that this identifies and validates these targets as potential therapeutic interventions for the treatment of individuals afflicted with Fragile X syndrome. Copyright © 2010 Elsevier B.V. All rights reserved.

Thin and open vessel windows for intra-vital fluorescence imaging of murine cochlear blood flow

PubMed Central

Shi, Xiaorui; Zhang, Fei; Urdang, Zachary; Dai, Min; Neng, Lingling; Zhang, Jinhui; Chen, Songlin; Ramamoorthy, Sripriya; Nuttall, Alfred L.

2014-01-01

Normal microvessel structure and function in the cochlea is essential for maintaining the ionic and metabolic homeostasis required for hearing function. Abnormal cochlear microcirculation has long been considered an etiologic factor in hearing disorders. A better understanding of cochlear blood flow (CoBF) will enable more effective amelioration of hearing disorders that result from aberrant blood flow. However, establishing the direct relationship between CoBF and other cellular events in the lateral wall and response to physio-pathological stress remains a challenge due to the lack of feasible interrogation methods and difficulty in accessing the inner ear. Here we report on new methods for studying the CoBF in a mouse model using a thin or open vessel-window in combination with fluorescence intra-vital microscopy (IVM). An open vessel-window enables investigation of vascular cell biology and blood flow permeability, including pericyte (PC) contractility, bone marrow cell migration, and endothelial barrier leakage, in wild type and fluorescent protein-labeled transgenic mouse models with high spatial and temporal resolution. Alternatively, the thin vessel-window method minimizes disruption of the homeostatic balance in the lateral wall and enables study CoBF under relatively intact physiological conditions. A thin vessel-window method can also be used for time-based studies of physiological and pathological processes. Although the small size of the mouse cochlea makes surgery difficult, the methods are sufficiently developed for studying the structural and functional changes in CoBF under normal and pathological conditions. PMID:24780131

Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse.

PubMed

Rayavarapu, Sree; Coley, William; Cakir, Erdinc; Jahnke, Vanessa; Takeda, Shin'ichi; Aoki, Yoshitsugu; Grodish-Dressman, Heather; Jaiswal, Jyoti K; Hoffman, Eric P; Brown, Kristy J; Hathout, Yetrib; Nagaraju, Kanneboyina

2013-05-01

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by a mutation in the dystrophin gene. DMD is characterized by progressive weakness of skeletal, cardiac, and respiratory muscles. The molecular mechanisms underlying dystrophy-associated muscle weakness and damage are not well understood. Quantitative proteomics techniques could help to identify disease-specific pathways. Recent advances in the in vivo labeling strategies such as stable isotope labeling in mouse (SILAC mouse) with (13)C6-lysine or stable isotope labeling in mammals (SILAM) with (15)N have enabled accurate quantitative analysis of the proteomes of whole organs and tissues as a function of disease. Here we describe the use of the SILAC mouse strategy to define the underlying pathological mechanisms in dystrophin-deficient skeletal muscle. Differential SILAC proteome profiling was performed on the gastrocnemius muscles of 3-week-old (early stage) dystrophin-deficient mdx mice and wild-type (normal) mice. The generated data were further confirmed in an independent set of mdx and normal mice using a SILAC spike-in strategy. A total of 789 proteins were quantified; of these, 73 were found to be significantly altered between mdx and normal mice (p < 0.05). Bioinformatics analyses using Ingenuity Pathway software established that the integrin-linked kinase pathway, actin cytoskeleton signaling, mitochondrial energy metabolism, and calcium homeostasis are the pathways initially affected in dystrophin-deficient muscle at early stages of pathogenesis. The key proteins involved in these pathways were validated by means of immunoblotting and immunohistochemistry in independent sets of mdx mice and in human DMD muscle biopsies. The specific involvement of these molecular networks early in dystrophic pathology makes them potential therapeutic targets. In sum, our findings indicate that SILAC mouse strategy has uncovered previously unidentified pathological pathways in mouse models of human skeletal muscle disease.

Identification of Disease Specific Pathways Using in Vivo SILAC Proteomics in Dystrophin Deficient mdx Mouse*

PubMed Central

Rayavarapu, Sree; Coley, William; Cakir, Erdinc; Jahnke, Vanessa; Takeda, Shin'ichi; Aoki, Yoshitsugu; Grodish-Dressman, Heather; Jaiswal, Jyoti K.; Hoffman, Eric P.; Brown, Kristy J.; Hathout, Yetrib; Nagaraju, Kanneboyina

2013-01-01

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by a mutation in the dystrophin gene. DMD is characterized by progressive weakness of skeletal, cardiac, and respiratory muscles. The molecular mechanisms underlying dystrophy-associated muscle weakness and damage are not well understood. Quantitative proteomics techniques could help to identify disease-specific pathways. Recent advances in the in vivo labeling strategies such as stable isotope labeling in mouse (SILAC mouse) with 13C6-lysine or stable isotope labeling in mammals (SILAM) with 15N have enabled accurate quantitative analysis of the proteomes of whole organs and tissues as a function of disease. Here we describe the use of the SILAC mouse strategy to define the underlying pathological mechanisms in dystrophin-deficient skeletal muscle. Differential SILAC proteome profiling was performed on the gastrocnemius muscles of 3-week-old (early stage) dystrophin-deficient mdx mice and wild-type (normal) mice. The generated data were further confirmed in an independent set of mdx and normal mice using a SILAC spike-in strategy. A total of 789 proteins were quantified; of these, 73 were found to be significantly altered between mdx and normal mice (p < 0.05). Bioinformatics analyses using Ingenuity Pathway software established that the integrin-linked kinase pathway, actin cytoskeleton signaling, mitochondrial energy metabolism, and calcium homeostasis are the pathways initially affected in dystrophin-deficient muscle at early stages of pathogenesis. The key proteins involved in these pathways were validated by means of immunoblotting and immunohistochemistry in independent sets of mdx mice and in human DMD muscle biopsies. The specific involvement of these molecular networks early in dystrophic pathology makes them potential therapeutic targets. In sum, our findings indicate that SILAC mouse strategy has uncovered previously unidentified pathological pathways in mouse models of human skeletal muscle disease. PMID:23297347

CD24-Positive Cells from Normal Adult Mouse Liver Are Hepatocyte Progenitor Cells

PubMed Central

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M.; Rao, Pulivarthi H.

2011-01-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45−, Ter119−) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes. PMID:21361791

CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells.

PubMed

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M; Rao, Pulivarthi H; Darlington, Gretchen J

2011-12-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45-, Ter119-) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes.

Brain perfusion SPECT in the mouse: normal pattern according to gender and age.

PubMed

Apostolova, Ivayla; Wunder, Andreas; Dirnagl, Ulrich; Michel, Roger; Stemmer, Nina; Lukas, Mathias; Derlin, Thorsten; Gregor-Mamoudou, Betina; Goldschmidt, Jürgen; Brenner, Winfried; Buchert, Ralph

2012-12-01

Regional cerebral blood flow (rCBF) is a useful surrogate marker of neuronal activity and a parameter of primary interest in the diagnosis of many diseases. The increasing use of mouse models spawns the demand for in vivo measurement of rCBF in the mouse. Small animal SPECT provides excellent spatial resolution at adequate sensitivity and is therefore a promising tool for imaging the mouse brain. This study evaluates the feasibility of mouse brain perfusion SPECT and assesses the regional pattern of normal Tc-99m-HMPAO uptake and the impact of age and gender. Whole-brain kinetics was compared between Tc-99m-HMPAO and Tc-99m-ECD using rapid dynamic planar scans in 10 mice. Assessment of the regional uptake pattern was restricted to the more suitable tracer, HMPAO. Two HMPAO SPECTs were performed in 18 juvenile mice aged 7.5 ± 1.5weeks, and in the same animals at young adulthood, 19.1 ± 4.0 weeks (nanoSPECT/CTplus, general purpose mouse apertures: 1.2kcps/MBq, 0.7mm FWHM). The 3-D MRI Digital Atlas Database of an adult C57BL/6J mouse brain was used for region-of-interest (ROI) analysis. SPECT images were stereotactically normalized using SPM8 and a custom made, left-right symmetric HMPAO template in atlas space. For testing lateral asymmetry, each SPECT was left-right flipped prior to stereotactical normalization. Flipped and unflipped SPECTs were compared by paired testing. Peak brain uptake was similar for ECD and HMPAO: 1.8 ± 0.2 and 2.1 ± 0.6 %ID (p=0.357). Washout after the peak was much faster for ECD than for HMPAO: 24 ± 7min vs. 4.6 ± 1.7h (p=0.001). The general linear model for repeated measures with gender as an intersubject factor revealed an increase in relative HMPAO uptake with age in the neocortex (p=0.018) and the hippocampus (p=0.012). A decrease was detected in the midbrain (p=0.025). Lateral asymmetry, with HMPAO uptake larger in the left hemisphere, was detected primarily in the neocortex, both at juvenile age (asymmetry index AI=2.7 ± 1.7%, p=0.000) and at young adult age (AI=2.4 ± 1.7%, p=0.000). Gender had no effect on asymmetry. Voxel-wise testing confirmed the ROI-based findings. In conclusion, high-resolution HMPAO SPECT is a promising technique for measuring rCBF in preclinical research. It indicates lateral asymmetry of rCBF in the mouse brain as well as age-related changes during late maturation. ECD is not suitable as tracer for brain SPECT in the mouse because of its fast clearance from tissue indicating an interspecies difference in esterase activity between mice and humans. Copyright © 2012 Elsevier Inc. All rights reserved.

Morphological and biochemical studies of the elastic fibres in the craniomandibular joint articular disc of the tight-skin mouse.

PubMed

O'dell, N L; Burlison, S K; Starcher, B C; Pennington, C B

1996-05-01

The tight-skin (TSK) mouse is characterized by the hyperplasia of loose connective tissues, and of excessive growth of cartilage and of bones including the mandible. Since the fibroelastic connective tissues of the craniomandibular joint (CMJ) are essential to the functions of this joint, the present histological study compared the presence and general distribution of elastic fibres in CMJ discal tissues of TSK and normal mice. The excised CMJs were processed for light microscopy. The tissues were fixed, demineralized, embedded in paraffin, sectioned and then stained with resorcin-fuchsin to demonstrate elastic fibres. There were no obvious histological differences in either the amount or the distribution of elastic fibres in the discs from the two groups. In both groups, elastic fibres were found in the disc and in many of the attachments of the disc to the mandible and squamosal bone. In addition to the morphological preparations, articular discs and samples of lung tissue were excised from other mice and subjected to a radioimmunoassay for desmosine in order to estimate the amounts of elastin in these tissues; the amount of elastin was significantly reduced in the TSK lung, but the amounts of elastin in the TSK and normal CMJ discal tissues were not significantly different statistically. These morphological and histochemical results suggest that the distribution and quantity of elastic fibres in the TSK mouse disc are not significantly different from those in the normal mouse articular disc. Moreover, these data may be interpreted to either suggest a differential effect on the elastic fibres in different TSK tissues, or to support the suggestion that abnormal degradation of elastic fibres may not be characteristic of the TSK mouse.

Analysis of mouse models carrying the I26T and R160C substitutions in the transcriptional repressor HESX1 as models for septo-optic dysplasia and hypopituitarism

PubMed Central

Sajedi, Ezat; Gaston-Massuet, Carles; Signore, Massimo; Andoniadou, Cynthia L.; Kelberman, Daniel; Castro, Sandra; Etchevers, Heather C.; Gerrelli, Dianne; Dattani, Mehul T.; Martinez-Barbera, Juan Pedro

2008-01-01

SUMMARY A homozygous substitution of the highly conserved isoleucine at position 26 by threonine (I26T) in the transcriptional repressor HESX1 has been associated with anterior pituitary hypoplasia in a human patient, with no forebrain or eye defects. Two individuals carrying a homozygous substitution of the conserved arginine at position 160 by cysteine (R160C) manifest septo-optic dysplasia (SOD), a condition characterised by pituitary abnormalities associated with midline telencephalic structure defects and optic nerve hypoplasia. We have generated two knock-in mouse models containing either the I26T or R160C substitution in the genomic locus. Hesx1I26T/I26T embryos show pituitary defects comparable with Hesx1−/− mouse mutants, with frequent occurrence of ocular abnormalities, although the telencephalon develops normally. Hesx1R160C/R160C mutants display forebrain and pituitary defects that are identical to those observed in Hesx1−/− null mice. We also show that the expression pattern of HESX1 during early human development is very similar to that described in the mouse, suggesting that the function of HESX1 is conserved between the two species. Together, these results suggest that the I26T mutation yields a hypomorphic allele, whereas R160C produces a null allele and, consequently, a more severe phenotype in both mice and humans. PMID:19093031

Deletion of lysophosphatidic acid receptor LPA1 reduces neurogenesis in the mouse dentate gyrus

PubMed Central

Matas-Rico, Elisa; García-Diaz, Beatriz; Llebrez-Zayas, Pedro; López-Barroso, Diana; Santín, Luis; Pedraza, Carmen; Smith-Fernández, Anibal; Fernández-Llebrez, Pedro; Tellez, Teresa; Redondo; Chun, Jerold; De Fonseca, Fernando Rodríguez; Estivill-Torrús, Guillermo

2013-01-01

Neurogenesis persists in certain regions of the adult brain including the subgranular zone of the hippocampal dentate gyrus wherein its regulation is essential, particularly in relation to learning, stress and modulation of mood. Lysophosphatidic acid (LPA) is an extracellular signaling phospholipid with important neural regulatory properties mediated by specific G protein-coupled receptors, LPA1-5. LPA1 is highly expressed in the developing neurogenic ventricular zone wherein it is required for normal embryonic neurogenesis, and, by extension may play a role in adult neurogenesis as well. By means of the analyses of a variant of the original LPA1-null mutant mouse, termed the Malaga variant or “maLPA1-null,” which has recently been reported to have defective neurogenesis within the embryonic cerebral cortex, we report here a role for LPA1 in adult hippocampal neurogenesis. Proliferation, differentiation and survival of newly formed neurons are defective in the absence of LPA1 under normal conditions and following exposure to enriched environment and voluntary exercise. Furthermore, analysis of trophic factors in maLPA1-null mice demonstrated alterations in brain-derived neurotrophic factor and insulin growth factor 1 levels after enrichment and exercise. Morphological analyses of doublecortin positive cells revealed the anomalous prevalence of bipolar cells in the subgranular zone, supporting the operation of LPA1 signaling pathways in normal proliferation, maturation and differentiation of neuronal precursors. PMID:18708146

Overexpression of c-jun, junB, or junD affects cell growth differently.

PubMed

Castellazzi, M; Spyrou, G; La Vista, N; Dangy, J P; Piu, F; Yaniv, M; Brun, G

1991-10-15

The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth.

Overexpression of c-jun, junB, or junD affects cell growth differently.

PubMed Central

Castellazzi, M; Spyrou, G; La Vista, N; Dangy, J P; Piu, F; Yaniv, M; Brun, G

1991-01-01

The coding sequences of murine c-jun, junB, or junD, which code for proteins with practically identical dimerization and DNA binding properties, were introduced into a nondefective retroviral vector, and the phenotype of primary avian fibroblasts chronically infected with each of these viruses was studied. Cells expressing c-jun grew in low-serum medium and developed into colonies in agar, two properties characteristic of in vitro transformation. Cells expressing junB grew in agar, with a reduced efficiency as compared to c-jun, but did not grow in low-serum medium. Finally, no effect of junD expression on cell growth was observed. These different phenotypes suggest that these three closely related transcription factors play distinct roles during normal cell growth. Analysis of c-jun deletion mutants and of c-jun/junB and c-jun/junD chimeric genes showed that the N-terminal portion (amino acids 2-168) of the c-Jun protein that is involved in transcriptional activation is required for efficient transformation. On the contrary, cells expressing a truncated mouse c-Jun lacking this N-terminal domain grew slower than normal embryo fibroblasts. The reduced growth rate may be related to the finding that expression of the intact or the truncated mouse c-jun repressed the endogenous avian c-Jun homologue, suggesting that functional c-Jun product is required for normal cell growth. Images PMID:1924349

No overt structural or functional changes associated with PEG-coated gold nanoparticles accumulation with acute exposure in the mouse heart.

PubMed

Yang, Chengzhi; Yang, Hui; Wu, Jimin; Meng, Zenghui; Xing, Rui; Tian, Aiju; Tian, Xin; Guo, Lijun; Zhang, Youyi; Nie, Guangjun; Li, Zijian

2013-10-24

In this study, we investigated the cardiac biodistribution of polyethylene glycol (PEG)-coated AuNPs and their effects on cardiac function, structure and inflammation in both normal and cardiac remodeling mice. The model of cardiac remodeling was induced by subcutaneously injection of isoproterenol (ISO), a non-selective beta-adrenergic agonist, for 7 days. After AuNPs were injected intravenously in mice for 7 consecutive days, Au content in different organs was determined quantitatively by inductively coupled plasma mass spectrometry (ICP-MS), cardiac function and structure were measured by echocardiography, cardiac fibrosis was examined with picrosirius red staining, the morphology of cardiomyocytes was observed with hematoxylin and eosin (H & E) staining. The accumulation of AuNPs in hearts did not affect cardiac function or induce cardiac hypertrophy, cardiac fibrosis and cardiac inflammation under normal physiological condition. Cardiac AuNPs content was 6-fold higher in the cardiac remodeling mouse than normal mice. However, the increased accumulation of AuNPs in the heart did not aggravate ISO-induced cardiac hypertrophy, cardiac fibrosis or cardiac inflammation. These observations suggest that PEG-coated AuNPs possess excellent biocompatibility under both physiological and pathological conditions. Thus, AuNPs may be safe for cardiac patients and hold great promise for further development for various biomedical applications. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A protocol to study ex vivo mouse working heart at human-like heart rate.

PubMed

Feng, Han-Zhong; Jin, Jian-Ping

2018-01-01

Genetically modified mice are widely used as experimental models to study human heart function and diseases. However, the fast rate of normal mouse heart at 400-600bpm limits its capacity of assessing kinetic parameters that are important for the physiology and pathophysiology of human heart that beats at a much slower rate (75-180bpm). To extend the value of mouse models, we established a protocol to study ex vivo mouse working hearts at a human-like heart rate. In the presence of 300μM lidocaine to lower pacemaker and conductive activities and prevent arrhythmia, a stable rate of 120-130bpm at 37°C is achieved for ex vivo mouse working hearts. The negative effects of decreased heart rate on force-frequency dependence and lidocaine as a myocardial depressant on intracellular calcium can be compensated by using a higher but still physiological level of calcium (2.75mM) in the perfusion media. Multiple parameters were studied to compare the function at the human-like heart rate with that of ex vivo mouse working hearts at the standard rate of 480bpm. The results showed that the conditions for slower heart rate in the presence of 300μM lidocaine did not have depressing effect on left ventricular pressure development, systolic and diastolic velocities and stroke volume with maintained positive inotropic and lusitropic responses to β-adrenergic stimulation. Compared with that at 480bpm, the human-like heart rate increased ventricular filling and end diastolic volume with enhanced Frank-Starling responses. Coronary perfusion was increased from longer relaxation time and interval between beats whereas cardiac efficiency was significantly improved. Although the intrinsic differences between mouse and human heart remain, this methodology for ex vivo mouse hearts to work at human-like heart rate extends the value of using genetically modified mouse models to study cardiac function and human heart diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Live dynamic analysis of mouse embryonic cardiogenesis with functional optical coherence tomography

NASA Astrophysics Data System (ADS)

Lopez, Andrew L.; Wang, Shang; Larina, Irina V.

2018-02-01

Hemodynamic load, contractile forces, and tissue elasticity are regulators of cardiac development and contribute to the mechanical homeostasis of the developing vertebrate heart. Congenital heart disease (CHD) is a prevalent condition in the United States that affects 8 in 1000 live births[1], and has been linked to disrupted cardiac biomechanics[2-4]. Therefore, it is important to understand how these forces integrate and regulate vertebrate cardiac development to inform clinical strategies to treat CHD early on by reintroducing proper mechanical load or modulating downstream factors that rely on mechanical signalling. Toward investigation of biomechanical regulation of mammalian cardiovascular dynamics and development, our methodology combines live mouse embryo culture protocols, state-of-the-art structural and functional Optical Coherence Tomography (OCT), second harmonic generation (SHG) microscopy, and computational analysis. Using these approaches, we assess functional aspects of the developing heart and characterize how they coincide with a determinant of tissue stiffness and main constituent of the extracellular matrix (ECM)—type I collagen. This work is bringing us closer to understanding how cardiac biomechanics change temporally and spatially during normal development, and how it regulates ECM to maintain mechanical homeostasis for proper function.

A mutation in Ccdc39 causes neonatal hydrocephalus with abnormal motile cilia development in mice.

PubMed

Abdelhamed, Zakia; Vuong, Shawn M; Hill, Lauren; Shula, Crystal; Timms, Andrew; Beier, David; Campbell, Kenneth; Mangano, Francesco T; Stottmann, Rolf W; Goto, June

2018-01-09

Pediatric hydrocephalus is characterized by an abnormal accumulation of cerebrospinal fluid (CSF) and is one of the most common congenital brain abnormalities. However, little is known about the molecular and cellular mechanisms regulating CSF flow in the developing brain. Through whole-genome sequencing analysis, we report that a homozygous splice site mutation in coiled-coil domain containing 39 ( Ccdc39 ) is responsible for early postnatal hydrocephalus in the progressive hydrocephal us ( prh ) mouse mutant. Ccdc39 is selectively expressed in embryonic choroid plexus and ependymal cells on the medial wall of the forebrain ventricle, and the protein is localized to the axoneme of motile cilia. The Ccdc39 prh/prh ependymal cells develop shorter cilia with disorganized microtubules lacking the axonemal inner arm dynein. Using high-speed video microscopy, we show that an orchestrated ependymal ciliary beating pattern controls unidirectional CSF flow on the ventricular surface, which generates bulk CSF flow in the developing brain. Collectively, our data provide the first evidence for involvement of Ccdc39 in hydrocephalus and suggest that the proper development of medial wall ependymal cilia is crucial for normal mouse brain development. © 2018. Published by The Company of Biologists Ltd.

PR-Set7 is degraded in a conditional Cul4A transgenic mouse model of lung cancer

DOE PAGES

Wang, Yang; Xu, Zhidong; Mao, Jian -Hua; ...

2015-06-01

Background and objective. Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. PR-Set7 (also known as Set8) is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20 (H4K20me1) to promote chromosome condensation and prevent DNA damage. Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage. This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage. Methods. We developed a new model of lung tumor developmentmore » in mice harboring a conditionally expressed allele of Cul4A. We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo. With this model, staining of PR-Set7 in the preneoplastic and tumor lesions in AdenoCre-induced mouse lungs was performed. Meanwhile we identified higher protein level changes of γ-tubulin and pericentrin by IHC. Results. The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A. We also identified higher levels of the proteins pericentrin and γ-tubulin in Cul4A mouse lungs induced by AdenoCre. Conclusion. PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.« less

Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride

PubMed Central

Batti, Laura; Mukhtarov, Marat; Audero, Enrica; Ivanov, Anton; Paolicelli, Rosa Chiara; Zurborg, Sandra; Gross, Cornelius; Bregestovski, Piotr; Heppenstall, Paul A.

2013-01-01

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo. PMID:23734096

PR-Set7 is degraded in a conditional Cul4A transgenic mouse model of lung cancer

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

Wang, Yang; Xu, Zhidong; Mao, Jian -Hua

Background and objective. Maintenance of genomic integrity is essential to ensure normal organismal development and to prevent diseases such as cancer. PR-Set7 (also known as Set8) is a cell cycle regulated enzyme that catalyses monomethylation of histone 4 at Lys20 (H4K20me1) to promote chromosome condensation and prevent DNA damage. Recent studies show that CRL4CDT2-mediated ubiquitylation of PR-Set7 leads to its degradation during S phase and after DNA damage. This might occur to ensure appropriate changes in chromosome structure during the cell cycle or to preserve genome integrity after DNA damage. Methods. We developed a new model of lung tumor developmentmore » in mice harboring a conditionally expressed allele of Cul4A. We have therefore used a mouse model to demonstrate for the first time that Cul4A is oncogenic in vivo. With this model, staining of PR-Set7 in the preneoplastic and tumor lesions in AdenoCre-induced mouse lungs was performed. Meanwhile we identified higher protein level changes of γ-tubulin and pericentrin by IHC. Results. The level of PR-Set7 down-regulated in the preneoplastic and adenocarcinomous lesions following over-expression of Cul4A. We also identified higher levels of the proteins pericentrin and γ-tubulin in Cul4A mouse lungs induced by AdenoCre. Conclusion. PR-Set7 is a direct target of Cul4A for degradation and involved in the formation of lung tumors in the conditional Cul4A transgenic mouse model.« less

Genetic Interactions Between Shox2 and Hox Genes During the Regional Growth and Development of the Mouse Limb

PubMed Central

Neufeld, Stanley J.; Wang, Fan; Cobb, John

2014-01-01

The growth and development of the vertebrate limb relies on homeobox genes of the Hox and Shox families, with their independent mutation often giving dose-dependent effects. Here we investigate whether Shox2 and Hox genes function together during mouse limb development by modulating their relative dosage and examining the limb for nonadditive effects on growth. Using double mRNA fluorescence in situ hybridization (FISH) in single embryos, we first show that Shox2 and Hox genes have associated spatial expression dynamics, with Shox2 expression restricted to the proximal limb along with Hoxd9 and Hoxa11 expression, juxtaposing the distal expression of Hoxa13 and Hoxd13. By generating mice with all possible dosage combinations of mutant Shox2 alleles and HoxA/D cluster deletions, we then show that their coordinated proximal limb expression is critical to generate normally proportioned limb segments. These epistatic interactions tune limb length, where Shox2 underexpression enhances, and Shox2 overexpression suppresses, Hox-mutant phenotypes. Disruption of either Shox2 or Hox genes leads to a similar reduction in Runx2 expression in the developing humerus, suggesting their concerted action drives cartilage maturation during normal development. While we furthermore provide evidence that Hox gene function influences Shox2 expression, this regulation is limited in extent and is unlikely on its own to be a major explanation for their genetic interaction. Given the similar effect of human SHOX mutations on regional limb growth, Shox and Hox genes may generally function as genetic interaction partners during the growth and development of the proximal vertebrate limb. PMID:25217052

Genetic interactions between Shox2 and Hox genes during the regional growth and development of the mouse limb.

PubMed

Neufeld, Stanley J; Wang, Fan; Cobb, John

2014-11-01

The growth and development of the vertebrate limb relies on homeobox genes of the Hox and Shox families, with their independent mutation often giving dose-dependent effects. Here we investigate whether Shox2 and Hox genes function together during mouse limb development by modulating their relative dosage and examining the limb for nonadditive effects on growth. Using double mRNA fluorescence in situ hybridization (FISH) in single embryos, we first show that Shox2 and Hox genes have associated spatial expression dynamics, with Shox2 expression restricted to the proximal limb along with Hoxd9 and Hoxa11 expression, juxtaposing the distal expression of Hoxa13 and Hoxd13. By generating mice with all possible dosage combinations of mutant Shox2 alleles and HoxA/D cluster deletions, we then show that their coordinated proximal limb expression is critical to generate normally proportioned limb segments. These epistatic interactions tune limb length, where Shox2 underexpression enhances, and Shox2 overexpression suppresses, Hox-mutant phenotypes. Disruption of either Shox2 or Hox genes leads to a similar reduction in Runx2 expression in the developing humerus, suggesting their concerted action drives cartilage maturation during normal development. While we furthermore provide evidence that Hox gene function influences Shox2 expression, this regulation is limited in extent and is unlikely on its own to be a major explanation for their genetic interaction. Given the similar effect of human SHOX mutations on regional limb growth, Shox and Hox genes may generally function as genetic interaction partners during the growth and development of the proximal vertebrate limb. Copyright © 2014 by the Genetics Society of America.

Characterization of motor units in behaving adult mice shows a wide primary range

PubMed Central

Ritter, Laura K.; Tresch, Matthew C.; Heckman, C. J.; Manuel, Marin

2014-01-01

The mouse is essential for genetic studies of motor function in both normal and pathological states. Thus it is important to consider whether the structure of motor output from the mouse is in fact analogous to that recorded in other animals. There is a striking difference in the basic electrical properties of mouse motoneurons compared with those in rats, cats, and humans. The firing evoked by injected currents produces a unique frequency-current (F-I) function that emphasizes recruitment of motor units at their maximum force. These F-I functions, however, were measured in anesthetized preparations that lacked two key components of normal synaptic input: high levels of synaptic noise and neuromodulatory inputs. Recent studies suggest that the alterations in the F-I function due to these two components are essential for recreating firing behavior of motor units in human subjects. In this study we provide the first data on firing patterns of motor units in the awake mouse, focusing on steady output in quiet stance. The resulting firing patterns did not match the predictions from the mouse F-I behaviors but instead revealed rate modulation across a remarkably wide range (10–60 Hz). The low end of the firing range may be due to changes in the F-I relation induced by synaptic noise and neuromodulatory inputs. The high end of the range may indicate that, unlike other species, quiet standing in the mouse involves recruitment of relatively fast-twitch motor units. PMID:24805075

Characterization of motor units in behaving adult mice shows a wide primary range.

PubMed

Ritter, Laura K; Tresch, Matthew C; Heckman, C J; Manuel, Marin; Tysseling, Vicki M

2014-08-01

The mouse is essential for genetic studies of motor function in both normal and pathological states. Thus it is important to consider whether the structure of motor output from the mouse is in fact analogous to that recorded in other animals. There is a striking difference in the basic electrical properties of mouse motoneurons compared with those in rats, cats, and humans. The firing evoked by injected currents produces a unique frequency-current (F-I) function that emphasizes recruitment of motor units at their maximum force. These F-I functions, however, were measured in anesthetized preparations that lacked two key components of normal synaptic input: high levels of synaptic noise and neuromodulatory inputs. Recent studies suggest that the alterations in the F-I function due to these two components are essential for recreating firing behavior of motor units in human subjects. In this study we provide the first data on firing patterns of motor units in the awake mouse, focusing on steady output in quiet stance. The resulting firing patterns did not match the predictions from the mouse F-I behaviors but instead revealed rate modulation across a remarkably wide range (10-60 Hz). The low end of the firing range may be due to changes in the F-I relation induced by synaptic noise and neuromodulatory inputs. The high end of the range may indicate that, unlike other species, quiet standing in the mouse involves recruitment of relatively fast-twitch motor units. Copyright © 2014 the American Physiological Society.

Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

PubMed Central

Krznar, Petra; Hörl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

2016-01-01

Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival. PMID:27176894

The endogenous preproglucagon system is not essential for gut growth homeostasis in mice.

PubMed

Wismann, Pernille; Barkholt, Pernille; Secher, Thomas; Vrang, Niels; Hansen, Henrik B; Jeppesen, Palle Bekker; Baggio, Laurie L; Koehler, Jacqueline A; Drucker, Daniel J; Sandoval, Darleen A; Jelsing, Jacob

2017-07-01

The prevalence of obesity and related co-morbidities is reaching pandemic proportions. Today, the most effective obesity treatments are glucagon-like peptide 1 (GLP-1) analogs and bariatric surgery. Interestingly, both intervention paradigms have been associated with adaptive growth responses in the gut; however, intestinotrophic mechanisms associated with or secondary to medical or surgical obesity therapies are poorly understood. Therefore, the objective of this study was to assess the local basal endogenous and pharmacological intestinotrophic effects of glucagon-like peptides and bariatric surgery in mice. We used in situ hybridization to provide a detailed and comparative anatomical map of the local distribution of GLP-1 receptor ( Glp1r ), GLP-2 receptor ( Glp2r ), and preproglucagon ( Gcg ) mRNA expression throughout the mouse gastrointestinal tract. Gut development in GLP-1R-, GLP-2R-, or GCG-deficient mice was compared to their corresponding wild-type controls, and intestinotrophic effects of GLP-1 and GLP-2 analogs were assessed in wild-type mice. Lastly, gut volume was determined in a mouse model of vertical sleeve gastrectomy (VSG). Comparison of Glp1r , Glp2r , and Gcg mRNA expression indicated a widespread, but distinct, distribution of these three transcripts throughout all compartments of the mouse gastrointestinal tract. While mice null for Glp1r or Gcg showed normal intestinal morphology, Glp2r -/- mice exhibited a slight reduction in small intestinal mucosa volume. Pharmacological treatment with GLP-1 and GLP-2 analogs significantly increased gut volume. In contrast, VSG surgery had no effect on intestinal morphology. The present study indicates that the endogenous preproglucagon system, exemplified by the entire GCG gene and the receptors for GLP-1 and GLP-2, does not play a major role in normal gut development in the mouse. Furthermore, elevation in local intestinal and circulating levels of GLP-1 and GLP-2 achieved after VSG has limited impact on intestinal morphometry. Hence, although exogenous treatment with GLP-1 and GLP-2 analogs enhances gut growth, the contributions of endogenously-secreted GLP-1 and GLP-2 to gut growth may be more modest and highly context-dependent.

N-myc regulates growth and fiber cell differentiation in lens development

PubMed Central

Cavalheiro, Gabriel R.; Matos-Rodrigues, Gabriel E.; Zhao, Yilin; Gomes, Anielle L.; Anand, Deepti; Predes, Danilo; de Lima, Silmara; Abreu, Jose G.; Zheng, Deyou; Lachke, Salil A.; Cvekl, Ales; Martins, Rodrigo A. P.

2017-01-01

Myc proto-oncogenes regulate diverse cellular processes during development, but their roles during morphogenesis of specific tissues are not fully understood. We found that c-myc regulates cell proliferation in mouse lens development and previous genome-wide studies suggested functional roles for N-myc in developing lens. Here, we examined the role of N-myc in mouse lens development. Genetic inactivation of N-myc in the surface ectoderm or lens vesicle impaired eye and lens growth, while "late" inactivation in lens fibers had no effect. Unexpectedly, defective growth of N-myc--deficient lenses was not associated with alterations in lens progenitor cell proliferation or survival. Notably, N-myc-deficient lens exhibited a delay in degradation of DNA in terminally differentiating lens fiber cells. RNA-sequencing analysis of N-myc--deficient lenses identified a cohort of down-regulated genes associated with fiber cell differentiation that included DNaseIIβ. Further, an integrated analysis of differentially expressed genes in N-myc-deficient lens using normal lens expression patterns of iSyTE, N-myc-binding motif analysis and molecular interaction data from the String database led to the derivation of an N-myc-based gene regulatory network in the lens. Finally, analysis of N-myc and c-myc double-deficient lens demonstrated that these Myc genes cooperate to drive lens growth prior to lens vesicle stage. Together, these findings provide evidence for exclusive and cooperative functions of Myc transcription factors in mouse lens development and identify novel mechanisms by which N-myc regulates cell differentiation during eye morphogenesis. PMID:28716713

p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas.

PubMed

Hendley, Audrey M; Provost, Elayne; Bailey, Jennifer M; Wang, Yue J; Cleveland, Megan H; Blake, Danielle; Bittman, Ross W; Roeser, Jeffrey C; Maitra, Anirban; Reynolds, Albert B; Leach, Steven D

2015-03-01

The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development. Copyright © 2014 Elsevier Inc. All rights reserved.

Sim1 is required for the migration and axonal projections of V3 interneurons in the developing mouse spinal cord.

PubMed

Blacklaws, Jake; Deska-Gauthier, Dylan; Jones, Christopher T; Petracca, Yanina L; Liu, Mingwei; Zhang, Han; Fawcett, James P; Glover, Joel C; Lanuza, Guillermo M; Zhang, Ying

2015-09-01

V3 spinal interneurons (INs) are a group of excitatory INs that play a crucial role in producing balanced and stable gaits in vertebrate animals. In the developing mouse spinal cord, V3 INs arise from the most ventral progenitor domain and form anatomically distinctive subpopulations in adult spinal cords. They are marked by the expression of transcription factor Sim1 postmitotically, but the function of Sim1 in V3 development remains unknown. Here, we used Sim1(Cre) ;tdTomato mice to trace the fate of V3 INs in a Sim1 mutant versus control genetic background during development. In Sim1 mutants, V3 INs are produced normally and maintain a similar position and organization as in wild types before E12.5. Further temporal analysis revealed that the V3 INs in the mutants failed to migrate properly to form V3 subgroups along the dorsoventral axis of the spinal cord. At birth, in the Sim1 mutant the number of V3 INs in the ventral subgroup was normal, but they were significantly reduced in the dorsal subgroup with a concomitant increase in the intermediate subgroup. Retrograde labeling at lumbar level revealed that loss of Sim1 led to a reduction in extension of contralateral axon projections both at E14.5 and P0 without affecting ipsilateral axon projections. These results demonstrate that Sim1 is essential for proper migration and the guidance of commissural axons of the spinal V3 INs. © 2015 Wiley Periodicals, Inc.

Hmx1 is required for the normal development of somatosensory neurons in the geniculate ganglion

PubMed Central

Quina, Lely A.; Tempest, Lynne; Hsu, Yun-Wei A.; Cox, Timothy C.; Turner, Eric E.

2012-01-01

Hmx1 is a variant homeodomain transcription factor expressed in the developing sensory nervous system, retina, and craniofacial mesenchyme. Recently, mutations at the Hmx1 locus have been linked to craniofacial defects in humans, rats, and mice, but its role in nervous system development is largely unknown. Here we show that Hmx1 is expressed in a subset of sensory neurons in the cranial and dorsal root ganglia which does not correspond to any specific sensory modality. Sensory neurons in the dorsal root and trigeminal ganglia of Hmx1dm/dm mouse embryos have no detectable Hmx1 protein, yet they undergo neurogenesis and express sensory subtype markers normally, demonstrating that Hmx1 is not globally required for the specification of sensory neurons from neural crest precursors. Loss of Hmx1 expression has no obvious effect on the early development of the trigeminal (V), superior (IX/X), or dorsal root ganglia neurons in which it is expressed, but results in marked defects in the geniculate (VII) ganglion. Hmx1dm/dm mouse embryos possess only a vestigial posterior auricular nerve, and general somatosensory neurons in the geniculate ganglion are greatly reduced by mid-gestation. Although Hmx1 is expressed in geniculate neurons prior to cell cycle exit, it does not appear to be required for neurogenesis, and the loss of geniculate neurons is likely to be the result of increased cell death. Fate mapping of neural crest-derived tissues indicates that Hmx1-expressing somatosensory neurons at different axial levels may be derived from either the neural crest or the neurogenic placodes. PMID:22586713

Altered procollagen gene expression in mid-gestational mouse excisional wounds.

PubMed

Goldberg, Stephanie R; Quirk, Gerald L; Sykes, Virginia W; Kordula, Tomasz; Lanning, David A

2007-11-01

Many pathologic conditions are characterized by excessive tissue contraction and scar formation. Previously, we developed a murine model of excisional wound healing in which mid-gestational wounds heal scarlessly compared with late-gestational wounds. We theorized that variations in procollagen gene expression may contribute to the scarless and rapid closure. Time-dated pregnant FVB strain mice underwent laparotomy and hysterotomy on embryonic days 15 (E15) and 18 (E18). Full-thickness, excisional wounds (3 mm) were made on each of 4 fetuses per doe and then harvested at 32, 48, or 72 h. Control tissue consisted of age-matched normal fetal skin. Procollagen types 1alpha1, 1alpha2, and 3 gene expressions were measured by real-time polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase. Trichrome staining was also performed. Procollagen 1alpha1 expression was decreased in E15 wounds at 32 h compared with their normal skin groups. Procollagen types 1alpha2 and 3 expressions were both increased in the E15 groups compared with the E18 groups at 48 h. At 72 h, the E15 wounds had a collagen density similar to the surrounding normal skin while E18 wounds exhibited increased collagen deposition in a disorganized pattern. This study demonstrates that the pattern of gene expression for types 1 and 3 collagen varies between mid- and late-gestational mouse excisional wounds. These alterations in procollagen expression may contribute to a pattern of collagen deposition in the mid-gestational fetuses that is more favorable for scarless healing with less type 1 and more type 3 collagen.

Using Genetic Mouse Models to Gain Insight into Glaucoma: Past Results and Future Possibilities

PubMed Central

Fernandes, Kimberly A.; Harder, Jeffrey M.; Williams, Pete A.; Rausch, Rebecca L.; Kiernan, Amy E.; Nair, K. Saidas; Anderson, Michael G.; John, Simon W.; Howell, Gareth R.; Libby, Richard T.

2015-01-01

While all forms of glaucoma are characterized by a specific pattern of retinal ganglion cell death, they are clinically divided into several distinct subclasses, including normal tension glaucoma, primary open angle glaucoma, congenital glaucoma, and secondary glaucoma. For each type of glaucoma there are likely numerous molecular pathways that control susceptibility to the disease. Given this complexity, a single animal model will never precisely model all aspects of all the different types of human glaucoma. Therefore, multiple animal models have been utilized to study glaucoma but more are needed. Because of the powerful genetic tools available to use in the laboratory mouse, it has proven to be a highly useful mammalian system for studying the pathophysiology of human disease. The similarity between human and mouse eyes coupled with the ability to use a combination of advanced cell biological and genetic tools in mice have led to a large increase in the number of studies using mice to model specific glaucoma phenotypes. Over the last decade, numerous new mouse models and genetic tools have emerged, providing important insight into the cell biology and genetics of glaucoma. In this review, we describe available mouse genetic models that can be used to study glaucoma-relevant disease/pathobiology. Furthermore, we discuss how these models have been used to gain insights into ocular hypertension (a major risk factor for glaucoma) and glaucomatous retinal ganglion cell death. Finally, the potential for developing new mouse models and using advanced genetic tools and resources for studying glaucoma are discussed. PMID:26116903

Characterization of kallikrein-related peptidase 4 glycosylations

PubMed Central

Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C.; Simmer, James P.

2012-01-01

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1–6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. PMID:22243251

Characterization of kallikrein-related peptidase 4 glycosylations.

PubMed

Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C; Simmer, James P

2011-12-01

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. © 2011 Eur J Oral Sci.

The Small GTPase Rif Is Dispensable for Platelet Filopodia Generation in Mice

PubMed Central

Goggs, Robert; Savage, Joshua S.; Mellor, Harry; Poole, Alastair W.

2013-01-01

Background Formation of filopodia and other shape change events are vital for platelet hemostatic function. The mechanisms regulating filopodia formation by platelets are incompletely understood however. In particular the small GTPase responsible for initiating filopodia formation by platelets remains elusive. The canonical pathway involving Cdc42 is not essential for filopodia formation in mouse platelets. The small GTPase Rif (RhoF) provides an alternative route to filopodia generation in other cell types and is expressed in both human and mouse platelets. Hypothesis/Objective We hypothesized that Rif might be responsible for generating filopodia by platelets and generated a novel knockout mouse model to investigate the functional role of Rif in platelets. Methodology/Principal Findings Constitutive RhoF−/− mice are viable and have normal platelet, leukocyte and erythrocyte counts and indices. RhoF−/− platelets form filopodia and spread normally on various agonist surfaces in static conditions and under arterial shear. In addition, RhoF−/− platelets have normal actin dynamics, are able to activate and aggregate normally and secrete from alpha and dense granules in response to collagen related peptide and thrombin stimulation. Conclusions The small GTPase Rif does not appear to be critical for platelet function in mice. Functional overlap between Rif and other small GTPases may be responsible for the non-essential role of Rif in platelets. PMID:23359340

Cystic Fibrosis Heterozygote Resistance to Cholera Toxin in the Cystic Fibrosis Mouse Model

NASA Astrophysics Data System (ADS)

Gabriel, Sherif E.; Brigman, Kristen N.; Koller, Beverly H.; Boucher, Richard C.; Stutts, M. Jackson

1994-10-01

The effect of the number of cystic fibrosis (CF) alleles on cholera toxin (CT)-induced intestinal secretion was examined in the CF mouse model. CF mice that expressed no CF transmembrane conductance regulator (CFTR) protein did not secrete fluid in response to CT. Heterozygotes expressed 50 percent of the normal amount of CFTR protein in the intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion in response to CT. This correlation between CFTR protein and CT-induced chloride ion and fluid secretion suggests that CF heterozygotes might possess a selective advantage of resistance to cholera.

University of California San Francisco (UCSF-2): Gene Expression Profiling of Normal Mouse Skin, Hras WT and Hras -/- | Office of Cancer Genomics

Cancer.gov

University of California San Francisco (UCSF-2): Gene Expression Profiling of Normal Mouse Skin, Hras WT and Hras -/- This data set contains the transcriptional profiles of 20 dorsal skin samples from eight-week-old mice. Mice were generated by crossing FVB/N to Mus spretus mice to generate F1 mice, and then crossing F1 mice back to the FVB/N strain. 10  FVB/N mice lacking Hras1 (aka HrasKO, Hras-/-) and 10  FVB/N mice with wild-type Hras1 were generated. Read the abstract.

Chimeric Mouse model to track the migration of bone marrow derived cells in glioblastoma following anti-angiogenic treatments.

PubMed

Achyut, B R; Shankar, Adarsh; Iskander, A S M; Ara, Roxan; Knight, Robert A; Scicli, Alfonso G; Arbab, Ali S

2016-01-01

Bone marrow derived cells (BMDCs) have been shown to contribute in the tumor development. In vivo animal models to investigate the role of BMDCs in tumor development are poorly explored. We established a novel chimeric mouse model using as low as 5 × 10(6) GFP+ BM cells in athymic nude mice, which resulted in >70% engraftment within 14 d. In addition, chimera was established in NOD-SCID mice, which displayed >70% with in 28 d. Since anti-angiogenic therapies (AAT) were used as an adjuvant against VEGF-VEGFR pathway to normalize blood vessels in glioblastoma (GBM), which resulted into marked hypoxia and recruited BMDCs to the tumor microenvironment (TME). We exploited chimeric mice in athymic nude background to develop orthotopic U251 tumor and tested receptor tyrosine kinase inhibitors and CXCR4 antagonist against GBM. We were able to track GFP+ BMDCs in the tumor brain using highly sensitive multispectral optical imaging instrument. Increased tumor growth associated with the infiltration of GFP+ BMDCs acquiring suppressive myeloid and endothelial phenotypes was seen in TME following treatments. Immunofluorescence study showed GFP+ cells accumulated at the site of VEGF, SDF1 and PDGF expression, and at the periphery of the tumors following treatments. In conclusion, we developed a preclinical chimeric model of GBM and phenotypes of tumor infiltrated BMDCs were investigated in context of AATs. Chimeric mouse model could be used to study detailed cellular and molecular mechanisms of interaction of BMDCs and TME in cancer.

Onlay bone augmentation on mouse calvarial bone using a hydroxyapatite/collagen composite material with total blood or platelet-rich plasma.

PubMed

Ohba, Seigo; Sumita, Yoshinori; Umebayashi, Mayumi; Yoshimura, Hitoshi; Yoshida, Hisato; Matsuda, Shinpei; Kimura, Hideki; Asahina, Izumi; Sano, Kazuo

2016-01-01

The aim of this study was to assess newly formed onlay bone on mouse calvarial bone using a new artificial bone material, a hydroxyapatite/collagen composite, with total blood or platelet-rich plasma. The hydroxyapatite/collagen composite material with normal saline, total blood or platelet-rich plasma was transplanted on mouse calvarial bone. The mice were sacrificed and the specimens were harvested four weeks after surgery. The newly formed bone area was measured on hematoxylin and eosin stained specimens using Image J software. The hydroxyapatite/collagen composite materials with total blood or platelet-rich plasma induced a significantly greater amount of newly formed bone than that with normal saline. Moreover, bone marrow was observed four weeks after surgery in the transplanted materials with total blood or platelet-rich plasma but not with normal saline. However, there were no significant differences in the amount of newly formed bone between materials used with total blood versus platelet-rich plasma. The hydroxyapatite/collagen composite material was valid for onlay bone augmentation and this material should be soaked in total blood or platelet-rich plasma prior to transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Olivocochlear neuron central anatomy is normal in alpha 9 knockout mice.

PubMed

Brown, M Christian; Vetter, Douglas E

2009-03-01

Olivocochlear (OC) neurons were studied in a transgenic mouse with deletion of the alpha 9 nicotinic acetylcholine receptor subunit. In this alpha 9 knockout mouse, the peripheral effects of OC stimulation are lacking and the peripheral terminals of OC neurons under outer hair cells have abnormal morphology. To account for this mouse's apparently normal hearing, it has been proposed to have central compensation via collateral branches to the cochlear nucleus. We tested this idea by staining OC neurons for acetylcholinesterase and examining their morphology in knockout mice, wild-type mice of the same background strain, and CBA/CaJ mice. Knockout mice had normal OC systems in terms of numbers of OC neurons, dendritic patterns, and numbers of branches to the cochlear nucleus. The branch terminations were mainly to edge regions and to a lesser extent the core of the cochlear nucleus, and were similar among the strains in terms of the distribution and staining density. These data demonstrate that there are no obvious changes in the central morphology of the OC neurons in alpha 9 knockout mice and make less attractive the idea that there is central compensation for deletion of the peripheral receptor in these mice.

Smooth muscle cell-specific knockout of androgen receptor: a new model for prostatic disease.

PubMed

Welsh, Michelle; Moffat, Lindsey; McNeilly, Alan; Brownstein, David; Saunders, Philippa T K; Sharpe, Richard M; Smith, Lee B

2011-09-01

Androgen-driven stromal-epithelial interactions play a key role in normal prostate development and function as well as in the progression of common prostatic diseases such as benign prostatic hyperplasia and prostate cancer. However, exactly how, and via which cell type, androgens mediate their effects in the adult prostate remains unclear. This study investigated the role for smooth muscle (SM) androgen signaling in normal adult prostate homeostasis and function using mice in which androgen receptor was selectively ablated from prostatic SM cells. In adulthood the knockout (KO) mice displayed a 44% reduction in prostate weight and exhibited histological abnormalities such as hyperplasia, inflammation, fibrosis, and reduced expression of epithelial, SM, and stem cell identify markers (e.g. p63 reduced by 27% and Pten by 31%). These changes emerged beyond puberty and were not explained by changes in serum hormones. Furthermore, in response to exogenous estradiol, adult KO mice displayed an 8.5-fold greater increase in prostate weight than controls and developed urinary retention. KO mice also demonstrated a reduced response to castration compared with controls. Together these results demonstrate that prostate SM cells are vital in mediating androgen-driven stromal-epithelial interactions in adult mouse prostates, determining cell identity and function and limiting hormone-dependent epithelial cell proliferation. This novel mouse model provides new insight into the possible role for SM androgen action in prostate disease.

Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis

PubMed Central

Leung, Lisa; Radulovich, Nikolina; Zhu, Chang-Qi; Wang, Dennis; To, Christine; Ibrahimov, Emin; Tsao, Ming-Sound

2013-01-01

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS G12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells. PMID:24386371

The Virtual Mouse Brain: A Computational Neuroinformatics Platform to Study Whole Mouse Brain Dynamics.

PubMed

Melozzi, Francesca; Woodman, Marmaduke M; Jirsa, Viktor K; Bernard, Christophe

2017-01-01

Connectome-based modeling of large-scale brain network dynamics enables causal in silico interrogation of the brain's structure-function relationship, necessitating the close integration of diverse neuroinformatics fields. Here we extend the open-source simulation software The Virtual Brain (TVB) to whole mouse brain network modeling based on individual diffusion magnetic resonance imaging (dMRI)-based or tracer-based detailed mouse connectomes. We provide practical examples on how to use The Virtual Mouse Brain (TVMB) to simulate brain activity, such as seizure propagation and the switching behavior of the resting state dynamics in health and disease. TVMB enables theoretically driven experimental planning and ways to test predictions in the numerous strains of mice available to study brain function in normal and pathological conditions.

Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

PubMed

Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

2011-05-01

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

Adamts18 deletion results in distinct developmental defects and provides a model for congenital disorders of lens, lung, and female reproductive tract development

PubMed Central

Ataca, Dalya; Caikovski, Marian; Piersigilli, Alessandra; Moulin, Alexandre; Benarafa, Charaf; Earp, Sarah E.; Guri, Yakir; Kostic, Corinne; Arsenivic, Yvan; Soininen, Raija; Apte, Suneel S.

2016-01-01

ABSTRACT The ADAMTS family comprises 19 secreted metalloproteinases that cleave extracellular matrix components and have diverse functions in numerous disease and physiological contexts. A number of them remain ‘orphan’ proteases and among them is ADAMTS18, which has been implicated in developmental eye disorders, platelet function and various malignancies. To assess in vivo function of ADAMTS18, we generated a mouse strain with inactivated Adamts18 alleles. In the C57Bl6/Ola background, Adamts18-deficient mice are born in a normal Mendelian ratio, and are viable but show a transient growth delay. Histological examination revealed a 100% penetrant eye defect resulting from leakage of lens material through the lens capsule occurring at embryonic day (E)13.5, when the lens grows rapidly. Adamts18-deficient lungs showed altered bronchiolar branching. Fifty percent of mutant females are infertile because of vaginal obstruction due to either a dorsoventral vaginal septum or imperforate vagina. The incidence of ovarian rete is increased in the mutant mouse strain. Thus, Adamts18 is essential in the development of distinct tissues and the new mouse strain is likely to be useful for investigating ADAMTS18 function in human disease, particularly in the contexts of infertility and carcinogenesis. PMID:27638769

Low sociability is associated with reduced size of the corpus callosum in the BALB/cJ inbred mouse strain.

PubMed

Fairless, Andrew H; Dow, Holly C; Toledo, Monica M; Malkus, Kristen A; Edelmann, Michele; Li, Hongzhe; Talbot, Konrad; Arnold, Steven E; Abel, Ted; Brodkin, Edward S

2008-09-16

The behavioral manifestations of autism, including reduced sociability (reduced tendency to seek social interaction), may be related to underdevelopment of the corpus callosum (CC). The BALB/cJ inbred mouse strain is a useful model system for testing the relationship between reduced sociability and CC underdevelopment. BALB/cJ mice show low levels of sociability, on average, but substantial intrastrain variability in sociability, as well as striking variability in CC development. This study tested the hypothesis that sociability is positively correlated with CC size within the BALB/cJ inbred strain. 30-day-old BALB/cJ and C57BL/6J mice were tested for sociability towards gonadectomized A/J stimulus mice in a social choice task. The size of the corpus callosum was measured histologically at the midsagittal plane. BALB/cJ mice showed a significant positive correlation between the tendency to sniff the stimulus mouse and size of the CC relative to brain weight. C57BL/6J mice showed consistently high levels of sociability and normal corpus callosum development. These results suggest that abnormal white matter structure is associated with deficits in sociability in BALB/cJ mice. Additional studies are warranted to elucidate the relationship between brain connectivity and sociability in this model system.

Mice with Inactivation of Aryl Hydrocarbon Receptor-Interacting Protein (Aip) Display Complete Penetrance of Pituitary Adenomas with Aberrant ARNT Expression

PubMed Central

Raitila, Anniina; Lehtonen, Heli J.; Arola, Johanna; Heliövaara, Elina; Ahlsten, Manuel; Georgitsi, Marianthi; Jalanko, Anu; Paetau, Anders; Aaltonen, Lauri A.; Karhu, Auli

2010-01-01

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been shown to predispose to pituitary adenoma predisposition, a condition characterized by growth hormone (GH)-secreting pituitary tumors. To study AIP-mediated tumorigenesis, we generated an Aip mouse model. Heterozygous mice developed normally but were prone to pituitary adenomas, in particular to those secreting GH. A complete loss of AIP was detected in these lesions, and full penetrance was reached at the age of 15 months. No excess of any other tumor type was found. Ki-67 analysis indicated that Aip-deficient tumors have higher proliferation rates compared with Aip-proficient tumors, suggesting a more aggressive disease. Similar to human AIP-deficient pituitary adenomas, immunohistochemical studies showed that expression of aryl hydrocarbon receptor nuclear translocator 1 or 2 (ARNT or ARNT2) protein was lost in the mouse tumors, suggesting that mechanisms of AIP-related tumorigenesis involve aberrant ARNT function. The Aip+/− mouse appears to be an excellent model for the respective human disease phenotype. This model constitutes a tool to further study AIP-associated pituitary tumorigenesis and may be potentially valuable in efforts to develop therapeutic strategies to treat pituitary adenomas. PMID:20709796

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

PubMed Central

Pennington, Kathleen A.; Schlitt, Jessica M.; Schulz, Laura C.

2012-01-01

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained for counting. PMID:22257865

Mouse models to study the interaction of risk factors for human liver cancer.

PubMed

Sell, Stewart

2003-11-15

Each of the risk factors for human liver cancer (aflatoxin exposure, hepatitis B virus-associated liver injury, p53 loss, p53ser249 mutation, and male sex) also increases the incidence of hepatocellular carcinoma (HCC) in mouse models of hepatocarcinogenesis. Neonatal mice, partially hepatectomized adult mice, and p53-deficient mice each have a higher hepatocyte proliferation rate, are less able to detoxify AFB1, and form more DNA adducts than do normal wild-type controls. However, transgenic hepatitis B surface antigen mice, expressing hepatitis B surface antigen under control of the albumin promoter (alb/psx), are able to detoxify AFB1 at the same level as do wild-type mice. Thus, AFB1-induced HCC development in neonatal mice and p53+/- mice may be due to "immature" carcinogen metabolism, whereas increased HCC in transgenic hepatitis B virus mice may be due to promotion effects of increased proliferation. Future studies will explore the effects of modifying factors on the development of HCC.

Generation of a high-fidelity antibody against nerve growth factor using library scanning mutagenesis and validation with structures of the initial and optimized Fab-antigen complexes

PubMed Central

La Porte, Sherry L; Eigenbrot, Charles; Ultsch, Mark; Ho, Wei-Hsien; Foletti, Davide; Forgie, Alison; Lindquist, Kevin C; Shelton, David L; Pons, Jaume

2014-01-01

Nerve growth factor (NGF) is indispensable during normal embryonic development and critical for the amplification of pain signals in adults. Intervention in NGF signaling holds promise for the alleviation of pain resulting from human diseases such as osteoarthritis, cancer and chronic lower back disorders. We developed a fast, high-fidelity method to convert a hybridoma-derived NGF-targeted mouse antibody into a clinical candidate. This method, termed Library Scanning Mutagenesis (LSM), resulted in the ultra-high affinity antibody tanezumab, a first-in-class anti-hyperalgesic specific for an NGF epitope. Functional and structural comparisons between tanezumab and the mouse 911 precursor antibody using neurotrophin-specific cell survival assays and X-ray crystal structures of both Fab-antigen complexes illustrated high fidelity retention of the NGF epitope. These results suggest the potential for wide applicability of the LSM method for optimization of well-characterized antibodies during humanization. PMID:24830649

Development of a functional thyroid model based on an organoid culture system.

PubMed

Saito, Yoshiyuki; Onishi, Nobuyuki; Takami, Hiroshi; Seishima, Ryo; Inoue, Hiroyoshi; Hirata, Yuki; Kameyama, Kaori; Tsuchihashi, Kenji; Sugihara, Eiji; Uchino, Shinya; Ito, Koichi; Kawakubo, Hirofumi; Takeuchi, Hiroya; Kitagawa, Yuko; Saya, Hideyuki; Nagano, Osamu

2018-03-04

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid-like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRAS Q61R ) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary.

PubMed

Park, E-H; Taketo, T

2003-12-01

When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.YTIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.YTIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.

Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta

PubMed Central

Poulter, James A.; Murillo, Gina; Brookes, Steven J.; Smith, Claire E. L.; Parry, David A.; Silva, Sandra; Kirkham, Jennifer; Inglehearn, Chris F.; Mighell, Alan J.

2014-01-01

Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid. PMID:24858907

seq-ImmuCC: Cell-Centric View of Tissue Transcriptome Measuring Cellular Compositions of Immune Microenvironment From Mouse RNA-Seq Data.

PubMed

Chen, Ziyi; Quan, Lijun; Huang, Anfei; Zhao, Qiang; Yuan, Yao; Yuan, Xuye; Shen, Qin; Shang, Jingzhe; Ben, Yinyin; Qin, F Xiao-Feng; Wu, Aiping

2018-01-01

The RNA sequencing approach has been broadly used to provide gene-, pathway-, and network-centric analyses for various cell and tissue samples. However, thus far, rich cellular information carried in tissue samples has not been thoroughly characterized from RNA-Seq data. Therefore, it would expand our horizons to better understand the biological processes of the body by incorporating a cell-centric view of tissue transcriptome. Here, a computational model named seq-ImmuCC was developed to infer the relative proportions of 10 major immune cells in mouse tissues from RNA-Seq data. The performance of seq-ImmuCC was evaluated among multiple computational algorithms, transcriptional platforms, and simulated and experimental datasets. The test results showed its stable performance and superb consistency with experimental observations under different conditions. With seq-ImmuCC, we generated the comprehensive landscape of immune cell compositions in 27 normal mouse tissues and extracted the distinct signatures of immune cell proportion among various tissue types. Furthermore, we quantitatively characterized and compared 18 different types of mouse tumor tissues of distinct cell origins with their immune cell compositions, which provided a comprehensive and informative measurement for the immune microenvironment inside tumor tissues. The online server of seq-ImmuCC are freely available at http://wap-lab.org:3200/immune/.

In vivo biometry in the mouse eye with low coherence interferometry.

PubMed

Schmucker, Christine; Schaeffel, Frank

2004-01-01

A major drawback of the mouse model of myopia is that the ocular dimensions cannot be measured in vivo, and that histological techniques post-mortem suffer from limited resolution. We have tested the potential of a newly developed technique, optical low coherence interferometry (OLCI), adapted for short measurement distances by Meditec, Carl Zeiss, Jena, Germany (the "ACMaster"). Using this technique, ocular biometry was performed in mice with normal vision and after deprivation of form vision. Axial eye length, corneal thickness and anterior chamber depth were measured in 23 mice, aged 25-53 days, and standard deviations from repeated measurements in the same eyes, as well as intra-individual and inter-individual variability were determined in different age groups. The data were compared to those from a preceding study in which biometrical data were obtained from frozen sections [Vision Res. 44 (2004) 1857]. Refractions were measured by automated infrared photorefraction. Mice had either normal visual exposure or were monocularly deprived of form vision for 14 days. Using OLCI, axial length could be determined with an average standard deviation of 8.0 +/- 2.9 microm, corneal thickness with 3.5 +/- 2.1 microm, and anterior chamber depth with 10.6 +/- 12.3 microm. Neither axial length, nor corneal thickness, nor anterior chamber depth were significantly different in left and right eyes of individual mice that had normal visual experience (mean absolute difference between axial lengths: 17 +/- 18 microm, between corneal thickness 5.1 +/- 4.8 microm, and between anterior chamber depths 16.7 +/- 14.8 microm). Compared to the variability that was previously found in frozen sections, the variability of axial length measurements with OLCI was 2.7 times less. After two weeks of form deprivation, OLCI revealed a significant axial elongation in the occluded eyes, compared to the contralateral fellow eyes (+38 +/- 36 microm or 1.16%, p = 0.045, n = 7, paired t-test). In this sample, no accompanying myopic shift was observed in the occluded eyes but this observation is not unexpected given the inherently variable responses of mouse eye growth to visual deprivation. OLCI had sufficient resolution in living mice to detect axial length changes in vivo that were equivalent to a dioptric change of 2 D. Using this technique, it was confirmed that mouse eyes respond to form deprivation by axial elongation, similar to the eyes of other animal models. The lack of a myopic shift in this sample, despite the axial elongation, demonstrates that biometric data are particularly important when the mouse eye is used as a model to study myopia.

Mouse cones require an arrestin for normal inactivation of phototransduction.

PubMed

Nikonov, Sergei S; Brown, Bruce M; Davis, Jason A; Zuniga, Freddi I; Bragin, Alvina; Pugh, Edward N; Craft, Cheryl M

2008-08-14

Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.

Mouse genetic corneal disease resulting from transgenic insertional mutagenesis

PubMed Central

Ramalho, J S; Gregory-Evans, K; Huxley, C; Seabra, M C

2004-01-01

Background/aims: To report the generation of a new mouse model for a genetically determined corneal abnormality that occurred in transgenesis experiments. Methods: Transgenic mice expressing mutant forms of Rab27a, a GTPase that has been implicated in the pathogenesis of choroideremia, were generated. Results: Only one transgenic line (T27aT15) exhibited an unexpected eye phenotype. T27aT15 mice developed corneal opacities, usually unilateral, and cataracts, resulting in some cases in phthisical eyes. Histologically, the corneal stroma was thickened and vacuolated, and both epithelium and endothelium were thinned. The posterior segment of the eye was also affected with abnormal pigmentation, vessel narrowing, and abnormal leakage of dye upon angiography but was histologically normal. Conclusion: Eye abnormality in T27aT15 mice results from random insertional mutagenesis of the transgene as it was only observed in one line. The corneal lesion observed in T27aT15 mice most closely resembles posterior polymorphous corneal dystrophy and might result from the disruption of the equivalent mouse locus. PMID:14977782

Eccentric localization of catalase to protect chromosomes from oxidative damages during meiotic maturation in mouse oocytes.

PubMed

Park, Yong Seok; You, Seung Yeop; Cho, Sungrae; Jeon, Hyuk-Joon; Lee, Sukchan; Cho, Dong-Hyung; Kim, Jae-Sung; Oh, Jeong Su

2016-09-01

The maintenance of genomic integrity and stability is essential for the survival of every organism. Unfortunately, DNA is vulnerable to attack by a variety of damaging agents. Oxidative stress is a major cause of DNA damage because reactive oxygen species (ROS) are produced as by-products of normal cellular metabolism. Cells have developed eloquent antioxidant defense systems to protect themselves from oxidative damage along with aerobic metabolism. Here, we show that catalase (CAT) is present in mouse oocytes to protect the genome from oxidative damage during meiotic maturation. CAT was expressed in the nucleus to form unique vesicular structures. However, after nuclear envelope breakdown, CAT was redistributed in the cytoplasm with particular focus at the chromosomes. Inhibition of CAT activity increased endogenous ROS levels, but did not perturb meiotic maturation. In addition, CAT inhibition produced chromosomal defects, including chromosome misalignment and DNA damage. Therefore, our data suggest that CAT is required not only to scavenge ROS, but also to protect DNA from oxidative damage during meiotic maturation in mouse oocytes.

A novel behavioral paradigm for assessing concept of nests in mice

PubMed Central

Kuang, Hui; Mei, Bing; Cui, Zhenzhong; Lin, Longnian; Tsien, Joe Z.

2013-01-01

Abstract concepts in the brain enable humans to efficiently and correctly recognize and categorize a seemingly infinite amount of objects and daily events. Such abstract generalization abilities are traditionally considered to be unique to humans and perhaps non-human primates. However, emerging neurophysiological recordings indicate the existence of neural correlates for the abstract concept of nests in the mouse brain. To facilitate the molecular and genetic analyses of concepts in the mouse model, we have developed a nest generalization test based on mice’s natural behavior. We show that inducible and forebrain-specific NMDA receptor knockout results in pronounced impairment in this test. Interestingly, this generalization deficit could be gradually compensated for over time by repeated experiences even in face of the continued deficit in object recognition memory. On the contrast, the forebrain-specific presenilin-1 knockout mice, which have subtle phenotypes, were normal in performing this test. Therefore, our study not only establishes a quantitative method for assessing the nest concept in mice, but also demonstrates its great potential in combining powerful mouse genetics for dissecting the molecular basis of concept formation in the brain. PMID:20350568

The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype

PubMed Central

BOOT-HANDFORD, R. P.; MICHAELIDIS, T. M.; HILLARBY, M. C.; ZAMBELLI, A.; DENTON, J.; HOYLAND, J. A.; FREEMONT, A. J.; GRANT, M. E.; WALLIS, G. A.

1998-01-01

Histological examination of long bones from 1-day-old bcl-2 knockout and age-matched control mice revealed no obvious differences in length of bone, growth plate architecture or stage of endochondral ossification. In 35-day-old bcl-2 knockout mice that are growth retarded or ‘dwarfed’, the proliferative zone of the growth plate appeared slightly thinner and the secondary centres of ossification less well developed than their age-matched wild-type controls. The most marked histological effects of bcl-2 ablation were on osteoblasts and bone. 35-day-old knockout mouse bones exhibited far greater numbers of osteoblasts than controls and the osteoblasts had a cuboidal phenotype in comparison with the normal flattened cell appearance. In addition, the collagen deposited by the osteoblasts in the bcl-2 knockout mouse bone was disorganized in comparison with control tissue and had a pseudo-woven appearance. The results suggest an important role for Bcl-2 in controlling osteoblast phenotype and bone deposition in vivo. PMID:10193316

A new mouse model of ARX dup24 recapitulates the patients' behavioral and fine motor alterations.

PubMed

Dubos, Aline; Meziane, Hamid; Iacono, Giovanni; Curie, Aurore; Riet, Fabrice; Martin, Christelle; Loaëc, Nadège; Birling, Marie-Christine; Selloum, Mohammed; Normand, Elisabeth; Pavlovic, Guillaume; Sorg, Tania; Stunnenberg, Henk G; Chelly, Jamel; Humeau, Yann; Friocourt, Gaëlle; Hérault, Yann

2018-06-15

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.

A new mouse model of ARX dup24 recapitulates the patients’ behavioral and fine motor alterations

PubMed Central

Dubos, Aline; Meziane, Hamid; Iacono, Giovanni; Curie, Aurore; Riet, Fabrice; Martin, Christelle; Loaëc, Nadège; Birling, Marie-Christine; Selloum, Mohammed; Normand, Elisabeth; Pavlovic, Guillaume; Sorg, Tania; Stunnenberg, Henk G; Chelly, Jamel; Humeau, Yann; Friocourt, Gaëlle; Hérault, Yann

2018-01-01

Abstract The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment. PMID:29659809

Lack of evidence that the XqYq pairing tips at meiosis in the mouse show hypersensitivity to DNAse I.

PubMed

Separovic, E R; Chandley, A C

1987-01-01

In situ nick translation procedures have been applied to meiotic metaphase I divisions of the normal and XY, Sxr mouse. Unlike in man, where the pairing tips of the XY bivalent show a special sensitivity to DNAse I nicking, no such sensitivity can be detected for either of these types of mouse. Hypersensitivity in the D-band equivalent region of the X chromosome does, however, exist, this site being early replicating in somatic cells and housing the X inactivation centre (Xce).

Induction of superficial cortical layer neurons from mouse embryonic stem cells by valproic acid.

PubMed

Juliandi, Berry; Abematsu, Masahiko; Sanosaka, Tsukasa; Tsujimura, Keita; Smith, Austin; Nakashima, Kinichi

2012-01-01

Within the developing mammalian cortex, neural progenitors first generate deep-layer neurons and subsequently more superficial-layer neurons, in an inside-out manner. It has been reported recently that mouse embryonic stem cells (mESCs) can, to some extent, recapitulate cortical development in vitro, with the sequential appearance of neurogenesis markers resembling that in the developing cortex. However, mESCs can only recapitulate early corticogenesis; superficial-layer neurons, which are normally produced in later developmental periods in vivo, are under-represented. This failure of mESCs to reproduce later corticogenesis in vitro implies the existence of crucial factor(s) that are absent or uninduced in existing culture systems. Here we show that mESCs can give rise to superficial-layer neurons efficiently when treated with valproic acid (VPA), a histone deacetylase inhibitor. VPA treatment increased the production of Cux1-positive superficial-layer neurons, and decreased that of Ctip2-positive deep-layer neurons. These results shed new light on the mechanisms of later corticogenesis. Copyright © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Tumor-homing peptides as tools for targeted delivery of payloads to the placenta

PubMed Central

King, Anna; Ndifon, Cornelia; Lui, Sylvia; Widdows, Kate; Kotamraju, Venkata R.; Agemy, Lilach; Teesalu, Tambet; Glazier, Jocelyn D.; Cellesi, Francesco; Tirelli, Nicola; Aplin, John D.; Ruoslahti, Erkki; Harris, Lynda K.

2016-01-01

The availability of therapeutics to treat pregnancy complications is severely lacking mainly because of the risk of causing harm to the fetus. As enhancement of placental growth and function can alleviate maternal symptoms and improve fetal growth in animal models, we have developed a method for targeted delivery of payloads to the placenta. We show that the tumor-homing peptide sequences CGKRK and iRGD bind selectively to the placental surface of humans and mice and do not interfere with normal development. Peptide-coated nanoparticles intravenously injected into pregnant mice accumulated within the mouse placenta, whereas control nanoparticles exhibited reduced binding and/or fetal transfer. We used targeted liposomes to efficiently deliver cargoes of carboxyfluorescein and insulin-like growth factor 2 to the mouse placenta; the latter significantly increased mean placental weight when administered to healthy animals and significantly improved fetal weight distribution in a well-characterized model of fetal growth restriction. These data provide proof of principle for targeted delivery of drugs to the placenta and provide a novel platform for the development of placenta-specific therapeutics. PMID:27386551

Gene Profiling in Experimental Models of Eye Growth: Clues to Myopia Pathogenesis

PubMed Central

Stone, Richard A.; Khurana, Tejvir S.

2010-01-01

To understand the complex regulatory pathways that underlie the development of refractive errors, expression profiling has evaluated gene expression in ocular tissues of well-characterized experimental models that alter postnatal eye growth and induce refractive errors. Derived from a variety of platforms (e.g. differential display, spotted microarrays or Affymetrix GeneChips), gene expression patterns are now being identified in species that include chicken, mouse and primate. Reconciling available results is hindered by varied experimental designs and analytical/statistical features. Continued application of these methods offers promise to provide the much-needed mechanistic framework to develop therapies to normalize refractive development in children. PMID:20363242

The Amyloid Precursor Protein (APP) Triplicated Gene Impairs Neuronal Precursor Differentiation and Neurite Development through Two Different Domains in the Ts65Dn Mouse Model for Down Syndrome*

PubMed Central

Trazzi, Stefania; Fuchs, Claudia; Valli, Emanuele; Perini, Giovanni; Bartesaghi, Renata; Ciani, Elisabetta

2013-01-01

Intellectual disability in Down syndrome (DS) appears to be related to severe proliferation impairment during brain development. Recent evidence shows that it is not only cellular proliferation that is heavily compromised in DS, but also cell fate specification and dendritic maturation. The amyloid precursor protein (APP), a gene that is triplicated in DS, plays a key role in normal brain development by influencing neural precursor cell proliferation, cell fate specification, and neuronal maturation. APP influences these processes via two separate domains, the APP intracellular domain (AICD) and the soluble secreted APP. We recently found that the proliferation impairment of neuronal precursors (NPCs) from the Ts65Dn mouse model for DS was caused by derangement of the Shh pathway due to overexpression of patched1(Ptch1), its inhibitory regulator. Ptch1 overexpression was related to increased levels within the APP/AICD system. The overall goal of this study was to determine whether APP contributes to neurogenesis impairment in DS by influencing in addition to proliferation, cell fate specification, and neurite development. We found that normalization of APP expression restored the reduced neuronogenesis, the increased astrogliogenesis, and the reduced neurite length of trisomic NPCs, indicating that APP overexpression underpins all aspects of neurogenesis impairment. Moreover, we found that two different domains of APP impair neuronal differentiation and maturation in trisomic NPCs. The APP/AICD system regulates neuronogenesis and neurite length through the Shh pathway, whereas the APP/secreted AP system promotes astrogliogenesis through an IL-6-associated signaling cascade. These results provide novel insight into the mechanisms underlying brain development alterations in DS. PMID:23740250

A novel ENU-induced mutation, peewee, causes dwarfism in the mouse

PubMed Central

Bon-Ryon, Lee; Kano, Kiyoshi; Young, Jay; John, Simon; Nishina, Patsy M; Naggert, Jurgen K; Naito, Kunihiko

2010-01-01

We identified a novel fertile, autosomal recessive mutation, called peewee and that results in dwarfing, in a region-specific ENU-induced mutagenesis. These mice at litter size were smaller those of other strains. Histological analysis revealed that the major organs appear normal, but abnormalities in cellular proliferation were observed in bone, liver and testis. Haplotype analysis localized the peewee gene to a 3.3-Mb region between D5Mit83 and D5Mit356.3. There are 18 genes in this linkage area, and we also performed in silico mapping using the PosMed℠ program, which searches for connections among keywords and genes in an interval, but no similar phenotype descriptions were found for these genes. In the peewee mutant compared to the normal, C57BL/6J mouse, only Slc10a4 expression was lower. Our preliminary mutation analysis examining the nucleotide sequence of three exons, two introns and an untranslated region of Slc10a4 did not find any sequence difference between the peewee mouse and the C57BL/6J mouse. Detailed analysis of peewee mice might provide novel molecular insights into the complex mechanisms regulating body growth. PMID:19513787

The wall traction induced by flowing red blood cells in model microvessels and its potential mechanotransduction

NASA Astrophysics Data System (ADS)

Freund, Jonathan; Vermot, Julien

2013-11-01

There is evidence in early embryonic development, even well before advective oxygen transport is important, that the presence of red bloods cells per se trigger essential steps of normal vascular development. For example, showed that sequestration of blood cells early in the development of a mouse, such that the hematocrit is reduced, suppresses normal vascular network development. Vascular development also provides a model for remodeling and angiogenesis. We consider the transient stresses associated with blood cells flowing in model microvessels of comparable diameter to those at early stages of development (6 μm to 12 μm). A detailed simulation tool is used to show that passing blood cells present a significant fluctuating traction signature on the vessel wall, well above the mean stresses. This is particularly pronounced for slow flows (<= 50 μm/s) or small diameters (<= 7 μm), for which root-mean-square wall traction fluctuations can exceed their mean. These events potentially present mechanotranduction triggers that direct development or remodeling. Attenuation of such fluctuating tractions by a viscoelastic endothelial glycocalyx layer is also considered. NSF supported.

Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

PubMed

Metildi, Cristina A; Kaushal, Sharmeela; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

2014-04-01

The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model. © 2013 Wiley Periodicals, Inc.

Intrauterine Growth Restriction Alters Mouse Intestinal Architecture during Development.

PubMed

Fung, Camille M; White, Jessica R; Brown, Ashley S; Gong, Huiyu; Weitkamp, Jörn-Hendrik; Frey, Mark R; McElroy, Steven J

2016-01-01

Infants with intrauterine growth restriction (IUGR) are at increased risk for neonatal and lifelong morbidities affecting multiple organ systems including the intestinal tract. The underlying mechanisms for the risk to the intestine remain poorly understood. In this study, we tested the hypothesis that IUGR affects the development of goblet and Paneth cell lineages, thus compromising the innate immunity and barrier functions of the epithelium. Using a mouse model of maternal thromboxane A2-analog infusion to elicit maternal hypertension and resultant IUGR, we tested whether IUGR alters ileal maturation and specifically disrupts mucus-producing goblet and antimicrobial-secreting Paneth cell development. We measured body weights, ileal weights and ileal lengths from birth to postnatal day (P) 56. We also determined the abundance of goblet and Paneth cells and their mRNA products, localization of cellular tight junctions, cell proliferation, and apoptosis to interrogate cellular homeostasis. Comparison of the murine findings with human IUGR ileum allowed us to verify observed changes in the mouse were relevant to clinical IUGR. At P14 IUGR mice had decreased ileal lengths, fewer goblet and Paneth cells, reductions in Paneth cell specific mRNAs, and decreased cell proliferation. These findings positively correlated with severity of IUGR. Furthermore, the decrease in murine Paneth cells was also seen in human IUGR ileum. IUGR disrupts the normal trajectory of ileal development, particularly affecting the composition and secretory products of the epithelial surface of the intestine. We speculate that this abnormal intestinal development may constitute an inherent "first hit", rendering IUGR intestine susceptible to further injury, infection, or inflammation.

Characterization of N-methyl-D-aspartate-evoked taurine release in the developing and adult mouse hippocampus.

PubMed

Saransaari, P; Oja, S S

2003-01-01

Taurine is an inhibitory amino acid acting as an osmoregulator and neuroromodulator in the brain, with neuroprotective properties. The ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) greatly potentiates taurine release from brain preparations in both normal and ischemic conditions, the effect being particularly marked in the developing hippocampus. We now characterized the regulation of NMDA-stimulated taurine release from hippocampal slices from adult (3-month-old) and developing (7-day-old) mouse using a superfusion system. The NMDA-stimulated taurine release was receptor-mediated in both adult and developing mouse hippocampus. In adults, only NO-generating compounds, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and hydroxylamine reduced the release, as did also NO synthase inhibitors, 7-nitroindazole and nitroarginine, indicating that the release is mediated by the NO/cGMP pathway. On the other hand, the regulation of the NMDA-evoked taurine release proved to be somewhat complex in the immature hippocampus. It was not affected by the NOergic compounds, but enhanced by the protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate and adenosine receptor A(1) agonists, N(6)-cyclohexyladenosine and R(-)N(6)-(2-phenylisopropyl)adenosine in a receptor-mediated manner. The activation of both ionotropic 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors and metabotropic glutamate group I receptors also enhanced the evoked release. The NMDA-receptor-stimulated taurine release could be a part of the neuroprotective properties of taurine, being important particularly under cell-damaging conditions in the developing hippocampus and hence preventing excitotoxicity.

Rod- and cone-driven responses in mice expressing human L-cone pigment

PubMed Central

Atorf, Jenny; Neitz, Maureen; Neitz, Jay

2015-01-01

The mouse is commonly used for studying retinal processing, primarily because it is amenable to genetic manipulation. To accurately study photoreceptor driven signals in the healthy and diseased retina, it is of great importance to isolate the responses of single photoreceptor types. This is not easily achieved in mice because of the strong overlap of rod and M-cone absorption spectra (i.e., maxima at 498 and 508 nm, respectively). With a newly developed mouse model (Opn1lwLIAIS) expressing a variant of the human L-cone pigment (561 nm) instead of the mouse M-opsin, the absorption spectra are substantially separated, allowing retinal physiology to be studied using silent substitution stimuli. Unlike conventional chromatic isolation methods, this spectral compensation approach can isolate single photoreceptor subtypes without changing the retinal adaptation. We measured flicker electroretinograms in these mutants under ketamine-xylazine sedation with double silent substitution (silent S-cone and either rod or M/L-cones) and obtained robust responses for both rods and (L-)cones. Small signals were yielded in wild-type mice, whereas heterozygotes exhibited responses that were generally intermediate to both. Fundamental response amplitudes and phase behaviors (as a function of temporal frequency) in all genotypes were largely similar. Surprisingly, isolated (L-)cone and rod response properties in the mutant strain were alike. Thus the LIAIS mouse warrants a more comprehensive in vivo assessment of photoreceptor subtype-specific physiology, because it overcomes the hindrance of overlapping spectral sensitivities present in the normal mouse. PMID:26245314

Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin.

PubMed

Takeo, Toru; Nakagata, Naomi

2011-11-01

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.

A Novel, Real-Time, In Vivo Mouse Retinal Imaging System.

PubMed

Butler, Mark C; Sullivan, Jack M

2015-11-01

To develop an efficient, low-cost instrument for robust real-time imaging of the mouse retina in vivo, and assess system capabilities by evaluating various animal models. Following multiple disappointing attempts to visualize the mouse retina during a subretinal injection using commercially available systems, we identified the key limitation to be inadequate illumination due to off axis illumination and poor optical train optimization. Therefore, we designed a paraxial illumination system for Greenough-type stereo dissecting microscope incorporating an optimized optical launch and an efficiently coupled fiber optic delivery system. Excitation and emission filters control spectral bandwidth. A color coupled-charged device (CCD) camera is coupled to the microscope for image capture. Although, field of view (FOV) is constrained by the small pupil aperture, the high optical power of the mouse eye, and the long working distance (needed for surgical manipulations), these limitations can be compensated by eye positioning in order to observe the entire retina. The retinal imaging system delivers an adjustable narrow beam to the dilated pupil with minimal vignetting. The optic nerve, vasculature, and posterior pole are crisply visualized and the entire retina can be observed through eye positioning. Normal and degenerative retinal phenotypes can be followed over time. Subretinal or intraocular injection procedures are followed in real time. Real-time, intravenous fluorescein angiography for the live mouse has been achieved. A novel device is established for real-time viewing and image capture of the small animal retina during subretinal injections for preclinical gene therapy studies.

Mitochondrial base excision repair in mouse synaptosomes during normal aging and in a model of Alzheimer’s disease

PubMed Central

Gredilla, Ricardo; Weissman, Lior; Yang, Jenq-Lin; Bohr, Vilhelm A.; Stevnsner, Tinna

2010-01-01

Brain aging is associated with synaptic decline and cognitive impairment. Increased levels of oxidative DNA base damage and accumulation of mitochondrial DNA (mtDNA) mutations or deletions lead to mitochondrial dysfunction, playing an important role in the aging process and the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD). In mitochondria, base excision repair (BER) is the main DNA repair pathway for base modifications such as deamination and oxidation, and constitutes an important mechanism to avoid accumulation of mtDNA mutations. Synaptic function is highly dependent on mitochondria, and in the current study we have investigated BER in synaptosomes of mouse brain during normal aging and in an AD model. Synaptosomes are isolated synapses in membranous structures produced by subcellular fractionation of brain tissue. They include the whole presynaptic terminal as well as portions of the postsynaptic terminal. Synaptosomes contain the molecular machinery necessary for uptake, storage, and release of neurotransmitters, including synaptic vesicles and mitochondria. BER activities were measured in synaptosomal fractions from young and old mice and from pre-symptomatic and symptomatic AD mice harboring mutated APP, Tau and PS1 (3xTgAD). During normal aging, a reduction in the BER capacity was observed in the synaptosomal fraction, which was associated with a decrease in the level of BER proteins. However, we did not observe changes between the synaptosomal BER activities of pre-symptomatic and symptomatic AD mice. Our findings suggest that the age-related reduction in BER capacity in the synaptosomal fraction might contribute to mitochondrial and synaptic dysfunction during aging. The development of AD-like pathology in the 3xTgAD mouse model was, however, not associated with deficiencies of the BER mechanisms in the synaptosomal fraction when the whole brain was analyzed. PMID:20708822

Histology Atlas of the Developing Mouse Hepatobiliary System with Emphasis on Embryonic Days 9.5-18.5

PubMed Central

Crawford, Laura Wilding; Foley, Julie F.; Elmore, Susan A.

2012-01-01

Animal model phenotyping, in utero exposure toxiciy studies, and investigation into causes of embryonic, fetal, or perinatal deaths have required pathologists to recognize and diagnose developmental disorders in spontaneous and engineered mouse models of disease. In mammals, the liver is the main site of hematopoiesis during fetal development, has endocrine and exocrine functions important for maintaining homeostasis in fetal and adult life; and performs other functions including waste detoxification, production and removal of glucose, glycogen storage, triglyceride and fatty acid processing, and serum protein production. Due to its role in many critical functions, alterations in the size, morphology, or function(s) of the liver often lead to embryonic lethality. Many publications and websites describe individual aspects of hepatobiliary development at defined stages. However, no single resource provides a detailed histological evaluation of H&E-stained sections of the developing murine liver and biliary systems using high-magnification and high-resolution color images. The work herein provides a histology atlas of hepatobiliary development between embryonic days 9.5-18.5. Although the focus of this work is normal hepatobiliary development, common defects in liver development are also described as a reference for pathologists who may be asked to phenotype mice with congenital, inherited, or treatment-related hepatobiliary defects. PMID:20805319

The Transcription Factor Atonal homolog 8 Regulates Gata4 and Friend of Gata-2 during Vertebrate Development

PubMed Central

Rawnsley, David R.; Xiao, Jiping; Lee, John S.; Liu, Xi; Mericko-Ishizuka, Patricia; Kumar, Vinayak; He, Jie; Basu, Arindam; Lu, MinMin; Lynn, Francis C.; Pack, Michael; Gasa, Rosa; Kahn, Mark L.

2013-01-01

GATA and Friend of GATA (FOG) form a transcriptional complex that plays a key role in cardiovascular development in both fish and mammals. In the present study we demonstrate that the basic helix-loop-helix transcription factor Atonal homolog 8 (Atoh8) is required for development of the heart in fish but not in mice. Genetic studies reveal that Atoh8 interacts specifically with Gata4 and Fog1 during development of the heart and swim bladder in the fish. Biochemical studies reveal that ATOH8, GATA4, and FOG2 associate in a single complex in vitro. In contrast to fish, ATOH8-deficient mice exhibit normal cardiac development and loss of ATOH8 does not alter cardiac development in Gata4+/− mice. This species difference in the role of ATOH8 is explained in part by LacZ and GFP reporter alleles that reveal restriction of Atoh8 expression to atrial but not ventricular myocardium in the mouse. Our findings identify ATOH8 as a novel regulator of GATA-FOG function that is required for cardiac development in the fish but not the mouse. Whether ATOH8 modulates GATA-FOG function at other sites or in more subtle ways in mammals is not yet known. PMID:23836893

Visualising Androgen Receptor Activity in Male and Female Mice

PubMed Central

Dart, D. Alwyn; Waxman, Jonathan; Aboagye, Eric O.; Bevan, Charlotte L.

2013-01-01

Androgens, required for normal development and fertility of males and females, have vital roles in the reproductive tract, brain, cardiovascular system, smooth muscle and bone. Androgens function via the androgen receptor (AR), a ligand-dependent transcription factor. To assay and localise AR activity in vivo we generated the transgenic “ARE-Luc” mouse, expressing a luciferase reporter gene under the control of activated endogenous AR. In vivo imaging of androgen-mediated luciferase activity revealed several strongly expressing tissues in the male mouse as expected and also in certain female tissues. In males the testes, prostate, seminal vesicles and bone marrow all showed high AR activity. In females, strong activity was seen in the ovaries, uterus, omentum tissue and mammary glands. In both sexes AR expression and activity was also found in salivary glands, the eye (and associated glands), adipose tissue, spleen and, notably, regions of the brain. Luciferase protein expression was found in the same cell layers as androgen receptor expression. Additionally, mouse AR expression and activity correlated well with AR expression in human tissues. The anti-androgen bicalutamide reduced luciferase signal in all tissues. Our model demonstrates that androgens can act in these tissues directly via AR, rather than exclusively via androgen aromatisation to estrogens and activation of the estrogen receptor. Additionally, it visually demonstrates the fundamental importance of AR signalling outside the normal role in the reproductive organs. This model represents an important tool for physiological and developmental analysis of androgen signalling, and for characterization of known and novel androgenic or antiandrogenic compounds. PMID:23940781

Thin and open vessel windows for intra-vital fluorescence imaging of murine cochlear blood flow.

PubMed

Shi, Xiaorui; Zhang, Fei; Urdang, Zachary; Dai, Min; Neng, Lingling; Zhang, Jinhui; Chen, Songlin; Ramamoorthy, Sripriya; Nuttall, Alfred L

2014-07-01

Normal microvessel structure and function in the cochlea is essential for maintaining the ionic and metabolic homeostasis required for hearing function. Abnormal cochlear microcirculation has long been considered an etiologic factor in hearing disorders. A better understanding of cochlear blood flow (CoBF) will enable more effective amelioration of hearing disorders that result from aberrant blood flow. However, establishing the direct relationship between CoBF and other cellular events in the lateral wall and response to physio-pathological stress remains a challenge due to the lack of feasible interrogation methods and difficulty in accessing the inner ear. Here we report on new methods for studying the CoBF in a mouse model using a thin or open vessel-window in combination with fluorescence intra-vital microscopy (IVM). An open vessel-window enables investigation of vascular cell biology and blood flow permeability, including pericyte (PC) contractility, bone marrow cell migration, and endothelial barrier leakage, in wild type and fluorescent protein-labeled transgenic mouse models with high spatial and temporal resolution. Alternatively, the thin vessel-window method minimizes disruption of the homeostatic balance in the lateral wall and enables study CoBF under relatively intact physiological conditions. A thin vessel-window method can also be used for time-based studies of physiological and pathological processes. Although the small size of the mouse cochlea makes surgery difficult, the methods are sufficiently developed for studying the structural and functional changes in CoBF under normal and pathological conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Immunocytochemical localisation of the nucleolar protein fibrillarin and RNA polymerase I during mouse early embryogenesis.

PubMed

Cuadros-Fernández, J M; Esponda, P

1996-02-01

We have employed immunocytochemical procedures to localise the nucleolar protein fibrillarin and the enzyme RNA polymerase I in the numerous dense fibrillar bodies (nucleolar precursor bodies) which appear in the nuclei of mammalian early embryos. The aim of this study was to search for relationships between the localisation of these proteins, the changes in the structure of the nucleolar precursor bodies and the resumption of rRNA gene transcription during mouse early embryogenesis. Three human autoimmune sera which recognised fibrillarin and a rabbit antiserum created against RNA polymerase I were employed for fluorescence and electron microscopic immunocytochemical assays. A statistical analysis was also applied. Immunocytochemistry revealed that fibrillarin and RNA polymerase I showed the same localisation in the nucleolar precursor bodies. These proteins were immunolocalised only from the late 2-cell stage onward. Fibrillarin was initially detected at the periphery of the nucleolar precursor bodies and the labelling gradually increased until the morula and blastocyst stages, where normally active nucleoli are found. The pattern of increase of fibrillarin during early embryogenesis shows a parallelism with the rise in rRNA gene transcription occurring during these embryonic stages, and a possible correlation between these two phenomena is suggested. Results demonstrated that nucleolar precursor bodies differ in their biochemical composition from the nucleolus and also from the prenucleolar bodies which appear during mitosis. When anti-fibrillarin antibodies were microinjected into the male pronucleus of mouse embryos to analyse the functions of fibrillarin during early development, they partially blocked the early development of mouse embryos and only 23.8% of injected embryos reach the blastocyst stage.

Studies of teratomas in mice: possibilities for the future production of animal models.

PubMed Central

Lehman, J. M.

1980-01-01

The murine teratoma-teratocarcinoma has become an interesting model for the study of neoplastic transformation, developmental biology, and possibly a useful system for genetic studies. These tumors arise spontaneously in 129 strain mice and can be induced in other strains by transplanting early embryos or portions of embryos into extrauterine sites. The majority of these tumors are benign, but some are capable of transplantation due to the presence of the stem cell, embryonal carcinoma, which is a multipotential cell able to proliferate and also differentiate into tissues and cell types representative of all the embryonic germ layers. It has been elegantly shown by transplantation of embryonal carcinoma cells into blastocysts which are then placed into a pseudopregnant mouse that a normal mouse is obtained composed of cells from the host blastocyst and also cells from the malignant embryonal carcinoma. Therefore, under this set of circumstances, embryonal carcinoma cells are induced to functionally differentiate into multiple cell and tissue types which are benign and able to contribute to the development of a mouse. The adaptation of the embryonal carcinoma cell to tissue culture has allowed the manipulation of these cells with subsequent selection of mutant cells which can be further transplanted into blastocysts to obtain a mouse which contains these mutant cells. If the mutant cells have populated the germ line, it may be possible to obtain a stock of mice with the lesion present in all cells. This system may be exploitable for studies in neoplasia, developmental biology, and with proper selection procedures, allow the development of new genetic strains of mice. PMID:7457573

Development and validation of LC-MS/MS method for quantitative determination of (-)-securinine in mouse plasma.

PubMed

Wabuyele, Simuli L; Wald, David; Xu, Yan

2014-06-01

(-)-Securinine (SE) is a major alkaloid found in plant Securinega suffruticosa, which has a wide range of pharmacological activities including anticancer, anti-parasitic and central nervous system stimulating effects, etc. To aid the pharmacological study of SE, we developed an LC-MS/MS method for quantitative determination of SE in mouse plasma. In this method, plasma samples were first prepared with salting-out assisted liquid-liquid extraction using cold acetonitrile (-20°C) and 2.00 M ammonium acetate. Separation of SE and the internal standard (IS) from sample matrix was achieved on a Gemini Nx C18 column using 40% acetonitrile and 60% 10.0mM ammonium acetate at a flow rate of 0.200 mL min(-1). Quantification of SE was accomplished with positive electrospray ionization tandem mass spectrometry using mass transitions m/z 218.1→84.1 for SE, and m/z 204.1→70.2 for the IS. This method has a lower limit of quantitation (LLOQ) of 0.600 ng mL(-1) and a linear calibration range up to 600 ng mL(-1) in mouse plasma. The intra- and inter-run accuracy (%RE) and precision (%CV) were ≤ ± 6% and 6%, respectively. The IS normalized matrix factors from six lots of plasma matrices ranged 0.92-1.07, and the recoveries of plasma SE were 99-109%. The validated method has been applied to the measurement of SE in plasma samples of a mouse study. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion.

PubMed

Eckardt, D; Theis, M; Degen, J; Ott, T; van Rijen, H V M; Kirchhoff, S; Kim, J-S; de Bakker, J M T; Willecke, K

2004-01-01

The gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development and in adult mice. Cx43 null mice (Cx43-/-) die perinatally due to cardiac malformation. In order to define the major functional role of Cx43 gap junction channels in adult mice and to circumvent perinatal death as well as direct or indirect compensation of Cx43 deficiency during development, we established a novel conditional Cx43 mouse mutant. To ablate Cx43 in adult mice in all cells that express Cx43 at a certain time, we targeted the 4-hydroxytamoxifen inducible Cre recombinase, Cre-ER(T), into the endogenous Cx43 locus. This approach left only one Cx43 coding region to be deleted upon induction of Cre-ER(T) activity. Highly efficient inducible ablation of Cx43 was shown in an embryonic stem cell test system and in adult mice. Although Cx43 protein was decreased in different tissues after induction of Cre-ER(T)-mediated recombination, cardiac abnormalities most likely account for death of those mice. Surface and telemetric ECG recordings revealed significant delay of ventricular activation and death during periods of bradyarrhythmia preceded by tachycardias. This novel approach of inducible ablation of Cx43 highlights the functional importance of normal activation of ventricular cardiomyocytes mediated by Cx43 gap junction channels in adult mouse heart to prevent initiation of fatal arrhythmias. The new mouse model should be useful for further analyses of molecular changes initiated by acute loss of Cx43 expression in various cell types.

Gpr177 regulates pulmonary vasculature development.

PubMed

Jiang, Ming; Ku, Wei-yao; Fu, Jiang; Offermanns, Stefan; Hsu, Wei; Que, Jianwen

2013-09-01

Establishment of the functional pulmonary vasculature requires intimate interaction between the epithelium and mesenchyme. Previous genetic studies have led to inconsistent conclusions about the contribution of epithelial Wnts to pulmonary vasculature development. This discrepancy is possibly due to the functional redundancy among different Wnts. Here, we use Shh-Cre to conditionally delete Gpr177 (the mouse ortholog of Drosophila Wntless, Wls), a chaperon protein important for the sorting and secretion of Wnt proteins. Deletion of epithelial Gpr177 reduces Wnt signaling activity in both the epithelium and mesenchyme, resulting in severe hemorrhage and abnormal vasculature, accompanied by branching defects and abnormal epithelial differentiation. We then used multiple mouse models to demonstrate that Wnt/β-catenin signaling is not only required for the proliferation and differentiation of mesenchyme, but also is important for the maintenance of smooth muscle cells through the regulation of the transcription factor Kruppel-like factor 2 (Klf2). Together, our studies define a novel mechanism by which epithelial Wnts regulate the normal development and maintenance of pulmonary vasculature. These findings provide insight into the pathobiology of congenital lung diseases, such as alveolar capillary dysplasia (ACD), that have abnormal alveolar development and dysmorphic pulmonary vasculature.

Fundamental properties of unperturbed haematopoiesis from stem cells in vivo.

PubMed

Busch, Katrin; Klapproth, Kay; Barile, Melania; Flossdorf, Michael; Holland-Letz, Tim; Schlenner, Susan M; Reth, Michael; Höfer, Thomas; Rodewald, Hans-Reimer

2015-02-26

Haematopoietic stem cells (HSCs) are widely studied by HSC transplantation into immune- and blood-cell-depleted recipients. Single HSCs can rebuild the system after transplantation. Chromosomal marking, viral integration and barcoding of transplanted HSCs suggest that very low numbers of HSCs perpetuate a continuous stream of differentiating cells. However, the numbers of productive HSCs during normal haematopoiesis, and the flux of differentiating progeny remain unknown. Here we devise a mouse model allowing inducible genetic labelling of the most primitive Tie2(+) HSCs in bone marrow, and quantify label progression along haematopoietic development by limiting dilution analysis and data-driven modelling. During maintenance of the haematopoietic system, at least 30% or ∼5,000 HSCs are productive in the adult mouse after label induction. However, the time to approach equilibrium between labelled HSCs and their progeny is surprisingly long, a time scale that would exceed the mouse's life. Indeed, we find that adult haematopoiesis is largely sustained by previously designated 'short-term' stem cells downstream of HSCs that nearly fully self-renew, and receive rare but polyclonal HSC input. By contrast, in fetal and early postnatal life, HSCs are rapidly used to establish the immune and blood system. In the adult mouse, 5-fluoruracil-induced leukopenia enhances the output of HSCs and of downstream compartments, thus accelerating haematopoietic flux. Label tracing also identifies a strong lineage bias in adult mice, with several-hundred-fold larger myeloid than lymphoid output, which is only marginally accentuated with age. Finally, we show that transplantation imposes severe constraints on HSC engraftment, consistent with the previously observed oligoclonal HSC activity under these conditions. Thus, we uncover fundamental differences between the normal maintenance of the haematopoietic system, its regulation by challenge, and its re-establishment after transplantation. HSC fate mapping and its linked modelling provide a quantitative framework for studying in situ the regulation of haematopoiesis in health and disease.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF-5-deficient mice.

PubMed

Maier, Jennifer A; Harfe, Brian D

2011-11-15

The transition of the mouse embryonic notochord into nuclei pulposi was determined ("fate mapped") in vivo in growth and differentiating factor-5 (GDF-5)-null mice using the Shhcre and R26R alleles. To determine whether abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5-null mice. The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5-null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5-null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or result from progressive postnatal degeneration of nuclei pulposi. Gdf-5 messenger RNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5-null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24-week-old mice. Our Gdf-5 messenger RNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate-mapping experiments revealed that notochord cells in Gdf-5-null mice correctly form nuclei pulposi. Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5-null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF5-deficient mice

PubMed Central

Maier, Jennifer A.; Harfe, Brian D.

2011-01-01

Study Design The transition of the mouse embryonic notochord into nuclei pulposi was determined (“fate mapped”) in vivo in GDF-5 null mice using the Shhcre and R26R alleles. Objective To determine if abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5 null mice. Summary of Background Data The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5 null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5 null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or resulted from progressive postnatal degeneration of nuclei pulposi. Methods Gdf-5 mRNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5 null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24 week old mice. Results Our Gdf-5 mRNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate mapping experiments revealed that notochord cells in Gdf-5 null mice correctly form nuclei pulposi. Conclusion Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5 null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects. PMID:21278629

Follistatin is a metastasis suppressor in a mouse model of HER2-positive breast cancer.

PubMed

Seachrist, Darcie D; Sizemore, Steven T; Johnson, Emhonta; Abdul-Karim, Fadi W; Weber Bonk, Kristen L; Keri, Ruth A

2017-06-05

Follistatin (FST) is an intrinsic inhibitor of activin, a member of the transforming growth factor-β superfamily of ligands. The prognostic value of FST and its family members, the follistatin-like (FSTL) proteins, have been studied in various cancers. However, these studies, as well as limited functional analyses of the FSTL proteins, have yielded conflicting results on the role of these proteins in disease progression. Furthermore, very few have been focused on FST itself. We assessed whether FST may be a suppressor of tumorigenesis and/or metastatic progression in breast cancer. Using publicly available gene expression data, we examined the expression patterns of FST and INHBA, a subunit of activin, in normal and cancerous breast tissue and the prognostic value of FST in breast cancer metastases, recurrence-free survival, and overall survival. The functional effects of activin and FST on in vitro proliferation, migration, and invasion of breast cancer cells were also examined. FST overexpression in an autochthonous mouse model of breast cancer was then used to assess the in vivo impact of FST on metastatic progression. Examination of multiple breast cancer datasets revealed that FST expression is reduced in breast cancers compared with normal tissue and that low FST expression predicts increased metastasis and reduced overall survival. FST expression was also reduced in a mouse model of HER2/Neu-induced metastatic breast cancer. We found that FST blocks activin-induced breast epithelial cell migration in vitro, suggesting that its loss may promote breast cancer aggressiveness. To directly determine if FST restoration could inhibit metastatic progression, we transgenically expressed FST in the HER2/Neu model. Although FST had no impact on tumor initiation or growth, it completely blocked the formation of lung metastases. These data indicate that FST is a bona fide metastasis suppressor in this mouse model and support future efforts to develop an FST mimetic to suppress metastatic progression.

The Transcription Factor Bright Plays a Role in Marginal Zone B Lymphocyte Development and Autoantibody Production

PubMed Central

Oldham, Athenia L.; Miner, Cathrine A.; Wang, Hong-Cheng; Webb, Carol F.

2011-01-01

Previous data suggested that constitutive expression of the transcription factor Bright (B cell regulator of immunoglobulin heavy chain transcription), normally tightly regulated during B cell differentiation, was associated with autoantibody production. Here we show that constitutive Bright expression results in skewing of mature B lineage subpopulations toward marginal zone cells at the expense of the follicular subpopulation. C57Bl/6 transgenic mice constitutively expressing Bright in B lineage cells generated autoantibodies that were not the result of global increases in immunoglobulin or of breaches in key tolerance checkpoints typically defective in other autoimmune mouse models. Rather, autoimmunity correlated with increased numbers of marginal zone B cells and alterations in the phenotype and gene expression profiles of lymphocytes within the follicular B cell compartment. These data suggest a novel role for Bright in the normal development of mature B cell subsets and in autoantibody production. PMID:21963220

Gastric cancer stem cells: A novel therapeutic target

PubMed Central

Singh, Shree Ram

2013-01-01

Gastric cancer remains one of the leading causes of global cancer mortality. Multipotent gastric stem cells have been identified in both mouse and human stomachs, and they play an essential role in the self-renewal and homeostasis of gastric mucosa. There are several environmental and genetic factors known to promote gastric cancer. In recent years, numerous in vitro and in vivo studies suggest that gastric cancer may originate from normal stem cells or bone marrow–derived mesenchymal cells, and that gastric tumors contain cancer stem cells. Cancer stem cells are believed to share a common microenvironment with normal niche, which play an important role in gastric cancer and tumor growth. This mini-review presents a brief overview of the recent developments in gastric cancer stem cell research. The knowledge gained by studying cancer stem cells in gastric mucosa will support the development of novel therapeutic strategies for gastric cancer. PMID:23583679

Nicotine Promotes Cholangiocarcinoma Growth in Xenograft Mice.

PubMed

Martínez, Allyson K; Jensen, Kendal; Hall, Chad; O'Brien, April; Ehrlich, Laurent; White, Tori; Meng, Fanyin; Zhou, Tianhao; Greene, John; Bernuzzi, Francesca; Invernizzi, Pietro; Dostal, David E; Lairmore, Terry; Alpini, Gianfranco; Glaser, Shannon S

2017-05-01

Nicotine, the main addictive substance in tobacco, is known to play a role in the development and/or progression of a number of malignant tumors. However, nicotine's involvement in the pathogenesis of cholangiocarcinoma is controversial. Therefore, we studied the effects of nicotine on the growth of cholangiocarcinoma cells in vitro and the progression of cholangiocarcinoma in a mouse xenograft model. The predominant subunit responsible for nicotine-mediated proliferation in normal and cancer cells, the α7 nicotinic acetylcholine receptor (α7-nAChR), was more highly expressed in human cholangiocarcinoma cell lines compared with normal human cholangiocytes. Nicotine also stimulated the proliferation of cholangiocarcinoma cell lines and promoted α7-nAChR-dependent activation of proliferation and phosphorylation of extracellular-regulated kinase in Mz-ChA-1 cells. In addition, nicotine and PNU282987 (α7-nAChR agonist) accelerated the growth of the cholangiocarcinoma tumors in our xenograft mouse model and increased fibrosis, proliferation of the tumor cells, and phosphorylation of extracellular-regulated kinase activation. Finally, α7-nAChR was expressed at significantly higher levels in human cholangiocarcinoma compared with normal human control liver samples. Taken together, results of this study suggest that nicotine acts through α7-nAChR and plays a novel role in the pathogenesis of cholangiocarcinoma. Furthermore, nicotine may act as a mitogen in cholestatic liver disease processes, thereby facilitating malignant transformation. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Deletion of Mecom in mouse results in early-onset spinal deformity and osteopenia.

PubMed

Juneja, Subhash C; Vonica, Alin; Zeiss, Caroline; Lezon-Geyda, Kimberly; Yatsula, Bogdan; Sell, David R; Monnier, Vincent M; Lin, Sharon; Ardito, Thomas; Eyre, David; Reynolds, David; Yao, Zhenqiang; Awad, Hani A; Yu, Hongbo; Wilson, Michael; Honnons, Sylvie; Boyce, Brendan F; Xing, Lianping; Zhang, Yi; Perkins, Archibald S

2014-03-01

Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues. Copyright © 2013 Elsevier Inc. All rights reserved.

Targeting Phosphatidylserine for Radioimmunotherapy of Breast Cancer Brain Metastasis

DTIC Science & Technology

2015-12-01

response. e. Correlate imaging findings with histological studies of vascular damage, tumor cell and endothelial cell apoptosis or necrosis and vascular ...phosphatidylserine (PS) is exposed exclusively on tumor vascular endothelium of brain metastases in mouse models. A novel PS-targeting antibody, PGN635... vascular endothelial cells in multi-focal brain metastases throughout the whole mouse brain. Vascular endothelium in normal brain tissues is negative

Memory and Learning--Using Mouse to Model Neurobiological and Behavioural Aspects of Down Syndrome and Assess Pharmacotherapeutics

ERIC Educational Resources Information Center

Gardiner, Katheleen

2009-01-01

Mouse models are a standard tool in the study of many human diseases, providing insights into the normal functions of a gene, how these are altered in disease and how they contribute to a disease process, as well as information on drug action, efficacy and side effects. Our knowledge of human genes, their genetics, functions, interactions and…

A different regional response by mouse oligodendrocyte progenitor cells (OPCs) to high-dose X-irradiation has consequences for repopulating OPC-depleted normal tissue.

PubMed

Irvine, Karen-Amanda; Blakemore, William F

2007-01-01

This study was designed to investigate whether the residual, dysfunctional oligodendrocyte progenitor cells (OPCs) observed following X-irradiation of the mouse spinal cord [D. M. Chari et al. (2003) Exp. Neurol., 198, 145-153], the presence of which prevented the endogenous repopulation of these areas from normal tissue, reflects a general response of OPCs in the mouse central nervous system (CNS) to X-irradiation. The brains of adult mice were exposed to 40 Gy of X-irradiation and the effect of X-irradiation on the OPCs was assessed up to 4 weeks post-irradiation using anti-NG2 antibodies. X-irradiation resulted in almost complete depletion of OPCs within the telencephalon (cortex, corpus callosum and hippocampus) by 7 days post-irradiation, which was followed by progressive repopulation of OPCs from non-irradiated areas of the cortex. By contrast, within the lower brain centres (the diencephalon and mesencephalon) OPC loss occurred much more slowly so that 26% of the OPCs still remained 4 weeks after X-irradiation. The consequence of this heterogeneous response to X-irradiation was that whereas transplanted and endogenous OPCs rapidly established themselves in the OPC-depleted telencephalon this did not occur in the areas where there was incomplete depletion of endogenous OPCs. Our findings confirm not only the requirement for almost complete OPC depletion in order to establish transplanted OPCs in normal tissue but also highlight a heterogeneity of progenitor populations in different areas of the mouse CNS.

A composite mouse model of aplastic anemia complicated with iron overload

PubMed Central

Wu, Dijiong; Wen, Xiaowen; Liu, Wenbin; Xu, Linlong; Ye, Baodong; Zhou, Yuhong

2018-01-01

Iron overload is commonly encountered during the course of aplastic anemia (AA), but no composite animal model has been developed yet, which hinders drug research. In the present study, the optimal dosage and duration of intraperitoneal iron dextran injection for the development of an iron overload model in mice were explored. A composite model of AA was successfully established on the principle of immune-mediated bone marrow failure. Liver volume, peripheral hemogram, bone marrow pathology, serum iron, serum ferritin, pathological iron deposition in multiple organs (liver, bone marrow, spleen), liver hepcidin, and bone morphogenetic protein 6 (BMP6), SMAD family member 4 (SMAD4) and transferrin receptor 2 (TfR2) mRNA expression levels were compared among the normal control, AA, iron overload and composite model groups to validate the composite model, and explore the pathogenesis and features of iron overload in this model. The results indicated marked increases in iron deposits, with significantly increased liver/body weight ratios as well as serum iron and ferritin in the iron overload and composite model groups as compared with the normal control and AA groups (P<0.05). There were marked abnormalities in iron regulation gene expression between the AA and composite model groups, as seen by the significant decrease of hepcidin expression in the liver (P<0.01) that paralleled the changes in BMP6, SMAD4, and TfR2. In summary, a composite mouse model with iron overload and AA was successfully established, and AA was indicated to possibly have a critical role in abnormal iron metabolism, which promoted the development of iron deposits. PMID:29434729

A composite mouse model of aplastic anemia complicated with iron overload.

PubMed

Wu, Dijiong; Wen, Xiaowen; Liu, Wenbin; Xu, Linlong; Ye, Baodong; Zhou, Yuhong

2018-02-01

Iron overload is commonly encountered during the course of aplastic anemia (AA), but no composite animal model has been developed yet, which hinders drug research. In the present study, the optimal dosage and duration of intraperitoneal iron dextran injection for the development of an iron overload model in mice were explored. A composite model of AA was successfully established on the principle of immune-mediated bone marrow failure. Liver volume, peripheral hemogram, bone marrow pathology, serum iron, serum ferritin, pathological iron deposition in multiple organs (liver, bone marrow, spleen), liver hepcidin, and bone morphogenetic protein 6 (BMP6), SMAD family member 4 (SMAD4) and transferrin receptor 2 (TfR2) mRNA expression levels were compared among the normal control, AA, iron overload and composite model groups to validate the composite model, and explore the pathogenesis and features of iron overload in this model. The results indicated marked increases in iron deposits, with significantly increased liver/body weight ratios as well as serum iron and ferritin in the iron overload and composite model groups as compared with the normal control and AA groups (P<0.05). There were marked abnormalities in iron regulation gene expression between the AA and composite model groups, as seen by the significant decrease of hepcidin expression in the liver (P<0.01) that paralleled the changes in BMP6, SMAD4, and TfR2. In summary, a composite mouse model with iron overload and AA was successfully established, and AA was indicated to possibly have a critical role in abnormal iron metabolism, which promoted the development of iron deposits.

Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

PubMed

Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

2003-01-01

We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

Deletion of OTX2 in neural ectoderm delays anterior pituitary development

PubMed Central

Mortensen, Amanda H.; Schade, Vanessa; Lamonerie, Thomas; Camper, Sally A.

2015-01-01

OTX2 is a homeodomain transcription factor that is necessary for normal head development in mouse and man. Heterozygosity for loss-of-function alleles causes an incompletely penetrant, haploinsufficiency disorder. Affected individuals exhibit a spectrum of features that range from developmental defects in eye and/or pituitary development to acephaly. To investigate the mechanism underlying the pituitary defects, we used different cre lines to inactivate Otx2 in early head development and in the prospective anterior and posterior lobes. Mice homozygous for Otx2 deficiency in early head development and pituitary oral ectoderm exhibit craniofacial defects and pituitary gland dysmorphology, but normal pituitary cell specification. The morphological defects mimic those observed in humans and mice with OTX2 heterozygous mutations. Mice homozygous for Otx2 deficiency in the pituitary neural ectoderm exhibited altered patterning of gene expression and ablation of FGF signaling. The posterior pituitary lobe and stalk, which normally arise from neural ectoderm, were extremely hypoplastic. Otx2 expression was intact in Rathke's pouch, the precursor to the anterior lobe, but the anterior lobe was hypoplastic. The lack of FGF signaling from the neural ectoderm was sufficient to impair anterior lobe growth, but not the differentiation of hormone-producing cells. This study demonstrates that Otx2 expression in the neural ectoderm is important intrinsically for the development of the posterior lobe and pituitary stalk, and it has significant extrinsic effects on anterior pituitary growth. Otx2 expression early in head development is important for establishing normal craniofacial features including development of the brain, eyes and pituitary gland. PMID:25315894

Immune dysregulation may contribute to disease pathogenesis in spinal muscular atrophy mice

PubMed Central

Deguise, Marc-Olivier; De Repentigny, Yves; McFall, Emily; Auclair, Nicole; Sad, Subash

2017-01-01

Abstract Spinal muscular atrophy (SMA) has long been solely considered a neurodegenerative disorder. However, recent work has highlighted defects in many other cell types that could contribute to disease aetiology. Interestingly, the immune system has never been extensively studied in SMA. Defects in lymphoid organs could exacerbate disease progression by neuroinflammation or immunodeficiency. Smn depletion led to severe alterations in the thymus and spleen of two different mouse models of SMA. The spleen from Smn depleted mice was dramatically smaller at a very young age and its histological architecture was marked by mislocalization of immune cells in the Smn2B/- model mice. In comparison, the thymus was relatively spared in gross morphology but showed many histological alterations including cortex thinning in both mouse models at symptomatic ages. Thymocyte development was also impaired as evidenced by abnormal population frequencies in the Smn2B/- thymus. Cytokine profiling revealed major changes in different tissues of both mouse models. Consistent with our observations, we found that survival motor neuron (Smn) protein levels were relatively high in lymphoid organs compared to skeletal muscle and spinal cord during postnatal development in wild type mice. Genetic introduction of one copy of the human SMN2 transgene was enough to rescue splenic and thymic defects in Smn2B/- mice. Thus, Smn is required for the normal development of lymphoid organs, and altered immune function may contribute to SMA disease pathogenesis. PMID:28108555

A step-wise approach for analysis of the mouse embryonic heart using 17.6 Tesla MRI

PubMed Central

Gabbay-Benziv, Rinat; Reece, E. Albert; Wang, Fang; Bar-Shir, Amnon; Harman, Chris; Turan, Ozhan M.; Yang, Peixin; Turan, Sifa

2018-01-01

Background The mouse embryo is ideal for studying human cardiac development. However, laboratory discoveries do not easily translate into clinical findings partially because of histological diagnostic techniques that induce artifacts and lack standardization. Aim To present a step-wise approach using 17.6 T MRI, for evaluation of mice embryonic heart and accurate identification of congenital heart defects. Subjects 17.5-embryonic days embryos from low-risk (non-diabetic) and high-risk (diabetic) model dams. Study design Embryos were imaged using 17.6 Tesla MRI. Three-dimensional volumes were analyzed using ImageJ software. Outcome measures Embryonic hearts were evaluated utilizing anatomic landmarks to locate the four-chamber view, the left- and right-outflow tracts, and the arrangement of the great arteries. Inter- and intra-observer agreement were calculated using kappa scores by comparing two researchers’ evaluations independently analyzing all hearts, blinded to the model, on three different, timed occasions. Each evaluated 16 imaging volumes of 16 embryos: 4 embryos from normal dams, and 12 embryos from diabetic dams. Results Inter-observer agreement and reproducibility were 0.779 (95% CI 0.653–0.905) and 0.763 (95% CI 0.605–0.921), respectively. Embryonic hearts were structurally normal in 4/4 and 7/12 embryos from normal and diabetic dams, respectively. Five embryos from diabetic dams had defects: ventricular septal defects (n = 2), transposition of great arteries (n = 2) and Tetralogy of Fallot (n = 1). Both researchers identified all cardiac lesions. Conclusion A step-wise approach for analysis of MRI-derived 3D imaging provides reproducible detailed cardiac evaluation of normal and abnormal mice embryonic hearts. This approach can accurately reveal cardiac structure and, thus, increases the yield of animal model in congenital heart defect research. PMID:27569369

Low levels of Survival Motor Neuron protein are sufficient for normal muscle function in the SMNΔ7 mouse model of SMA.

PubMed

Iyer, Chitra C; McGovern, Vicki L; Murray, Jason D; Gombash, Sara E; Zaworski, Phillip G; Foust, Kevin D; Janssen, Paul M L; Burghes, Arthur H M

2015-11-01

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons. SMA is caused by deletion or mutation of the Survival Motor Neuron 1 (SMN1) gene and retention of the SMN2 gene. The loss of SMN1 results in reduced levels of the SMN protein. SMN levels appear to be particularly important in motor neurons; however SMN levels above that produced by two copies of SMN2 have been suggested to be important in muscle. Studying the spatial requirement of SMN is important in both understanding how SMN deficiency causes SMA and in the development of effective therapies. Using Myf5-Cre, a muscle-specific Cre driver, and the Cre-loxP recombination system, we deleted mouse Smn in the muscle of mice with SMN2 and SMNΔ7 transgenes in the background, thus providing low level of SMN in the muscle. As a reciprocal experiment, we restored normal levels of SMN in the muscle with low SMN levels in all other tissues. We observed that decreasing SMN in the muscle has no phenotypic effect. This was corroborated by muscle physiology studies with twitch force, tetanic and eccentric contraction all being normal. In addition, electrocardiogram and muscle fiber size distribution were also normal. Replacement of Smn in muscle did not rescue SMA mice. Thus the muscle does not appear to require high levels of SMN above what is produced by two copies of SMN2 (and SMNΔ7). © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Concordance between isolated cleft palate in mice and alterations within a region including the gene encoding the [beta][sub 3] subunit of the type A [gamma]-aminobutyric acid receptor

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

Culiat, C.T.; Stubbs, L.; Nicholls, R.D.

1993-06-01

Genetic and molecular analyses of a number of radiation-induced deletion mutations of the pink-eyed dilution (p) locus in mouse chromosome 7 have identified a specific interval on the genetic map associated with a neonatally lethal mutation that results in cleft palate. This interval, closely linked and distal to p, and bracketed by the genes encoding the [alpha][sub 5] and [beta][sub 3] subunits of the type A [gamma]-aminobutyric acid receptor (Gabra5 and Gabrb3, respectively), contains a gene(s) (cp1; cleft palate 1) necessary for normal palate development. The cp1 interval extends from the distal breakpoint of the prenatally lethal p[sup 83FBFo] deletionmore » to the Gabrb3 locus. Among 20 p deletions tested, there was complete concordance between alterations at the Gabrb3 transcription unit and inability to complement the cleft-palate defect. These mapping data, along with previously described in vivo and in vitro teratological effects of [gamma]-aminobutyric acid or its agonists on palate development, suggest the possibility that a particular type A [gamma]-aminobutyric acid receptor that includes the [beta][sub 3] subunit may be necessary for normal palate development. The placement of the cp1 gene within a defined segment of the larger D15S12h (p)-D15S9h-1 interval in the mouse suggests that the highly homologous region of the human genome, 15q11-q13, be evaluated for a role(s) in human fetal facial development. 29 refs., 4 figs., 1 tab.« less

Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model

PubMed Central

Katikaneni, Ranjitha; Ponnapakkam, Tulasi; Matsushita, Osamu; Sakon, Joshua; Gensure, Robert

2014-01-01

Alopecia is a psychologically devastating complication of chemotherapy for which there is currently no effective therapy. PTH-CBD is a collagen-targeted parathyroid hormone analog that has shown promise as a therapy for alopecia disorders. To compare the efficacy of prophylactic versus therapeutic administration of PTH-CBD in chemotherapy-induced alopecia using a mouse model that mimics the cyclic chemotherapy dosing used clinically. C57BL/6J mice were treated with a single subcutaneous injection of PTH-CBD (320 mcg/kg) or vehicle control before or after hair loss developing from three courses of cyclophosphamide chemotherapy (50–150 mg/kg/week). Mice receiving chemotherapy alone developed hair loss and depigmentation over 6–12 months. Mice pretreated with PTH-CBD did not develop these changes and maintained a normal-appearing coat. Mice treated with PTH-CBD after development of hair loss showed a partial recovery. Observations of hair loss were confirmed quantitatively by gray scale analysis. Histological examination showed that in mice receiving chemotherapy alone, there were small, dystrophic hair follicles mostly in the catagen phase. Mice receiving PTH-CBD before chemotherapy showed a mix of normal-appearing telogen and anagen hair follicles with no evidence of dystrophy. Mice receiving PTH-CBD therapy after chemotherapy showed intermediate histological features. PTH-CBD was effective in both the prevention and the treatment of chemotherapy-induced alopecia in mice, but pretreatment appears to result in a better cosmetic outcome. PTH-CBD shows promise as an agent in the prevention of this complication of chemotherapy and improving the quality of life for cancer patients. PMID:24025564

Treatment and prevention of chemotherapy-induced alopecia with PTH-CBD, a collagen-targeted parathyroid hormone analog, in a non-depilated mouse model.

PubMed

Katikaneni, Ranjitha; Ponnapakkam, Tulasi; Matsushita, Osamu; Sakon, Joshua; Gensure, Robert

2014-01-01

Alopecia is a psychologically devastating complication of chemotherapy for which there is currently no effective therapy. PTH-CBD is a collagen-targeted parathyroid hormone analog that has shown promise as a therapy for alopecia disorders. This study compared the efficacy of prophylactic versus therapeutic administration of PTH-CBD in chemotherapy-induced alopecia using a mouse model that mimics the cyclic chemotherapy dosing used clinically. C57BL/6J mice were treated with a single subcutaneous injection of PTH-CBD (320 mcg/kg) or vehicle control before or after hair loss developing from three courses of cyclophosphamide chemotherapy (50-150 mg/kg/week). Mice receiving chemotherapy alone developed hair loss and depigmentation over 6-12 months. Mice pretreated with PTH-CBD did not develop these changes and maintained a normal-appearing coat. Mice treated with PTH-CBD after development of hair loss showed a partial recovery. Observations of hair loss were confirmed quantitatively by gray scale analysis. Histological examination showed that in mice receiving chemotherapy alone, there were small, dystrophic hair follicles mostly in the catagen phase. Mice receiving PTH-CBD before chemotherapy showed a mix of normal-appearing telogen and anagen hair follicles with no evidence of dystrophy. Mice receiving PTH-CBD therapy after chemotherapy showed intermediate histological features. PTH-CBD was effective in both the prevention and the treatment of chemotherapy-induced alopecia in mice, but pretreatment appears to result in a better cosmetic outcome. PTH-CBD shows promise as an agent in the prevention of this complication of chemotherapy and improving the quality of life for cancer patients.

Dynamic changes in leptin distribution in the progression from ovum to blastocyst of the pre-implantation mouse embryo

PubMed Central

Schulz, Laura C.; Roberts, R. Michael

2011-01-01

The hormone leptin, which is primarily produced by adipose tissue, is a critical permissive factor for multiple reproductive events in the mouse, including implantation. In the CD1 strain, maternally-derived leptin from the oocyte becomes differentially distributed among blastomeres of pre-implantation embryos to create a polarized pattern, a feature consistent with a model of development in which blastomeres are biased towards a particular fate as early as the 2-cell stage. Here, we have confirmed that embryonic leptin is of maternal origin and re-examined leptin distribution in two distinct strains in which embryos were derived after either normal ovulation or superovulation. A polarized pattern of leptin distribution was found in the majority of both CD1 and CF1 embryos (79.1 % and 76.9 %, respectively) collected following superovulation, but was reduced, particularly in CF1 embryos (29.8 %; p < 0.0001), after natural ovulation. The difference in leptin asymmetries in the CF1 strain arose between ovulation and the first cleavage division, and was not affected by removal of the zona pellucida. Presence or absence of leptin polarization was not linked to differences in ability of embryos to develop normally to blastocyst. In the early blastocyst, leptin was confined subcortically to trophectoderm but upon blastocoel expansion it was lost from cells. Throughout development leptin co-localized with LRP2, a multi-ligand transport protein, and its patterning resembled that noted for the maternal-effect proteins OOEP, NLRP5, and PADI6, suggesting that it is a component of the subcortical maternal complex with as yet unknown significance in pre-implantation development. PMID:21444625

Heterogeneity of the tumor vasculature.

PubMed

Nagy, Janice A; Chang, Sung-Hee; Shih, Shou-Ching; Dvorak, Ann M; Dvorak, Harold F

2010-04-01

The blood vessels supplying tumors are strikingly heterogeneous and differ from their normal counterparts with respect to organization, structure, and function. Six distinctly different tumor vessel types have been identified, and much has been learned about the steps and mechanisms by which they form. Four of the six vessel types (mother vessels, capillaries, glomeruloid microvascular proliferations, and vascular malformations) develop from preexisting normal venules and capillaries by angiogenesis. The two remaining vessel types (feeder arteries and draining veins) develop from arterio-venogenesis, a parallel, poorly understood process that involves the remodeling of preexisting arteries and veins. All six of these tumor vessel types can be induced to form sequentially in normal mouse tissues by an adenoviral vector expressing vascular endothelial growth factor (VEGF)-A164. Current antiangiogenic cancer therapies directed at VEGF-A or its receptors have been of only limited benefit to cancer patients, perhaps because they target only the endothelial cells of the tumor blood vessel subset that requires exogenous VEGF-A for maintenance. A goal of future work is to identify therapeutic targets on tumor blood vessel endothelial cells that have lost this requirement. Thieme Medical Publishers.

Heterogeneity of the Tumor Vasculature

PubMed Central

Nagy, Janice A.; Chang, Sung-Hee; Shih, Shou-Ching; Dvorak, Ann M.; Dvorak, Harold F.

2012-01-01

The blood vessels supplying tumors are strikingly heterogeneous and differ from their normal counterparts with respect to organization, structure, and function. Six distinctly different tumor vessel types have been identified, and much has been learned about the steps and mechanisms by which they form. Four of the six vessel types (mother vessels, capillaries, glomeruloid microvascular proliferations, and vascular malformations) develop from preexisting normal venules and capillaries by angiogenesis. The two remaining vessel types (feeder arteries and draining veins) develop from arterio-venogenesis, a parallel, poorly understood process that involves the remodeling of preexisting arteries and veins. All six of these tumor vessel types can be induced to form sequentially in normal mouse tissues by an adenoviral vector expressing vascular endothelial growth factor (VEGF)-A164. Current antiangiogenic cancer therapies directed at VEGF-A or its receptors have been of only limited benefit to cancer patients, perhaps because they target only the endothelial cells of the tumor blood vessel subset that requires exogenous VEGF-A for maintenance. A goal of future work is to identify therapeutic targets on tumor blood vessel endothelial cells that have lost this requirement. PMID:20490982

Transgenic mice: an irreplaceable tool for the study of mammalian development and biology.

PubMed

Babinet, C

2000-11-01

Stable integration into the mouse genome of exogenous genetic information, i.e., the creation of transgenic mice, has become a privileged way of analyzing gene function in normal development and pathology. Both gene addition and gene replacement may be performed. This has allowed, in particular, the creation of mice in which precise mutations are introduced into a given gene. Furthermore, in recent years, strategies that induce the expression of a mutation in a given type of cell and/or at a given time in development have been developed. Thus, the transgenic methodology affords a unique and irreplaceable tool for the study of mammalian development and biology and for the creation of animal models for human genetic diseases.

The expression and crucial roles of BMP signaling in development of smooth muscle progenitor cells in the mouse embryonic gut.

PubMed

Torihashi, Shigeko; Hattori, Takako; Hasegawa, Hirotaka; Kurahashi, Masaaki; Ogaeri, Takunori; Fujimoto, Toyoshi

2009-03-01

Bone morphogenetic protein (BMP) signaling is essential for normal development of the gastrointestinal (GI) tract. BMPs also play multiple roles in vascular smooth muscle cells; however, the BMP signaling in the development of the GI musculature remains to be clarified. We investigated the expression of BMPs and their receptors in mouse embryonic GI tracts by immunohistochemistry and in situ hybridization. We demonstrated that BMP2, BMP receptor Ib and BMP receptor II were expressed in the smooth muscle progenitors from E12 to E13 for the first time. BMP signaling on smooth muscle differentiation was examined by implantation of agarose beads soaked with BMPs in the in vitro developmental model that is gut-like structures from mouse embryonic stem (ES) cells. BMP2 rather than BMP4 beads enhanced smooth muscle differentiation, and increased gut-like structures showing spontaneous contractions and expressing intensive alpha-smooth muscle actin immunoreactivity. This increase was confirmed by up-regulation of SM22 mRNA shown by real-time PCR. By addition of noggin beads or noggin to the medium at BMP2 bead implantation, the ratio of contractive gut-like structures decreased. Implantation of BMP2 beads at EB7 (EB--embryoid bodies) (corresponding to E12 or E13 of mouse embryo) showed the highest effects and up-regulation of transcription factors msx-1 after 24h. This increase was blocked by noggin, and msx-1 decreased to almost the control level after 60 h. BMP2 beads at EB7 increased platelet-derived growth factor-A (PDGF-A) in the differentiating smooth muscle cells. We have recently reported that PDGF-A is expressed in the developing inner circular smooth muscle and is crucial for the longitudinal smooth muscle differentiation. Taken together, BMP signaling was expressed for a short window in the smooth muscle progenitors and the signal, especially BMP2, plays an essential role in smooth muscle differentiation in cooperation with PDGF signaling.

Mast cells play no role in the pathogenesis of postoperative ileus induced by intestinal manipulation.

PubMed

Gomez-Pinilla, Pedro J; Farro, Giovanna; Di Giovangiulio, Martina; Stakenborg, Nathalie; Némethova, Andrea; de Vries, Annick; Liston, Adrian; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Rodewald, Hans-Reimwer; Boeckxstaens, Guy E; Matteoli, Gianluca

2014-01-01

Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3(Cre/+) , devoid of mast cells but with intact Kit signaling. The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. Kit(W-sh/W-sh) and Cpa3(Cre/+) mice, and by use of the mast cell stabilizer cromolyn. Kit(W-sh/W-sh) mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3(Cre/+) mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3(Cre/+) mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3(+/+) ). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI. Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.

Mast Cells Play No Role in the Pathogenesis of Postoperative Ileus Induced by Intestinal Manipulation

PubMed Central

Gomez-Pinilla, Pedro J.; Farro, Giovanna; Di Giovangiulio, Martina; Stakenborg, Nathalie; Némethova, Andrea; de Vries, Annick; Liston, Adrian; Feyerabend, Thorsten B.; Rodewald, Hans-Reimwer; Boeckxstaens, Guy E.; Matteoli, Gianluca

2014-01-01

Introduction Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3Cre/+, devoid of mast cells but with intact Kit signaling. Design The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. KitW-sh/W-sh and Cpa3Cre/+ mice, and by use of the mast cell stabilizer cromolyn. Results KitW-sh/W-sh mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3Cre/+ mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3Cre/+ mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3+/+). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI. Conclusions Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI. PMID:24416383

Radiation-induced lung fibrosis in a tumor-bearing mouse model is associated with enhanced Type-2 immunity.

PubMed

Chen, Jing; Wang, Yacheng; Mei, Zijie; Zhang, Shimin; Yang, Jie; Li, Xin; Yao, Ye; Xie, Conghua

2016-03-01

Lung fibrosis may be associated with Type-2 polarized inflammation. Herein, we aim to investigate whether radiation can initiate a Type-2 immune response and contribute to the progression of pulmonary fibrosis in tumor-bearing animals. We developed a tumor-bearing mouse model with Lewis lung cancer to receive either radiation therapy alone or radiation combined with Th1 immunomodulator unmethylated cytosine-phosphorothioate-guanine containing oligodeoxynucleotide (CpG-ODN). The Type-2 immune phenotype in tumors and the histological grade of lung fibrosis were evaluated in mice sacrificed three weeks after irradiation. Mouse lung tissues were analyzed for hydroxyproline and the expression of Type-1/Type-2 key transcription factors (T-bet/GATA-3). The concentration of Type-1/Type-2 cytokines in serum was measured by cytometric bead array. Lung fibrosis was observed to be more serious in tumor-bearing mice than in normal mice post-irradiation. The fibrosis score in irradiated tumor-bearing mice on Day 21 was 4.33 ± 0.82, which was higher than that of normal mice (2.00 ± 0.63; P < 0.05). Hydroxyproline and GATA-3 expression were increased in the lung tissues of tumor-bearing mice following irradiation. CpG-ODN attenuated fibrosis by markedly decreasing GATA-3 expression. Serum IL-13 and IL-5 were elevated, whereas INF-γ and IL-12 expression were decreased in irradiated tumor-bearing mice. These changes were reversed after CpG-ODN treatment. Thus, Type-2 immunity in tumors appeared to affect the outcome of radiation damage and might be of interest for future studies on developing approaches in which Type-1-related immunotherapy and radiotherapy are used in combination. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

The forkhead box m1 transcription factor is essential for embryonic development of pulmonary vasculature.

PubMed

Kim, Il-Man; Ramakrishna, Sneha; Gusarova, Galina A; Yoder, Helena M; Costa, Robert H; Kalinichenko, Vladimir V

2005-06-10

Transgenic and gene knock-out studies demonstrated that the mouse Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is essential for hepatocyte entry into mitosis during liver development, regeneration, and liver cancer. Targeted deletion of Foxm1 gene in mice produces an embryonic lethal phenotype due to severe abnormalities in the development of liver and heart. In this study, we show for the first time that Foxm1(-/-) lungs exhibit severe hypertrophy of arteriolar smooth muscle cells and defects in the formation of peripheral pulmonary capillaries as evidenced by significant reduction in platelet endothelial cell adhesion molecule 1 staining of the distal lung. Consistent with these findings, significant reduction in proliferation of the embryonic Foxm1(-/-) lung mesenchyme was found, yet proliferation levels were normal in the Foxm1-deficient epithelial cells. Severe abnormalities of the lung vasculature in Foxm1(-/-) embryos were associated with diminished expression of the transforming growth factor beta receptor II, a disintegrin and metalloprotease domain 17 (ADAM-17), vascular endothelial growth factor receptors, Polo-like kinase 1, Aurora B kinase, laminin alpha4 (Lama4), and the Forkhead Box f1 transcription factor. Cotransfection studies demonstrated that Foxm1 stimulates transcription of the Lama4 promoter, and this stimulation requires the Foxm1 binding sites located between -1174 and -1145 bp of the mouse Lama4 promoter. In summary, development of mouse lungs depends on the Foxm1 transcription factor, which regulates expression of genes essential for mesenchyme proliferation, extracellular matrix remodeling, and vasculogenesis.

DNA double-strand break repair of blood lymphocytes and normal tissues analysed in a preclinical mouse model: implications for radiosensitivity testing.

PubMed

Rübe, Claudia E; Grudzenski, Saskia; Kühne, Martin; Dong, Xiaorong; Rief, Nicole; Löbrich, Markus; Rübe, Christian

2008-10-15

Radiotherapy is an effective cancer treatment, but a few patients suffer severe radiation toxicities in neighboring normal tissues. There is increasing evidence that the variable susceptibility to radiation toxicities is caused by the individual genetic predisposition, by subtle mutations, or polymorphisms in genes involved in cellular responses to ionizing radiation. Double-strand breaks (DSB) are the most deleterious form of radiation-induced DNA damage, and DSB repair deficiencies lead to pronounced radiosensitivity. Using a preclinical mouse model, the highly sensitive gammaH2AX-foci approach was tested to verify even subtle, genetically determined DSB repair deficiencies known to be associated with increased normal tissue radiosensitivity. By enumerating gammaH2AX-foci in blood lymphocytes and normal tissues (brain, lung, heart, and intestine), the induction and repair of DSBs after irradiation with therapeutic doses (0.1-2 Gy) was investigated in repair-proficient and repair-deficient mouse strains in vivo and blood samples irradiated ex vivo. gammaH2AX-foci analysis allowed to verify the different DSB repair deficiencies; even slight impairments caused by single polymorphisms were detected similarly in both blood lymphocytes and solid tissues, indicating that DSB repair measured in lymphocytes is valid for different and complex organs. Moreover, gammaH2AX-foci analysis of blood samples irradiated ex vivo was found to reflect repair kinetics measured in vivo and, thus, give reliable information about the individual DSB repair capacity. gammaH2AX analysis of blood and tissue samples allows to detect even minor genetically defined DSB repair deficiencies, affecting normal tissue radiosensitivity. Future studies will have to evaluate the clinical potential to identify patients more susceptible to radiation toxicities before radiotherapy.

A mouse model of Salmonella typhi infection

PubMed Central

Mathur, Ramkumar; Oh, Hyunju; Zhang, Dekai; Park, Sung-Gyoo; Seo, Jin; Koblansky, Alicia; Hayden, Matthew S.; Ghosh, Sankar

2012-01-01

Salmonella spp. are gram-negative flagellated bacteria that can cause food and water-borne gastroenteritis and typhoid fever in humans. We now report that flagellin from Salmonella spp. is recognized in mouse intestine by Toll-like receptor 11 (TLR11). Absence of TLR11 renders mice more susceptible to infection by S. typhimurium, with increased dissemination of the bacteria and enhanced lethality. Unlike S. typhimurium, S. typhi, a human obligatory pathogen that causes typhoid fever, is normally unable to infect mice. TLR11 is expressed in mice but not in humans, and remarkably, we find that tlr11−/− mice are efficiently infected with orally-administered S. typhi. We also find that tlr11−/− mice can be immunized against S. typhi. Therefore, tlr11−/− mice represent the first small animal model for the study of the immune response to S. typhi, and for the development of vaccines against this important human pathogen. PMID:23101627

An Ethyl-Nitrosourea-Induced Point Mutation in Phex Causes Exon Skipping, X-Linked Hypophosphatemia, and Rickets

PubMed Central

Carpinelli, Marina R.; Wicks, Ian P.; Sims, Natalie A.; O’Donnell, Kristy; Hanzinikolas, Katherine; Burt, Rachel; Foote, Simon J.; Bahlo, Melanie; Alexander, Warren S.; Hilton, Douglas J.

2002-01-01

We describe the clinical, genetic, biochemical, and molecular characterization of a mouse that arose in the first generation (G1) of a random mutagenesis screen with the chemical mutagen ethyl-nitrosourea. The mouse was observed to have skeletal abnormalities inherited with an X-linked dominant pattern of inheritance. The causative mutation, named Skeletal abnormality 1 (Ska1), was shown to be a single base pair mutation in a splice donor site immediately following exon 8 of the Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome) gene. This point mutation caused skipping of exon 8 from Phex mRNA, hypophosphatemia, and features of rickets. This experimentally induced phenotype mirrors the human condition X-linked hypophosphatemia; directly confirms the role of Phex in phosphate homeostasis, normal skeletal development, and rickets; and illustrates the power of mutagenesis in exploring animal models of human disease. PMID:12414538

An ethyl-nitrosourea-induced point mutation in phex causes exon skipping, x-linked hypophosphatemia, and rickets.

PubMed

Carpinelli, Marina R; Wicks, Ian P; Sims, Natalie A; O'Donnell, Kristy; Hanzinikolas, Katherine; Burt, Rachel; Foote, Simon J; Bahlo, Melanie; Alexander, Warren S; Hilton, Douglas J

2002-11-01

We describe the clinical, genetic, biochemical, and molecular characterization of a mouse that arose in the first generation (G(1)) of a random mutagenesis screen with the chemical mutagen ethyl-nitrosourea. The mouse was observed to have skeletal abnormalities inherited with an X-linked dominant pattern of inheritance. The causative mutation, named Skeletal abnormality 1 (Ska1), was shown to be a single base pair mutation in a splice donor site immediately following exon 8 of the Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome) gene. This point mutation caused skipping of exon 8 from Phex mRNA, hypophosphatemia, and features of rickets. This experimentally induced phenotype mirrors the human condition X-linked hypophosphatemia; directly confirms the role of Phex in phosphate homeostasis, normal skeletal development, and rickets; and illustrates the power of mutagenesis in exploring animal models of human disease.

Nicotine Inhibits Memory CTL Programming

PubMed Central

Sun, Zhifeng; Smyth, Kendra; Garcia, Karla; Mattson, Elliot; Li, Lei; Xiao, Zhengguo

2013-01-01

Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development. PMID:23844169

Molecular basis of pharmacotherapies for cognition in Down syndrome

PubMed Central

Gardiner, Katheleen J.

2009-01-01

Intellectual disability (ID) in Down syndrome (DS) ranges from low normal to severely impaired, and has a significant impact on the quality of life of the individuals affected and their families. Because the incidence of DS remains at approximately one in 700 live births and the life span is now >50 years, development of pharmacotherapies for cognitive deficits is an important goal. DS is due to an extra copy of human chromosome 21 and has often been considered too complex a genetic abnormality to be amenable to intervention. However, recent successes in rescuing learning/memory impairments in a mouse model of DS suggest that this negative outlook may not be justified. In this article, we first review the DS phenotype, chromosome 21 gene content and mouse models, and then discuss recent successes and remaining challenges in the identification of targets for and preclinical evaluation of potential therapeutics. PMID:19963286

Transposable elements become active and mobile in the genomes of aging mammalian somatic tissues.

PubMed

De Cecco, Marco; Criscione, Steven W; Peterson, Abigail L; Neretti, Nicola; Sedivy, John M; Kreiling, Jill A

2013-12-01

Transposable elements (TEs) were discovered by Barbara McClintock in maize and have since been found to be ubiquitous in all living organisms. Transposition is mutagenic and organisms have evolved mechanisms to repress the activity of their endogenous TEs. Transposition in somatic cells is very low, but recent evidence suggests that it may be derepressed in some cases, such as cancer development. We have found that during normal aging several families of retrotransposable elements (RTEs) start being transcribed in mouse tissues. In advanced age the expression culminates in active transposition. These processes are counteracted by calorie restriction (CR), an intervention that slows down aging. Retrotransposition is also activated in age-associated, naturally occurring cancers in the mouse. We suggest that somatic retrotransposition is a hitherto unappreciated aging process. Mobilization of RTEs is likely to be an important contributor to the progressive dysfunction of aging cells.

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

Kvon, Evgeny Z.; Kamneva, Olga K.; Melo, Uirá S.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. In this paper, we identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologsmore » caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution.« less

Computer-aided detection and quantification of endolymphatic hydrops within the mouse cochlea in vivo using optical coherence tomography

NASA Astrophysics Data System (ADS)

Liu, George S.; Kim, Jinkyung; Applegate, Brian E.; Oghalai, John S.

2017-07-01

Diseases that cause hearing loss and/or vertigo in humans such as Meniere's disease are often studied using animal models. The volume of endolymph within the inner ear varies with these diseases. Here, we used a mouse model of increased endolymph volume, endolymphatic hydrops, to develop a computer-aided objective approach to measure endolymph volume from images collected in vivo using optical coherence tomography. The displacement of Reissner's membrane from its normal position was measured in cochlear cross sections. We validated our computer-aided measurements with manual measurements and with trained observer labels. This approach allows for computer-aided detection of endolymphatic hydrops in mice, with test performance showing sensitivity of 91% and specificity of 87% using a running average of five measurements. These findings indicate that this approach is accurate and reliable for classifying endolymphatic hydrops and quantifying endolymph volume.

MRI evidence of endolymphatic impermeability to the gadolinium molecule in the in vivo mouse inner ear at 9.4 tesla.

PubMed

Counter, S Allen; Nikkhou, Sahar; Brené, Stefan; Damberg, Peter; Sierakowiak, Adam; Klason, Tomas; Berglin, Cecilia Engmér; Laurell, Göran

2013-01-01

Previous in vivo experimental magnetic resonance imaging (MRI) investigations of the mammalian inner ear at 4.7 Tesla have indicated that intravenously injected gadolinium (Gd) penetrates the perilymphatic labyrinth, but not the endolymphatic membranous labyrinth. In the present study, high field MRI at 9.4T was used to visualize the in vivo mouse vestibulo-cochlea system, and to determine whether the endolymphatic system is permeable to a Gd complex. A 9.4 T Varian magnet equipped with a 12 cm inner diameter gradient system with maximum gradient strength of 600 mT/m, a millipede coil (Varian design) and a Gd contrast agent were used for image acquisition in the normal C57 BL-6 mouse. High-resolution 2D and 3D images of the mouse cochlea were acquired within 80 minutes following intravenous injection of Gd. Gd initially permeated the perilymphatic scala tympani and scala vestibuli, and permitted visualization of both cochlear turns from base to apex. The superior, inferior and lateral semicircular canals were subsequently visualized in 3 planes. The membranous endolymphatic labyrinth was impermeable to intravenously injected Gd, and thus showed no apparent uptake of Gd at 9.4T. The 9.4T field strength MRI permitted acquisition of high resolution images of anatomical and physiological features of the normal, wild type mouse perilymphatic inner ear in vivo, and provided further evidence that the endolymphatic system is impermeable to intravenously injected Gd.

Sensitivity of housekeeping genes in the suprachiasmatic nucleus of the mouse brain to diet and the daily light-dark cycle.

PubMed

Cleal, Jane K; Shepherd, James N; Shearer, Jasmine L; Bruce, Kimberley D; Cagampang, Felino R

2014-08-05

The endogenous timing system within the suprachiasmatic nuclei (SCN) of the hypothalamus drives the cyclic expression of the clock molecules across the 24h day-night cycle controlling downstream molecular pathways and physiological processes. The developing fetal clock system is sensitive to the environment and physiology of the pregnant mother and as such disruption of this system could lead to altered physiology in the offspring. Characterizing the gene profiles of the endogenous molecular clock system by quantitative reverse transcription polymerase chain reaction is dependent on normalization by appropriate housekeeping genes (HKGs). However, many HKGs commonly used as internal controls, although stably expressed under control conditions, can vary significantly in their expression under certain experimental conditions. Here we analyzed the expression of 10 classic HKG across the 24h light-dark cycle in the SCN of mouse offspring exposed to normal chow or a high fat diet during early development and in postnatal life. We found that the HKGs glyceraldehyde-3-phosphate dehydrogenase, beta actin and adenosine triphosphate synthase subunit to be the most stably expressed genes in the SCN regardless of diet or time within the 24h light-dark cycle, and are therefore suitable to be used as internal controls. However SCN samples collected during the light and dark periods did show differences in expression and as such the timing of collection should be considered when carrying out gene expression studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire.

PubMed

Dashtsoodol, Nyambayar; Shigeura, Tomokuni; Ozawa, Ritsuko; Harada, Michishige; Kojo, Satoshi; Watanabe, Takashi; Koseki, Haruhiko; Nakayama, Manabu; Ohara, Osamu; Taniguchi, Masaru

2016-01-01

Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells.

Regulatory effect of Bcl-2 in ultraviolet radiation-induced apoptosis of the mouse crystalline lens

PubMed Central

DONG, YUCHEN; ZHENG, YAJUAN; XIAO, JUN; ZHU, CHAO; ZHAO, MEISHENG

2016-01-01

The aim of the present study was to analyze the role of Bcl-2 during the process of apoptosis in the mouse crystalline lens. In total, 12 normal mice served as the control group and 12 Bcl-2 knockout (K.O) mice served as the experimental group. The mouse crystalline lens was sampled for the detection of Bcl-2 and caspase-3 expression following exposure to ultraviolet (UV) radiation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine Bcl-2 expression in the groups of normal mice receiving UV radiation or not receiving UV radiation. Samples of the murine crystalline lens were microscopically harvested and analyzed using western blotting. Apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, caspase 3 activity was examined using enzyme-linked immunosorbent assay kits, and RT-qPCR was used to analyze caspase-3 expression levels. The results of the present study demonstrated that there was no statistically significant difference in the level of Bcl-2 gene transcription between the two groups. In addition, UV radiation did not change the macrostructure of the crystalline lens in the group of normal mice or the group of Bcl-2 K.O mice. The results of the TUNEL assay indicated that the normal-UV group exhibited a more significant apoptosis level compared with the Bcl-2 K.O-UV group. Furthermore, the mRNA expression level of caspase-3 in the normal-UV group was significantly higher compared with the normal-nonUV group (P<0.05), while the levels in the Bcl-2 K.O-UV group were significantly higher compared with the Bcl-2 K.O and normal-nonUV groups (P<0.05). In addition, the mRNA expression level of caspase-3 was significantly higher in the normal-UV, as compared with the Bcl-2 K.O-UV group (P<0.05), and the variation trends in caspase-3 activity were consistent. In conclusion, the results of the present study demonstrated that Bcl-2 may have an important role in the promotion of UV-induced apoptosis in the crystalline lens. PMID:26998022

Regulatory effect of Bcl-2 in ultraviolet radiation-induced apoptosis of the mouse crystalline lens.

PubMed

Dong, Yuchen; Zheng, Yajuan; Xiao, Jun; Zhu, Chao; Zhao, Meisheng

2016-03-01

The aim of the present study was to analyze the role of Bcl-2 during the process of apoptosis in the mouse crystalline lens. In total, 12 normal mice served as the control group and 12 Bcl-2 knockout (K.O) mice served as the experimental group. The mouse crystalline lens was sampled for the detection of Bcl-2 and caspase-3 expression following exposure to ultraviolet (UV) radiation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to determine Bcl-2 expression in the groups of normal mice receiving UV radiation or not receiving UV radiation. Samples of the murine crystalline lens were microscopically harvested and analyzed using western blotting. Apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, caspase 3 activity was examined using enzyme-linked immunosorbent assay kits, and RT-qPCR was used to analyze caspase-3 expression levels. The results of the present study demonstrated that there was no statistically significant difference in the level of Bcl-2 gene transcription between the two groups. In addition, UV radiation did not change the macrostructure of the crystalline lens in the group of normal mice or the group of Bcl-2 K.O mice. The results of the TUNEL assay indicated that the normal-UV group exhibited a more significant apoptosis level compared with the Bcl-2 K.O-UV group. Furthermore, the mRNA expression level of caspase-3 in the normal-UV group was significantly higher compared with the normal-nonUV group (P<0.05), while the levels in the Bcl-2 K.O-UV group were significantly higher compared with the Bcl-2 K.O and normal-nonUV groups (P<0.05). In addition, the mRNA expression level of caspase-3 was significantly higher in the normal-UV, as compared with the Bcl-2 K.O-UV group (P<0.05), and the variation trends in caspase-3 activity were consistent. In conclusion, the results of the present study demonstrated that Bcl-2 may have an important role in the promotion of UV-induced apoptosis in the crystalline lens.

STRIP1, a core component of STRIPAK complexes, is essential for normal mesoderm migration in the mouse embryo.

PubMed

Bazzi, Hisham; Soroka, Ekaterina; Alcorn, Heather L; Anderson, Kathryn V

2017-12-19

Regulated mesoderm migration is necessary for the proper morphogenesis and organ formation during embryonic development. Cell migration and its dependence on the cytoskeleton and signaling machines have been studied extensively in cultured cells; in contrast, remarkably little is known about the mechanisms that regulate mesoderm cell migration in vivo. Here, we report the identification and characterization of a mouse mutation in striatin-interacting protein 1 ( Strip1 ) that disrupts migration of the mesoderm after the gastrulation epithelial-to-mesenchymal transition (EMT). STRIP1 is a core component of the biochemically defined mammalian striatin-interacting phosphatases and kinase (STRIPAK) complexes that appear to act through regulation of protein phosphatase 2A (PP2A), but their functions in mammals in vivo have not been examined. Strip1 -null mutants arrest development at midgestation with profound disruptions in the organization of the mesoderm and its derivatives, including a complete failure of the anterior extension of axial mesoderm. Analysis of cultured mesoderm explants and mouse embryonic fibroblasts from null mutants shows that the mesoderm migration defect is correlated with decreased cell spreading, abnormal focal adhesions, changes in the organization of the actin cytoskeleton, and decreased velocity of cell migration. The results show that STRIPAK complexes are essential for cell migration and tissue morphogenesis in vivo. Copyright © 2017 the Author(s). Published by PNAS.

Adamts18 deletion results in distinct developmental defects and provides a model for congenital disorders of lens, lung, and female reproductive tract development.

PubMed

Ataca, Dalya; Caikovski, Marian; Piersigilli, Alessandra; Moulin, Alexandre; Benarafa, Charaf; Earp, Sarah E; Guri, Yakir; Kostic, Corinne; Arsenijevic, Yvan; Soininen, Raija; Apte, Suneel S; Brisken, Cathrin

2016-11-15

The ADAMTS family comprises 19 secreted metalloproteinases that cleave extracellular matrix components and have diverse functions in numerous disease and physiological contexts. A number of them remain 'orphan' proteases and among them is ADAMTS18, which has been implicated in developmental eye disorders, platelet function and various malignancies. To assess in vivo function of ADAMTS18, we generated a mouse strain with inactivated Adamts18 alleles. In the C57Bl6/Ola background, Adamts18-deficient mice are born in a normal Mendelian ratio, and are viable but show a transient growth delay. Histological examination revealed a 100% penetrant eye defect resulting from leakage of lens material through the lens capsule occurring at embryonic day (E)13.5, when the lens grows rapidly. Adamts18-deficient lungs showed altered bronchiolar branching. Fifty percent of mutant females are infertile because of vaginal obstruction due to either a dorsoventral vaginal septum or imperforate vagina. The incidence of ovarian rete is increased in the mutant mouse strain. Thus, Adamts18 is essential in the development of distinct tissues and the new mouse strain is likely to be useful for investigating ADAMTS18 function in human disease, particularly in the contexts of infertility and carcinogenesis. © 2016. Published by The Company of Biologists Ltd.

Disordered vertebral and rib morphology in pudgy mice. Structural relationships to human scoliosis.

PubMed

Shapiro, Frederic

2016-01-01

Normal and abnormal vertebral development have been studied over the past 200 years at increasing levels of resolution as techniques for biological investigation have improved. Disordered development of the axial skeleton from the early embryonic period on leads to structurally malformed vertebrae and intervertebral discs and ribs causing the severe deformities of scoliosis, kyphosis, and kyphoscoliosis. Developmental malformation of the axial skeleton therefore has led to considerable biological and clinical interest. This work will detail our studies on the structural deformities of the vertebral column and adjacent ribs in the pudgy mouse [1] caused by mutations in the delta-like 3 (Dll3) gene of the Notch family [2]. While gene abnormalities in the pudgy mouse have been outlined, there has been no in-depth assessment of the histopathology of the pudgy vertebral and rib abnormalities that this study will provide. In addition, although congenital scoliosis has been recognized as a clinical problem since the mid-nineteenth century (1800s) [3] and accurately defined by radiography since the early twentieth century (1900s) [4-6], there have been few detailed histopathologic studies of human cases. We will also relate our histopathologic findings in the pudgy mouse to the histopathology of human vertebral and rib malformations in clinical cases of congenital scoliosis, one of which we defined in detail previously [7].

Aging, Breast Cancer and the Mouse Model

DTIC Science & Technology

2005-05-01

architecture and function of the of normal human cells in culture ( Hayflick , 1965). This limit surrounding tissue and stimulate (or inhibit) the... LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON OF ABSTRACT OF PAGES USAMRMC a. REPORT b. ABSTRACT c. THIS PAGE 19b. TELEPHONE NUMBER (include area U...mammary cancers in the mouse and that these tumors have strikingly similar histology. Nonetheless, several limitations exists to this model system and

Role of Inflammation in MPTP-Induced Dopaminergic Neuronal Death

DTIC Science & Technology

2008-12-01

treated mouse . We found that indeed both microglia and astrocytes are activated in the SNpc, that certain enzymes, such as NADPH oxidase and...different time points in the MPTP mouse model of PD using both normal and NADPH oxidase -deficient mice was the plan. This included assessing...superoxide radical can be produced in several different ways. First of all, DA itself is metabolized by monoamine oxidase (MAO), an outer

Antibody response to Giardia muris trophozoites in mouse intestine.

PubMed

Heyworth, M F

1986-05-01

The protozoan parasite Giardia muris colonizes the mouse small intestinal lumen. This parasite is cleared immunologically from the intestine of normal mice. In contrast, T-lymphocyte-deficient (nude) mice have an impaired immunological response to G. muris and become chronically infected. In the present study, trophozoites were harvested from the intestinal lumen of immunocompetent BALB/c mice and nude mice and examined for surface-bound mouse immunoglobulins by immunofluorescence microscopy. Immunoglobulin A (IgA) and IgG, but not IgM, were detected on trophozoites obtained from BALB/c mice, from day 10 of the infection onwards. Trophozoites from nude mice showed very little evidence of surface-bound mouse immunoglobulin at any time during the 5-week period immediately following infection of these animals with G. muris cysts. Intestinal G. muris infection was cleared by the BALB/c mice but not by the nude animals. The data suggest that parasite-specific IgA and IgG bind to G. muris trophozoites in the intestinal lumen of immunocompetent BALB/c mice. Intestinal antibodies that bind to trophozoite surfaces are likely to play an important part in the clearance of G. muris infection by immunocompetent mice. The inability of nude mice to clear this infection at a normal rate is likely to be due to impairment of Giardia-specific intestinal antibody production.

Tissue Gene Expression Analysis Using Arrayed Normalized cDNA Libraries

PubMed Central

Eickhoff, Holger; Schuchhardt, Johannes; Ivanov, Igor; Meier-Ewert, Sebastian; O'Brien, John; Malik, Arif; Tandon, Neeraj; Wolski, Eryk-Witold; Rohlfs, Elke; Nyarsik, Lajos; Reinhardt, Richard; Nietfeld, Wilfried; Lehrach, Hans

2000-01-01

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference. [The sequence data described in this paper have been submitted to the EMBL data library under accession nos. AL360374–AL36537.] PMID:10958641

Prostate and mammary adenocarcinoma in transgenic mice carrying a rat C3(1) simian virus 40 large tumor antigen fusion gene.

PubMed Central

Maroulakou, I G; Anver, M; Garrett, L; Green, J E

1994-01-01

A transgenic mouse model for prostate and mammary cancer has been developed in mice containing a recombinant gene expressing the simian virus 40 early-region transforming sequences under the regulatory control of the rat prostatic steroid binding protein [C3(1)] gene. Male transgenic mice develop prostatic hyperplasia in early life that progresses to adenoma or adenocarcinoma in most animals surviving to longer than 7 months of age. Prostate cancer metastases to lung have been observed. Female animals from the same founder lines generally develop mammary hyperplasia by 3 months of age with subsequent development of mammary adenocarcinoma by 6 months of age in 100% of the animals. The development of tumors correlates with the expression of the transgene as determined by Northern blot and immunohistochemical analyses. The results of these experiments demonstrate that the C3(1) regulatory region used in these experiments is useful for targeting expression to the prostate and mammary gland. To our knowledge, this experimental system is the first reported transgenic mouse model for prostate cancer. These transgenic animals offer the opportunity to study hormone response elements in vivo and the multistage progression from normal tissue to carcinoma in the prostate and mammary glands. Images PMID:7972041

Adrenocorticotropic hormone affects nonapoptotic cell death of undifferentiated germ cells in the fetal mouse testis: in vivo study by exo utero transplantation of corticotropic tumor cells into embryos.

PubMed

Nimura, Masayuki; Udagawa, Jun; Otani, Hiroki

2008-06-01

Adrenocorticotropic hormone (ACTH) has been suggested to have possible roles in the fetal testes, one of the organs that express its specific receptors, melanocortin type 2 and 5 receptors (MC2R and MC5R), during the fetal period. We investigated the effect of ACTH on the cells in the testis cord of the fetal mouse testis by inducing ACTH-secreting AtT20 tumor cells in mouse fetuses. We first identified that mouse testicular germ cells at embryonic day (E) 16.5 and E18.5 spermatogonia were entirely CDH1 (E-cadherin)-positive by immunohistochemistry. We next performed AtT20-cell transplantation into the mouse fetus at E12.5, and analyzed ACTH effects on the development of fetal male mouse germ cells that express MC2R and MC5R at E16.5 and E18.5. The spermatogonia in the testis of AtT20-implanted embryos exhibited morphological changes, including pyknotic nuclei and swollen cytoplasm. In the AtT20-implanted embryos, the number of spermatogonia per unit area of the testis cord was significantly lower, but there were more pyknotic spermatogonia than in the controls. Single-stranded DNA-positive (apoptotic) and histone H3-positive (mitotic) spermatogonia were rarely observed and their numbers did not significantly differ in the two groups. Anti-Müllerian hormone (AMH)-positive Sertoli cells, another cell type that constitutes the fetal testis cord but does not express MC2R or MC5R, showed no apparent morphological changes compared with controls, nor were their numbers in the two groups significantly different between the two groups. These results suggest that ACTH, via MC2R and/or MC5R, may be involved in the nonapoptotic cell death of fetal mouse spermatogonia that is observed during the normal perinatal period.

Unexpected effects of different genetic backgrounds on identification of genomic rearrangements via whole-genome next generation sequencing.

PubMed

Chen, Zhangguo; Gowan, Katherine; Leach, Sonia M; Viboolsittiseri, Sawanee S; Mishra, Ameet K; Kadoishi, Tanya; Diener, Katrina; Gao, Bifeng; Jones, Kenneth; Wang, Jing H

2016-10-21

Whole genome next generation sequencing (NGS) is increasingly employed to detect genomic rearrangements in cancer genomes, especially in lymphoid malignancies. We recently established a unique mouse model by specifically deleting a key non-homologous end-joining DNA repair gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in germinal center B cells. This mouse model spontaneously develops mature B cell lymphomas (termed G1XP lymphomas). Here, we attempt to employ whole genome NGS to identify novel structural rearrangements, in particular inter-chromosomal translocations (CTXs), in these G1XP lymphomas. We sequenced six lymphoma samples, aligned our NGS data with mouse reference genome (in C57BL/6J (B6) background) and identified CTXs using CREST algorithm. Surprisingly, we detected widespread CTXs in both lymphomas and wildtype control samples, majority of which were false positive and attributable to different genetic backgrounds. In addition, we validated our NGS pipeline by sequencing multiple control samples from distinct tissues of different genetic backgrounds of mouse (B6 vs non-B6). Lastly, our studies showed that widespread false positive CTXs can be generated by simply aligning sequences from different genetic backgrounds of mouse. We conclude that mapping and alignment with reference genome might not be a preferred method for analyzing whole-genome NGS data obtained from a genetic background different from reference genome. Given the complex genetic background of different mouse strains or the heterogeneity of cancer genomes in human patients, in order to minimize such systematic artifacts and uncover novel CTXs, a preferred method might be de novo assembly of personalized normal control genome and cancer cell genome, instead of mapping and aligning NGS data to mouse or human reference genome. Thus, our studies have critical impact on the manner of data analysis for cancer genomics.

Development of the mouse cochlea database (MCD).

PubMed

Santi, Peter A; Rapson, Ian; Voie, Arne

2008-09-01

The mouse cochlea database (MCD) provides an interactive, image database of the mouse cochlea for learning its anatomy and data mining of its resources. The MCD website is hosted on a centrally maintained, high-speed server at the following URL: (http://mousecochlea.umn.edu). The MCD contains two types of image resources, serial 2D image stacks and 3D reconstructions of cochlear structures. Complete image stacks of the cochlea from two different mouse strains were obtained using orthogonal plane fluorescence optical microscopy (OPFOS). 2D images of the cochlea are presented on the MCD website as: viewable images within a stack, 2D atlas of the cochlea, orthogonal sections, and direct volume renderings combined with isosurface reconstructions. In order to assess cochlear structures quantitatively, "true" cross-sections of the scala media along the length of the basilar membrane were generated by virtual resectioning of a cochlea orthogonal to a cochlear structure, such as the centroid of the basilar membrane or the scala media. 3D images are presented on the MCD website as: direct volume renderings, movies, interactive QuickTime VRs, flythrough, and isosurface 3D reconstructions of different cochlear structures. 3D computer models can also be used for solid model fabrication by rapid prototyping and models from different cochleas can be combined to produce an average 3D model. The MCD is the first comprehensive image resource on the mouse cochlea and is a new paradigm for understanding the anatomy of the cochlea, and establishing morphometric parameters of cochlear structures in normal and mutant mice.

Localization of cholinergic innervation and neurturin receptors in adult mouse heart and expression of the neurturin gene.

PubMed

Mabe, Abigail M; Hoard, Jennifer L; Duffourc, Michelle M; Hoover, Donald B

2006-10-01

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor alpha2 (GFRalpha2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRalpha2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.

A Novel, Real-Time, In Vivo Mouse Retinal Imaging System

PubMed Central

Butler, Mark C.; Sullivan, Jack M.

2015-01-01

Purpose To develop an efficient, low-cost instrument for robust real-time imaging of the mouse retina in vivo, and assess system capabilities by evaluating various animal models. Methods Following multiple disappointing attempts to visualize the mouse retina during a subretinal injection using commercially available systems, we identified the key limitation to be inadequate illumination due to off axis illumination and poor optical train optimization. Therefore, we designed a paraxial illumination system for Greenough-type stereo dissecting microscope incorporating an optimized optical launch and an efficiently coupled fiber optic delivery system. Excitation and emission filters control spectral bandwidth. A color coupled-charged device (CCD) camera is coupled to the microscope for image capture. Although, field of view (FOV) is constrained by the small pupil aperture, the high optical power of the mouse eye, and the long working distance (needed for surgical manipulations), these limitations can be compensated by eye positioning in order to observe the entire retina. Results The retinal imaging system delivers an adjustable narrow beam to the dilated pupil with minimal vignetting. The optic nerve, vasculature, and posterior pole are crisply visualized and the entire retina can be observed through eye positioning. Normal and degenerative retinal phenotypes can be followed over time. Subretinal or intraocular injection procedures are followed in real time. Real-time, intravenous fluorescein angiography for the live mouse has been achieved. Conclusions A novel device is established for real-time viewing and image capture of the small animal retina during subretinal injections for preclinical gene therapy studies. PMID:26551329

Automated gait analysis in the open-field test for laboratory mice.

PubMed

Leroy, Toon; Silva, Mitchell; D'Hooge, Rudi; Aerts, Jean-Marie; Berckmans, Daniel

2009-02-01

In this article, an automated and accurate mouse observation method, based on a conventional test for motor function evaluation, is outlined. The proposed measurement technique was integrated in a regular open-field test, where the trajectory and locomotion of a free-moving mouse were measured simultaneously. The system setup consisted of a transparent cage and a camera placed below it with its lens pointing upward, allowing for images to be captured from underneath the cage while the mouse was walking on the transparent cage floor. Thus, additional information was obtained about the position of the limbs of the mice for gait reconstruction. In a first step, the camera was calibrated as soon as it was fixed in place. A linear calibration factor, relating distances in image coordinates to real-world dimensions, was determined. In a second step, the mouse was located and its body contour segmented from the image by subtracting a previously taken "background" image of the empty cage from the camera image. In a third step, the movement of the mouse was analyzed and its speed estimated from its location in the past few images. If the speed was above a 1-sec threshold, the mouse was recognized to be running, and the image was further processed for footprint recognition. In a fourth step, color filtering was applied within the recovered mouse region to measure the position of the mouse's paws, which were visible in the image as small pink spots. Paws that were detected at the same location in a number of subsequent images were kept as footprints-that is, paws in contact with the cage floor. The footprints were classified by their position relative to the mouse's outline as corresponding to the front left or right paw or the hind left or right paw. Finally, eight parameters were calculated from the footprint pattern to describe the locomotion of the mouse: right/left overlap, front/hind base, right/left front limb stride, and right/left hind limb stride. As an application, the system was tested using normal mice and mice displaying pentobarbital-induced ataxia. The footprint parameters measured using the proposed system showed differences of 10% to 20% between normal and ataxic mice.

Deletion of ameloblastin exon 6 is associated with amelogenesis imperfecta.

PubMed

Poulter, James A; Murillo, Gina; Brookes, Steven J; Smith, Claire E L; Parry, David A; Silva, Sandra; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

2014-10-15

Amelogenesis imperfecta (AI) describes a heterogeneous group of inherited dental enamel defects reflecting failure of normal amelogenesis. Ameloblastin (AMBN) is the second most abundant enamel matrix protein expressed during amelogenesis. The pivotal role of AMBN in amelogenesis has been confirmed experimentally using mouse models. However, no AMBN mutations have been associated with human AI. Using autozygosity mapping and exome sequencing, we identified genomic deletion of AMBN exon 6 in a second cousin consanguineous family with three of the six children having hypoplastic AI. The genomic deletion corresponds to an in-frame deletion of 79 amino acids, shortening the protein from 447 to 368 residues. Exfoliated primary teeth (unmatched to genotype) were available from family members. The most severely affected had thin, aprismatic enamel (similar to that reported in mice homozygous for Ambn lacking exons 5 and 6). Other teeth exhibited thicker but largely aprismatic enamel. One tooth had apparently normal enamel. It has been suggested that AMBN may function in bone development. No clinically obvious bone or other co-segregating health problems were identified in the family investigated. This study confirms for the first time that AMBN mutations cause non-syndromic human AI and that mouse models with disrupted Ambn function are valid. © The Author 2014. Published by Oxford University Press.

The oculocerebrorenal syndrome gene product is a 105-kD protein localized to the Golgi complex.

PubMed Central

Olivos-Glander, I M; Jänne, P A; Nussbaum, R L

1995-01-01

The oculocerebrorenal syndrome of Lowe (OCRL) is a multisystem disorder affecting the lens, kidney, and CNS. The predicted amino acid sequence of the OCRL gene, OCRL-1, was used to develop antibodies against the OCRL-1 protein. Western blot analysis using affinity-purified serum against the amino terminus of the OCRL-1 gene product (ocrl-1) demonstrates a single protein of 105 kD in fibroblasts of a normal individual that is absent in fibroblasts of an OCRL patient who lacks OCRL-1 transcript. A single protein with the same electrophoretic mobility is found by western analysis in various human cultured cell lines, and approximately the same size protein is also found in all mouse tissues tested. Northern analysis of various human and mouse tissues demonstrate that OCRL-1 transcript is expressed in nearly all tissues examined. By immunofluorescence, the ocrl-1 antibody stains a juxtanuclear region in normal fibroblast cells, while no specific staining is evident in the OCRL patient who produces no transcript. Colocalization of the ocrl-1 protein to the Golgi complex was demonstrated using a known monoclonal antibody against a Golgi-specific coat protein, beta-COP (beta coatomer protein). Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7573041

A Computer Program for Practical Semivariogram Modeling and Ordinary Kriging: A Case Study of Porosity Distribution in an Oil Field

NASA Astrophysics Data System (ADS)

Mert, Bayram Ali; Dag, Ahmet

2017-12-01

In this study, firstly, a practical and educational geostatistical program (JeoStat) was developed, and then example analysis of porosity parameter distribution, using oilfield data, was presented. With this program, two or three-dimensional variogram analysis can be performed by using normal, log-normal or indicator transformed data. In these analyses, JeoStat offers seven commonly used theoretical variogram models (Spherical, Gaussian, Exponential, Linear, Generalized Linear, Hole Effect and Paddington Mix) to the users. These theoretical models can be easily and quickly fitted to experimental models using a mouse. JeoStat uses ordinary kriging interpolation technique for computation of point or block estimate, and also uses cross-validation test techniques for validation of the fitted theoretical model. All the results obtained by the analysis as well as all the graphics such as histogram, variogram and kriging estimation maps can be saved to the hard drive, including digitised graphics and maps. As such, the numerical values of any point in the map can be monitored using a mouse and text boxes. This program is available to students, researchers, consultants and corporations of any size free of charge. The JeoStat software package and source codes available at: http://www.jeostat.com/JeoStat_2017.0.rar.

Application study of the optical biopsy system for small experimental animals

NASA Astrophysics Data System (ADS)

Sato, Hidetoshi; Suzuki, Toshiaki; Morita, Shin-ichi; Maruyama, Atsushi; Shimosegawa, Toru; Matsuura, Yuji; Kanai, Gen'ichi; Ura, Nobuo; Masutani, Koji; Ozaki, Yukihiro

2008-02-01

An optical biopsy system for small experimental animals has been developed. The system includes endoscope probe, portable probe and two kinds of miniaturized Raman probes. The micro Raman probe (MRP) is made of optical fibers and the ball lens hollow optical fiber Raman probe (BHRP) is made of hollow fiber. The former has large focal depth and suitable to measure average spectra of subsurface tissue. The latter has rather small focal depth and it is possible to control focal length by selecting ball lens attached at the probe head. It is suitable to survey materials at the fixed depth in the tissue. The system is applied to study various small animal cancer models, such as esophagus and stomach rat models and subcutaneous mouse models of pancreatic cancers. In the studies of subcutaneous tumor model mouse, it is suggested that protein conformational changes occur in the tumor tissue within few minutes after euthanasia of the mouse. No more change is observed for the following ten minutes. Any alterations in the molecular level are not observed in normal skin, muscle tissues. Since the change completes in such a short time, it is suggested that this phenomenon caused by termination of blood circulation.

Morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse.

PubMed

Fletcher, Sue; Honeyman, Kaite; Fall, Abbie M; Harding, Penny L; Johnsen, Russell D; Steinhaus, Joshua P; Moulton, Hong M; Iversen, Patrick L; Wilton, Stephen D

2007-09-01

Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterized by an absence of functional protein, whereas Becker muscular dystrophy, commonly caused by in-frame deletions, shows synthesis of partially functional protein. Anti-sense oligonucleotides can induce specific exon removal during processing of the dystrophin primary transcript, while maintaining or restoring the reading frame, and thereby overcome protein-truncating mutations. The mdx mouse has a non-sense mutation in exon 23 of the dystrophin gene that precludes functional dystrophin production, and this model has been used in the development of treatment strategies for dystrophinopathies. A phosphorodiamidate morpholino oligomer (PMO) has previously been shown to exclude exon 23 from the dystrophin gene transcript and induce dystrophin expression in the mdxmouse, in vivo and in vitro. In this report, a cell-penetrating peptide (CPP)-conjugated oligomer targeted to the mouse dystrophin exon 23 donor splice site was administered to mdxmice by intraperitoneal injection. We demonstrate dystrophin expression and near-normal muscle architecture in all muscles examined, except for cardiac muscle. The CPP greatly enhanced uptake of the PMO, resulting in widespread dystrophin expression.

Quantitative and Qualitative Changes in V-J α Rearrangements During Mouse Thymocytes Differentiation

PubMed Central

Pasqual, Nicolas; Gallagher, Maighréad; Aude-Garcia, Catherine; Loiodice, Mélanie; Thuderoz, Florence; Demongeot, Jacques; Ceredig, Rod; Marche, Patrice Noël; Jouvin-Marche, Evelyne

2002-01-01

Knowledge of the complete nucleotide sequence of the mouse TCRAD locus allows an accurate determination V-J rearrangement status. Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR α chain in normal mouse thymocytes during development. These respective qualitative and quantitative approaches give rise to four major points describing the control of gene rearrangements. (a) The V-J recombination pattern is not random during ontogeny and generates a limited TCR α repertoire; (b) V-J rearrangement control is intrinsic to the thymus; (c) each V gene rearranges to a set of contiguous J segments with a gaussian-like frequency; (d) there are more rearrangements involving V genes at the 3′ side than 5′ end of V region. Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways. These results provide a new insight into TCR α repertoire size and suggest a scenario for V usage during differentiation. PMID:12417627

Characterization of Oct4-GFP transgenic mice as a model to study the effect of environmental estrogens on the maturation of male germ cells by using flow cytometry.

PubMed

Porro, Valentina; Pagotto, Romina; Harreguy, María Belén; Ramírez, Sofía; Crispo, Martina; Santamaría, Clarisa; Luque, Enrique H; Rodríguez, Horacio A; Bollati-Fogolín, Mariela

2015-11-01

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17α-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development. Copyright © 2015 Elsevier Ltd. All rights reserved.

The fibronectin leucine-rich repeat transmembrane protein Flrt2 is required in the epicardium to promote heart morphogenesis

PubMed Central

Müller, Pari-Sima; Schulz, Ramona; Maretto, Silvia; Costello, Ita; Srinivas, Shankar; Bikoff, Elizabeth; Robertson, Elizabeth

2011-01-01

The epicardium, the outermost tissue layer that envelops the developing heart and provides essential trophic signals for the myocardium, derives from the pro-epicardial organ (PEO). Two of the three members of the Flrt family of transmembrane glycoproteins, Flrt2 and Flrt3, are strongly co-expressed in the PEO. However, beginning at around day 10 of mouse development, following attachment and outgrowth, Flrt3 is selectively downregulated, and only Flrt2 is exclusively expressed in the fully delaminated epicardium. The present gene-targeting experiments demonstrate that mouse embryos lacking Flrt2 expression arrest at mid-gestation owing to cardiac insufficiency. The defects in integrity of the epicardial sheet and disturbed organization of the underlying basement membrane closely resemble those described in Flrt3-deficient embryos that fail to maintain cell-cell contacts in the anterior visceral endoderm (AVE) signalling centre that normally establishes the A-P axis. Using in vitro and in vivo reconstitution assays, we demonstrate that Flrt2 and Flrt3 are functionally interchangeable. When acting alone, either of these proteins is sufficient to rescue functional activities in the AVE and the developing epicardium. PMID:21350012

SIRT1 Activity Is Linked to Its Brain Region-Specific Phosphorylation and Is Impaired in Huntington’s Disease Mice

PubMed Central

Tulino, Raffaella; Benjamin, Agnesska C.; Jolinon, Nelly; Smith, Donna L.; Chini, Eduardo N.; Carnemolla, Alisia; Bates, Gillian P.

2016-01-01

Huntington’s disease (HD) is a neurodegenerative disorder for which there are no disease-modifying treatments. SIRT1 is a NAD+-dependent protein deacetylase that is implicated in maintaining neuronal health during development, differentiation and ageing. Previous studies suggested that the modulation of SIRT1 activity is neuroprotective in HD mouse models, however, the mechanisms controlling SIRT1 activity are unknown. We have identified a striatum-specific phosphorylation-dependent regulatory mechanism of SIRT1 induction under normal physiological conditions, which is impaired in HD. We demonstrate that SIRT1 activity is down-regulated in the brains of two complementary HD mouse models, which correlated with altered SIRT1 phosphorylation levels. This SIRT1 impairment could not be rescued by the ablation of DBC1, a negative regulator of SIRT1, but was linked to changes in the sub-cellular distribution of AMPK-α1, a positive regulator of SIRT1 function. This work provides insights into the regulation of SIRT1 activity with the potential for the development of novel therapeutic strategies. PMID:26815359

ROLE OF NRF2 IN THE OXIDATIVE STRESS-DEPENDENT HYPERTENSION ASSOCIATED WITH THE DEPLETION OF DJ-1

PubMed Central

Cuevas, Santiago; Yang, Yu; Konkalmatt, Prasad; Asico, Laureano; Feranil, Jun; Jones, John; Villar, Van Anthony; Armando, Ines; Jose, Pedro A.

2015-01-01

Renal dopamine 2 receptor dysfunction is associated with oxidative stress and high blood pressure. We have reported that DJ-1, an oxidative stress response protein, is positively regulated by dopamine 2 receptor in the kidney. The transcription factor Nrf2 regulates the expression of several antioxidant genes. We tested the hypothesis that Nrf2 is involved in the renal DJ-1-mediated inhibition of reactive oxygen species production. We have reported that silencing dopamine 2 receptor in mouse renal proximal tubule cells decreases the expression of DJ-1. We now report that silencing DJ-1 or dopamine 2 receptor in mouse proximal tubule cells and mouse kidneys, decreases Nrf2 expression and activity and increases reactive oxygen species production; blood pressure is also increased in mice in which renal DJ-1 or dopamine 2 receptor is silenced. DJ-1−/− mice have decreased renal Nrf2 expression and activity, and increased nitro-tyrosine levels an dopamine 2 receptor d blood pressure. Silencing Nrf2 in mouse proximal tubule cells does not alter the expression of DJ-1 or dopamine 2 receptor, indicating that Nrf2 is downstream of dopamine 2 receptor and DJ-1. A Nrf2 inducer, bardoxolone, normalizes the systolic blood pressure and renal malondialdehyde levels in DJ-1−/− mice without affecting them in their wild-type littermates. Because Nrf2 ubiquitination is increased in DJ-1−/− mice, we conclude that the protective effect of DJ-1 on renal oxidative stress is mediated, in part, by preventing Nrf2 degradation. Moreover, renal dopamine 2 receptor and DJ-1 are necessary for normal Nrf2 activity to keep a normal redox balance and blood pressure. PMID:25895590

Type II iodothyronine deiodinase provides intracellular 3,5,3'-triiodothyronine to normal and regenerating mouse skeletal muscle.

PubMed

Marsili, Alessandro; Tang, Dan; Harney, John W; Singh, Prabhat; Zavacki, Ann Marie; Dentice, Monica; Salvatore, Domenico; Larsen, P Reed

2011-11-01

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T(3) under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T(4)-to-T(3) conversion increases during differentiation in C(2)C(12) myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given (125)I-T(4) and (131)I-T(3), the intracellular (125)I-T(3)/(131)I-T(3) ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the (125)I-T(3)/(131)I-T(3) ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in (125)I-T(3)/(131)I-T(3) ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of (125)I-T(3) is doubled after skeletal muscle injury. Thus, D2-mediated T(4)-to-T(3) conversion generates significant intracellular T(3) in normal mouse skeletal muscle, with the increased T(3) required for muscle regeneration being provided by increased D2 synthesis, not by T(3) from the circulation.

Type II iodothyronine deiodinase provides intracellular 3,5,3′-triiodothyronine to normal and regenerating mouse skeletal muscle

PubMed Central

Marsili, Alessandro; Tang, Dan; Harney, John W.; Singh, Prabhat; Zavacki, Ann Marie; Dentice, Monica; Salvatore, Domenico

2011-01-01

The FoxO3-dependent increase in type II deiodinase (D2), which converts the prohormone thyroxine (T4) to 3,5,3′-triiodothyronine (T3), is required for normal mouse skeletal muscle differentiation and regeneration. This implies a requirement for an increase in D2-generated intracellular T3 under these conditions, which has not been directly demonstrated despite the presence of D2 activity in skeletal muscle. We directly show that D2-mediated T4-to-T3 conversion increases during differentiation in C2C12 myoblast and primary cultures of mouse neonatal skeletal muscle precursor cells, and that blockade of D2 eliminates this. In adult mice given 125I-T4 and 131I-T3, the intracellular 125I-T3/131I-T3 ratio is significantly higher than in serum in both the D2-expressing cerebral cortex and the skeletal muscle of wild-type, but not D2KO, mice. In D1-expressing liver and kidney, the 125I-T3/131I-T3 ratio does not differ from that in serum. Hypothyroidism increases D2 activity, and in agreement with this, the difference in 125I-T3/131I-T3 ratio is increased further in hypothyroid wild-type mice but not altered in the D2KO. Notably, in wild-type but not in D2KO mice, the muscle production of 125I-T3 is doubled after skeletal muscle injury. Thus, D2-mediated T4-to-T3 conversion generates significant intracellular T3 in normal mouse skeletal muscle, with the increased T3 required for muscle regeneration being provided by increased D2 synthesis, not by T3 from the circulation. PMID:21771965

Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

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

Cuneo, Kyle C.; Mito, Jeffrey K.; Javid, Melodi P.

2013-05-01

Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activatedmore » fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.« less

The dynamics of gene expression changes in a mouse model of oral tumorigenesis may help refine prevention and treatment strategies in patients with oral cancer.

PubMed

Foy, Jean-Philippe; Tortereau, Antonin; Caulin, Carlos; Le Texier, Vincent; Lavergne, Emilie; Thomas, Emilie; Chabaud, Sylvie; Perol, David; Lachuer, Joël; Lang, Wenhua; Hong, Waun Ki; Goudot, Patrick; Lippman, Scott M; Bertolus, Chloé; Saintigny, Pierre

2016-06-14

A better understanding of the dynamics of molecular changes occurring during the early stages of oral tumorigenesis may help refine prevention and treatment strategies. We generated genome-wide expression profiles of microdissected normal mucosa, hyperplasia, dysplasia and tumors derived from the 4-NQO mouse model of oral tumorigenesis. Genes differentially expressed between tumor and normal mucosa defined the "tumor gene set" (TGS), including 4 non-overlapping gene subsets that characterize the dynamics of gene expression changes through different stages of disease progression. The majority of gene expression changes occurred early or progressively. The relevance of these mouse gene sets to human disease was tested in multiple datasets including the TCGA and the Genomics of Drug Sensitivity in Cancer project. The TGS was able to discriminate oral squamous cell carcinoma (OSCC) from normal oral mucosa in 3 independent datasets. The OSCC samples enriched in the mouse TGS displayed high frequency of CASP8 mutations, 11q13.3 amplifications and low frequency of PIK3CA mutations. Early changes observed in the 4-NQO model were associated with a trend toward a shorter oral cancer-free survival in patients with oral preneoplasia that was not seen in multivariate analysis. Progressive changes observed in the 4-NQO model were associated with an increased sensitivity to 4 different MEK inhibitors in a panel of 51 squamous cell carcinoma cell lines of the areodigestive tract. In conclusion, the dynamics of molecular changes in the 4-NQO model reveal that MEK inhibition may be relevant to prevention and treatment of a specific molecularly-defined subgroup of OSCC.

TGFbeta type II receptor signaling controls Schwann cell death and proliferation in developing nerves.

PubMed

D'Antonio, Maurizio; Droggiti, Anna; Feltri, M Laura; Roes, Jürgen; Wrabetz, Lawrence; Mirsky, Rhona; Jessen, Kristján R

2006-08-16

During development, Schwann cell numbers are precisely adjusted to match the number of axons. It is essentially unknown which growth factors or receptors carry out this important control in vivo. Here, we tested whether the type II transforming growth factor (TGF) beta receptor has a role in this process. We generated a conditional knock-out mouse in which the type II TGFbeta receptor is specifically ablated only in Schwann cells. Inactivation of the receptor, evident at least from embryonic day 18, resulted in suppressed Schwann cell death in normally developing and injured nerves. Notably, the mutants also showed a strong reduction in Schwann cell proliferation. Consequently, Schwann cell numbers in wild-type and mutant nerves remained similar. Lack of TGFbeta signaling did not appear to affect other processes in which TGFbeta had been implicated previously, including myelination and response of adult nerves to injury. This is the first in vivo evidence for a growth factor receptor involved in promoting Schwann cell division during development and the first genetic evidence for a receptor that controls normal developmental Schwann cell death.

Identification of Pro-Differentiation p53 Target Genes and Evaluation of Expression in Normal and Malignant Mammary Gland

DTIC Science & Technology

2008-04-01

genes such as c -myc and Klf-4, frequently upregulated in tumors also have been shown to establish and preserve the ES cell phenotype and the rapid...proliferation of ES cells in culture. More importantly, the introduction of these four factors (Oct3/4, Sox-2, c -myc and Klf-4) into mouse embryonic or...GTA-3’; mouse nanog: sense 5’-AAG TAC CTC AGC CTC CAG CA-3’, antisense 5’-CGT AAG GCT GCA GAA AGT GC-3’; mouse c -myc: sense 5’-CAC CAT GCC CCT CAA CGT

Regulation of the expression of GARP/latent-TGF-β1 complexes on mouse T cells and their role in Regulatory T Cell and Th17 differentiation1

PubMed Central

Edwards, Justin P.; Fujii, Hodaka; Zhou, Angela X.; Creemers, John; Unutmaz, Derya; Shevach, Ethan M.

2013-01-01

GARP/LRRC32 has previously been defined as a marker of activated human regulatory T-cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation, but in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4+ T cells results in lack of expression of latent-TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of T conventional cells in vitro. Activated Tregs expressing GARP/latent-TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17 producing cells and Treg is preferentially induced by Tregs expressing the latent-TGF-β1/GARP complex on their cell surface rather than by secreted latent-TGF-β1. PMID:23645881

Regulation of the expression of GARP/latent TGF-β1 complexes on mouse T cells and their role in regulatory T cell and Th17 differentiation.

PubMed

Edwards, Justin P; Fujii, Hodaka; Zhou, Angela X; Creemers, John; Unutmaz, Derya; Shevach, Ethan M

2013-06-01

GARP/LRRC32 was defined as a marker of activated human regulatory T cells (Tregs) that is responsible for surface localization of latent TGF-β1. We find that GARP and latent TGF-β1 are also found on mouse Tregs activated via TCR stimulation; however, in contrast to human Tregs, GARP is also expressed at a low level on resting Tregs. The expression of GARP can be upregulated on mouse Tregs by IL-2 or IL-4 exposure in the absence of TCR signaling. GARP is expressed at a low level on Tregs within the thymus, and Treg precursors from the thymus concomitantly express GARP and Foxp3 upon exposure to IL-2. The expression of GARP is independent of TGF-β1 and TGF-β1 loading into GARP and is independent of furin-mediated processing of pro-TGF-β1 to latent TGF-β1. Specific deletion of GARP in CD4(+) T cells results in lack of expression of latent TGF-β1 on activated Tregs. GARP-deficient Tregs develop normally, are present in normal numbers in peripheral tissues, and are fully competent suppressors of the activation of conventional T cells in vitro. Activated Tregs expressing GARP/latent TGF-β1 complexes are potent inducers of Th17 differentiation in the presence of exogenous IL-6 and inducers of Treg in the presence of IL-2. Induction of both Th17-producing cells and Tregs is caused preferentially by Tregs expressing the latent TGF-β1/GARP complex on their cell surface rather than by secreted latent TGF-β1.

CD24 expression does not affect dopamine neuronal survival in a mouse model of Parkinson's disease.

PubMed

Stott, Simon R W; Hayat, Shaista; Carnwath, Tom; Garas, Shaady; Sleeman, Jonathan P; Barker, Roger A

2017-01-01

Parkinson's disease (PD) is a progressive neurodegenerative condition that is characterised by the loss of specific populations of neurons in the brain. The mechanisms underlying this selective cell death are unknown but by using laser capture microdissection, the glycoprotein, CD24 has been identified as a potential marker of the populations of cells that are affected in PD. Using in situ hybridization and immunohistochemistry on sections of mouse brain, we confirmed that CD24 is robustly expressed by many of these subsets of cells. To determine if CD24 may have a functional role in PD, we modelled the dopamine cell loss of PD in Cd24 mutant mice using striatal delivery of the neurotoxin 6-OHDA. We found that Cd24 mutant mice have an anatomically normal dopamine system and that this glycoprotein does not modulate the lesion effects of 6-OHDA delivered into the striatum. We then undertook in situ hybridization studies on sections of human brain and found-as in the mouse brain-that CD24 is expressed by many of the subsets of the cells that are vulnerable in PD, but not those of the midbrain dopamine system. Finally, we sought to determine if CD24 is required for the neuroprotective effect of Glial cell-derived neurotrophic factor (GDNF) on the dopaminergic nigrostriatal pathway. Our results indicate that in the absence of CD24, there is a reduction in the protective effects of GDNF on the dopaminergic fibres in the striatum, but no difference in the survival of the cell bodies in the midbrain. While we found no obvious role for CD24 in the normal development and maintenance of the dopaminergic nigrostriatal system in mice, it may have a role in mediating the neuroprotective aspects of GDNF in this system.

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

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

PubMed Central

Bortolussi, Giulia; Zentilin, Lorena; Baj, Gabriele; Giraudi, Pablo; Bellarosa, Cristina; Giacca, Mauro; Tiribelli, Claudio; Muro, Andrés F.

2012-01-01

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer. PMID:22094718

A male-specific QTL for social interaction behavior in mice mapped with automated pattern detection by a hidden Markov model incorporated into newly developed freeware.

PubMed

Arakawa, Toshiya; Tanave, Akira; Ikeuchi, Shiho; Takahashi, Aki; Kakihara, Satoshi; Kimura, Shingo; Sugimoto, Hiroki; Asada, Nobuhiko; Shiroishi, Toshihiko; Tomihara, Kazuya; Tsuchiya, Takashi; Koide, Tsuyoshi

2014-08-30

Owing to their complex nature, social interaction tests normally require the observation of video data by a human researcher, and thus are difficult to use in large-scale studies. We previously established a statistical method, a hidden Markov model (HMM), which enables the differentiation of two social states ("interaction" and "indifference"), and three social states ("sniffing", "following", and "indifference"), automatically in silico. Here, we developed freeware called DuoMouse for the rapid evaluation of social interaction behavior. This software incorporates five steps: (1) settings, (2) video recording, (3) tracking from the video data, (4) HMM analysis, and (5) visualization of the results. Using DuoMouse, we mapped a genetic locus related to social interaction. We previously reported that a consomic strain, B6-Chr6C(MSM), with its chromosome 6 substituted for one from MSM/Ms, showed more social interaction than C57BL/6 (B6). We made four subconsomic strains, C3, C5, C6, and C7, each of which has a shorter segment of chromosome 6 derived from B6-Chr6C, and conducted social interaction tests on these strains. DuoMouse indicated that C6, but not C3, C5, and C7, showed higher interaction, sniffing, and following than B6, specifically in males. The data obtained by human observation showed high concordance to those from DuoMouse. The results indicated that the MSM-derived chromosomal region present in C6-but not in C3, C5, and C7-associated with increased social behavior. This method to analyze social interaction will aid primary screening for difference in social behavior in mice. Copyright © 2014 Elsevier B.V. All rights reserved.

ATRX has a critical and conserved role in mammalian sexual differentiation

PubMed Central

2011-01-01

Background X-linked alpha thalassemia, mental retardation syndrome in humans is a rare recessive disorder caused by mutations in the ATRX gene. The disease is characterised by severe mental retardation, mild alpha-thalassemia, microcephaly, short stature, facial, skeletal, genital and gonadal abnormalities. Results We examined the expression of ATRX and ATRY during early development and gonadogenesis in two distantly related mammals: the tammar wallaby (a marsupial) and the mouse (a eutherian). This is the first examination of ATRX and ATRY in the developing mammalian gonad and fetus. ATRX and ATRY were strongly expressed in the developing male and female gonad respectively, of both species. In testes, ATRY expression was detected in the Sertoli cells, germ cells and some interstitial cells. In the developing ovaries, ATRX was initially restricted to the germ cells, but was present in the granulosa cells of mature ovaries from the primary follicle stage onwards and in the corpus luteum. ATRX mRNA expression was also examined outside the gonad in both mouse and tammar wallaby whole embryos. ATRX was detected in the developing limbs, craniofacial elements, neural tissues, tail and phallus. These sites correspond with developmental deficiencies displayed by ATR-X patients. Conclusions There is a complex expression pattern throughout development in both mammals, consistent with many of the observed ATR-X syndrome phenotypes in humans. The distribution of ATRX mRNA and protein in the gonads was highly conserved between the tammar and the mouse. The expression profile within the germ cells and somatic cells strikingly overlaps with that of DMRT1, suggesting a possible link between these two genes in gonadal development. Taken together, these data suggest that ATRX has a critical and conserved role in normal development of the testis and ovary in both the somatic and germ cells, and that its broad roles in early mammalian development and gonadal function have remained unchanged for over 148 million years of mammalian evolution. PMID:21672208

ATRX has a critical and conserved role in mammalian sexual differentiation.

PubMed

Huyhn, Kim; Renfree, Marilyn B; Graves, Jennifer A; Pask, Andrew J

2011-06-14

X-linked alpha thalassemia, mental retardation syndrome in humans is a rare recessive disorder caused by mutations in the ATRX gene. The disease is characterised by severe mental retardation, mild alpha-thalassemia, microcephaly, short stature, facial, skeletal, genital and gonadal abnormalities. We examined the expression of ATRX and ATRY during early development and gonadogenesis in two distantly related mammals: the tammar wallaby (a marsupial) and the mouse (a eutherian). This is the first examination of ATRX and ATRY in the developing mammalian gonad and fetus. ATRX and ATRY were strongly expressed in the developing male and female gonad respectively, of both species. In testes, ATRY expression was detected in the Sertoli cells, germ cells and some interstitial cells. In the developing ovaries, ATRX was initially restricted to the germ cells, but was present in the granulosa cells of mature ovaries from the primary follicle stage onwards and in the corpus luteum. ATRX mRNA expression was also examined outside the gonad in both mouse and tammar wallaby whole embryos. ATRX was detected in the developing limbs, craniofacial elements, neural tissues, tail and phallus. These sites correspond with developmental deficiencies displayed by ATR-X patients. There is a complex expression pattern throughout development in both mammals, consistent with many of the observed ATR-X syndrome phenotypes in humans. The distribution of ATRX mRNA and protein in the gonads was highly conserved between the tammar and the mouse. The expression profile within the germ cells and somatic cells strikingly overlaps with that of DMRT1, suggesting a possible link between these two genes in gonadal development. Taken together, these data suggest that ATRX has a critical and conserved role in normal development of the testis and ovary in both the somatic and germ cells, and that its broad roles in early mammalian development and gonadal function have remained unchanged for over 148 million years of mammalian evolution.

Therapeutic Role of Bmi-1 Inhibitors in Eliminating Prostate Tumor Stem Cells

DTIC Science & Technology

2015-10-01

antitumor activity in mouse xenografts did not exert toxic effects on normal tissues. BMI-1 targeted therapy when combined with taxotere resulted in...utilizing zebrafish xenografts (Sabaawy Lab) and prostate cancer cell lines (Bertino Lab), and 3) Confirmation of the antitumor activity of C-209...in mouse xenografts alone and upon combination with taxotere (Bertino Lab). The following tasks from the approved SOW were performed to achieve the

Angiogenic Signaling in Living Breast Tumor Models

DTIC Science & Technology

2010-06-01

harmonic generation imaging of the diseased state osteogenesis imperfecta : experiment and simulation,” Biophys. J. 94(11), 4504–4514 (2008). 3. O...biopsies, mouse models of breast cancer, and dermis from mouse models of Osteogenesis Imperfecta (OIM) [1–5,7]. The F/B ratio revealed the length scale of...interest in discriminating skin with Osteogenesis Imperfecta [2] from normal dermis [2] and SHG F/B ratio measurements have been used to help determine

A comparative study of the characterization of miR-155 in knockout mice

PubMed Central

Zhang, Dong; Cui, Yongchun; Li, Bin; Luo, Xiaokang; Li, Bo; Tang, Yue

2017-01-01

miR-155 is one of the most important miRNAs and plays a very important role in numerous biological processes. However, few studies have characterized this miRNA in mice under normal physiological conditions. We aimed to characterize miR-155 in vivo by using a comparative analysis. In our study, we compared miR-155 knockout (KO) mice with C57BL/6 wild type (WT) mice in order to characterize miR-155 in mice under normal physiological conditions using many evaluation methods, including a reproductive performance analysis, growth curve, ultrasonic estimation, haematological examination, and histopathological analysis. These analyses showed no significant differences between groups in the main evaluation indices. The growth and development were nearly normal for all mice and did not differ between the control and model groups. Using a comparative analysis and a summary of related studies published in recent years, we found that miR-155 was not essential for normal physiological processes in 8-week-old mice. miR-155 deficiency did not affect the development and growth of naturally ageing mice during the 42 days after birth. Thus, studying the complex biological functions of miR-155 requires the further use of KO mouse models. PMID:28278287

Selective Deletion of Sodium Salt Taste during Development Leads to Expanded Terminal Fields of Gustatory Nerves in the Adult Mouse Nucleus of the Solitary Tract.

PubMed

Sun, Chengsan; Hummler, Edith; Hill, David L

2017-01-18

Neuronal activity plays a key role in the development of sensory circuits in the mammalian brain. In the gustatory system, experimental manipulations now exist, through genetic manipulations of specific taste transduction processes, to examine how specific taste qualities (i.e., basic tastes) impact the functional and structural development of gustatory circuits. Here, we used a mouse knock-out model in which the transduction component used to discriminate sodium salts from other taste stimuli was deleted in taste bud cells throughout development. We used this model to test the hypothesis that the lack of activity elicited by sodium salt taste impacts the terminal field organization of nerves that carry taste information from taste buds to the nucleus of the solitary tract (NST) in the medulla. The glossopharyngeal, chorda tympani, and greater superficial petrosal nerves were labeled to examine their terminal fields in adult control mice and in adult mice in which the α-subunit of the epithelial sodium channel was conditionally deleted in taste buds (αENaC knockout). The terminal fields of all three nerves in the NST were up to 2.7 times greater in αENaC knock-out mice compared with the respective field volumes in control mice. The shapes of the fields were similar between the two groups; however, the density and spread of labels were greater in αENaC knock-out mice. Overall, our results show that disruption of the afferent taste signal to sodium salts disrupts the normal age-dependent "pruning" of all terminal fields, which could lead to alterations in sensory coding and taste-related behaviors. Neural activity plays a major role in the development of sensory circuits in the mammalian brain. To date, there has been no direct test of whether taste-elicited neural activity has a role in shaping central gustatory circuits. However, recently developed genetic tools now allow an assessment of how specific taste stimuli, in this case sodium salt taste, play a role in the maturation of the terminal fields in the mouse brainstem. We found that the specific deletion of sodium salt taste during development produced terminal fields in adults that were dramatically larger than in control mice, demonstrating for the first time that sodium salt taste-elicited activity is necessary for the normal maturation of gustatory inputs into the brain. Copyright © 2017 the authors 0270-6474/17/370660-13$15.00/0.

Selective Deletion of Sodium Salt Taste during Development Leads to Expanded Terminal Fields of Gustatory Nerves in the Adult Mouse Nucleus of the Solitary Tract

PubMed Central

Sun, Chengsan; Hummler, Edith

2017-01-01

Neuronal activity plays a key role in the development of sensory circuits in the mammalian brain. In the gustatory system, experimental manipulations now exist, through genetic manipulations of specific taste transduction processes, to examine how specific taste qualities (i.e., basic tastes) impact the functional and structural development of gustatory circuits. Here, we used a mouse knock-out model in which the transduction component used to discriminate sodium salts from other taste stimuli was deleted in taste bud cells throughout development. We used this model to test the hypothesis that the lack of activity elicited by sodium salt taste impacts the terminal field organization of nerves that carry taste information from taste buds to the nucleus of the solitary tract (NST) in the medulla. The glossopharyngeal, chorda tympani, and greater superficial petrosal nerves were labeled to examine their terminal fields in adult control mice and in adult mice in which the α-subunit of the epithelial sodium channel was conditionally deleted in taste buds (αENaC knockout). The terminal fields of all three nerves in the NST were up to 2.7 times greater in αENaC knock-out mice compared with the respective field volumes in control mice. The shapes of the fields were similar between the two groups; however, the density and spread of labels were greater in αENaC knock-out mice. Overall, our results show that disruption of the afferent taste signal to sodium salts disrupts the normal age-dependent “pruning” of all terminal fields, which could lead to alterations in sensory coding and taste-related behaviors. SIGNIFICANCE STATEMENT Neural activity plays a major role in the development of sensory circuits in the mammalian brain. To date, there has been no direct test of whether taste-elicited neural activity has a role in shaping central gustatory circuits. However, recently developed genetic tools now allow an assessment of how specific taste stimuli, in this case sodium salt taste, play a role in the maturation of the terminal fields in the mouse brainstem. We found that the specific deletion of sodium salt taste during development produced terminal fields in adults that were dramatically larger than in control mice, demonstrating for the first time that sodium salt taste-elicited activity is necessary for the normal maturation of gustatory inputs into the brain. PMID:28100747

PARTITIONING OF BENZENE IN SICKLE CELL BLOOD AND NORMAL BLOOD AND IN THE BLOOD OF THE GUINEA PIG, MOUSE, AND RAT

EPA Science Inventory

Abstract
Benzene was used as a prototypic volatile organic chemical to ascertain whether the insoluble form of hemoglobin (HbS) from subjects with homozygous sickle cell disease (SCD) has a greater VOC carrying capacity than hemoglobin (Hb) from normal subjects (HbA) which is ...

Musical Electroacupuncture May Be a Better Choice than Electroacupuncture in a Mouse Model of Alzheimer's Disease.

PubMed

Jiang, Jing; Liu, Gang; Shi, Suhua; Li, Zhigang

2016-01-01

Objectives . To compare musical electroacupuncture and electroacupuncture in a mouse model of Alzheimer's disease. Methods . In this study, 7.5-month-old male senescence-accelerated mouse prone 8 (SAMP8) mice were used as an Alzheimer's disease animal model. In the normal control paradigm, 7.5-month-old male SAMR1 mice were used as the blank control group (N group). After 15 days of treatment, using Morris water maze test, micro-PET, and immunohistochemistry, the differences among the musical electroacupuncture (MEA), electroacupuncture (EA), Alzheimer's disease (AD), and normal (N) groups were assessed. Results . The Morris water maze test, micro-PET, and immunohistochemistry revealed that MEA and EA therapies could improve spatial learning and memory ability, glucose metabolism level in the brain, and A β amyloid content in the frontal lobe, compared with the AD group ( P < 0.05). Moreover, MEA therapy performed better than EA treatment in decreasing amyloid-beta levels in the frontal lobe of mice with AD. Conclusion . MEA therapy may be superior to EA in treating Alzheimer's disease as demonstrated in SAMP8 mice.

Musical Electroacupuncture May Be a Better Choice than Electroacupuncture in a Mouse Model of Alzheimer's Disease

PubMed Central

Jiang, Jing; Liu, Gang

2016-01-01

Objectives. To compare musical electroacupuncture and electroacupuncture in a mouse model of Alzheimer's disease. Methods. In this study, 7.5-month-old male senescence-accelerated mouse prone 8 (SAMP8) mice were used as an Alzheimer's disease animal model. In the normal control paradigm, 7.5-month-old male SAMR1 mice were used as the blank control group (N group). After 15 days of treatment, using Morris water maze test, micro-PET, and immunohistochemistry, the differences among the musical electroacupuncture (MEA), electroacupuncture (EA), Alzheimer's disease (AD), and normal (N) groups were assessed. Results. The Morris water maze test, micro-PET, and immunohistochemistry revealed that MEA and EA therapies could improve spatial learning and memory ability, glucose metabolism level in the brain, and Aβ amyloid content in the frontal lobe, compared with the AD group (P < 0.05). Moreover, MEA therapy performed better than EA treatment in decreasing amyloid-beta levels in the frontal lobe of mice with AD. Conclusion. MEA therapy may be superior to EA in treating Alzheimer's disease as demonstrated in SAMP8 mice. PMID:27974974

Wnt-Responsive Cancer Stem Cells Are Located Close to Distorted Blood Vessels and Not in Hypoxic Regions in a p53-Null Mouse Model of Human Breast Cancer

PubMed Central

Landua, John D.; Bu, Wen; Wei, Wei; Li, Fuhai; Wong, Stephen T.C.; Dickinson, Mary E.; Rosen, Jeffrey M.; Lewis, Michael T.

2014-01-01

Cancer stem cells (CSCs, or tumor-initiating cells) may be responsible for tumor formation in many types of cancer, including breast cancer. Using high-resolution imaging techniques, we analyzed the relationship between a Wnt-responsive, CSC-enriched population and the tumor vasculature using p53-null mouse mammary tumors transduced with a lentiviral Wnt signaling reporter. Consistent with their localization in the normal mammary gland, Wnt-responsive cells in tumors were enriched in the basal/myoepithelial population and generally located in close proximity to blood vessels. The Wnt-responsive CSCs did not colocalize with the hypoxia-inducible factor 1α-positive cells in these p53-null basal-like tumors. Average vessel diameter and vessel tortuosity were increased in p53-null mouse tumors, as well as in a human tumor xenograft as compared with the normal mammary gland. The combined strategy of monitoring the fluorescently labeled CSCs and vasculature using high-resolution imaging techniques provides a unique opportunity to study the CSC and its surrounding vasculature. PMID:24797826

Transplantation Immunity in the Isologous Mouse Radiation Chimaera

PubMed Central

Bridges, J. B.; Loutit, J. F.; Micklem, H. S.

1960-01-01

The survival of skin homo- and heterografts on isologous CBA mouse chimaeras has been investigated. Homografts usually persist for considerably longer than on normal unirradiated mice. Immunization of the host against the appropriate foreign antigens before irradiation neither reduces nor increases the duration of this persistence. When an irradiated non-immune host is restored with bone marrow from an immunized donor, a measure of immunity is transferred. If adult spleen cells from normal or immunized donors are added to the restorative inoculum, strongly antigenic foreign skins are shed with something like normal rapidity, but weakly antigenic skins may be retained for 100 days or more, and even indefinitely. Heterografts do not enjoy a span of survival comparable with that of homografts. These findings are discussed, and it is concluded that two factors are of importance in the prolongation of graft survival: (1) A weakening of the mechanism by which antigens are recognized as foreign, (2) an overall central depression of the immune response. ImagesPLATE IPLATE IIPLATE III PMID:13804388

Surface immunoglobulin on cultured foetal mouse thymocytes

PubMed Central

Haustein, D.; Mandel, T. E.

1979-01-01

Organ cultures of 14–15 day foetal mouse thymus were used as a source of non-neoplastic differentiating T cells, free of contaminating B cells. Viable cells obtained from such cultured thymuses were radio-iodinated and immunoglobulins (Ig) were isolated by co-precipitation from the 125I-labelled cell-surface proteins released during 1 h of incubation at 37°. The precipitates, both reduced and unreduced, were then analysed by polyacrylamide gel electrophoresis. The unreduced material migrated in a 5% gel as a single peak with a mobility slightly faster than that of mouse IgG. After reduction, however, two peaks were obtained (in a 10% gel), one corresponding in migration to mouse light chain and the other which moved slightly faster than mouse μ chain. This pattern was identical with that previously seen for both surface Ig of normal mouse thymocytes and neoplastic T lymphoma cells. Uncultured, 15 day foetal thymocytes did not produce any detectable co-precipitated cell surface material. Ig detected in these experiments was therefore produced during in vitro culture by non-neoplastic T cells in a system free of contaminating B cells and mouse serum proteins. PMID:315364

Mlh1 deficiency in normal mouse colon mucosa associates with chromosomally unstable colon cancer

PubMed Central

Pussila, Marjaana; Törönen, Petri; Einarsdottir, Elisabet; Katayama, Shintaro; Krjutškov, Kaarel; Holm, Liisa; Kere, Juha; Peltomäki, Päivi; Mäkinen, Markus J; Linden, Jere; Nyström, Minna

2018-01-01

Abstract Colorectal cancer (CRC) genome is unstable and different types of instabilities, such as chromosomal instability (CIN) and microsatellite instability (MSI) are thought to reflect distinct cancer initiating mechanisms. Although 85% of sporadic CRC reveal CIN, 15% reveal mismatch repair (MMR) malfunction and MSI, the hallmarks of Lynch syndrome with inherited heterozygous germline mutations in MMR genes. Our study was designed to comprehensively follow genome-wide expression changes and their implications during colon tumorigenesis. We conducted a long-term feeding experiment in the mouse to address expression changes arising in histologically normal colonic mucosa as putative cancer preceding events, and the effect of inherited predisposition (Mlh1+/−) and Western-style diet (WD) on those. During the 21-month experiment, carcinomas developed mainly in WD-fed mice and were evenly distributed between genotypes. Unexpectedly, the heterozygote (B6.129-Mlh1tm1Rak) mice did not show MSI in their CRCs. Instead, both wildtype and heterozygote CRC mice showed a distinct mRNA expression profile and shortage of several chromosomal segregation gene-specific transcripts (Mlh1, Bub1, Mis18a, Tpx2, Rad9a, Pms2, Cenpe, Ncapd3, Odf2 and Dclre1b) in their colon mucosa, as well as an increased mitotic activity and abundant numbers of unbalanced/atypical mitoses in tumours. Our genome-wide expression profiling experiment demonstrates that cancer preceding changes are already seen in histologically normal colon mucosa and that decreased expressions of Mlh1 and other chromosomal segregation genes may form a field-defect in mucosa, which trigger MMR-proficient, chromosomally unstable CRC. PMID:29701748

Absence of Colony Stimulation Factor-1 Receptor Results in Loss of Microglia, Disrupted Brain Development and Olfactory Deficits

PubMed Central

Etgen, Anne M.; Dobrenis, Kostantin; Pollard, Jeffrey W.

2011-01-01

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1op) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure. PMID:22046273

Mice null for Frizzled4 (Fzd4-/-) are infertile and exhibit impaired corpora lutea formation and function.

PubMed

Hsieh, Minnie; Boerboom, Derek; Shimada, Masayuki; Lo, Yuet; Parlow, Albert F; Luhmann, Ulrich F O; Berger, Wolfgang; Richards, JoAnne S

2005-12-01

Previous studies showed that transcripts encoding specific Wnt ligands and Frizzled receptors including Wnt4, Frizzled1 (Fzd1), and Frizzled4 (Fzd4) were expressed in a cell-specific manner in the adult mouse ovary. Overlapping expression of Wnt4 and Fzd4 mRNA in small follicles and corpora lutea led us to hypothesize that the infertility of mice null for Fzd4 (Fzd4-/-) might involve impaired follicular growth or corpus luteum formation. Analyses at defined stages of reproductive function indicate that immature Fzd4-/- mouse ovaries contain follicles at many stages of development and respond to exogenous hormone treatments in a manner similar to their wild-type littermates, indicating that the processes controlling follicular development and follicular cell responses to gonadotropins are intact. Adult Fzd4-/- mice also exhibit normal mating behavior and ovulate, indicating that endocrine events controlling these processes occur. However, Fzd4-/- mice fail to become pregnant and do not produce offspring. Histological and functional analyses of ovaries from timed mating pairs at Days 1.5-7.5 postcoitus (p.c.) indicate that the corpora lutea of the Fzd4-/- mice do not develop normally. Expression of luteal cell-specific mRNAs (Lhcgr, Prlr, Cyp11a1 and Sfrp4) is reduced, luteal cell morphology is altered, and markers of angiogenesis and vascular formation (Efnb1, Efnb2, Ephb4, Vegfa, Vegfc) are low in the Fzd4-/- mice. Although a recently identified, high-affinity FZD4 ligand Norrin (Norrie disease pseudoglioma homolog) is expressed in the ovary, adult Ndph-/- mice contain functional corpora lutea and do not phenocopy Fzd4-/- mice. Thus, Fzd4 appears to impact the formation of the corpus luteum by mechanisms that more closely phenocopy Prlr null mice.

Vestibular Evoked Myogenic Potentials in Normal Mice and Phex Mice With Spontaneous Endolymphatic Hydrops

PubMed Central

Sheykholeslami, Kianoush; Megerian, Cliff A.; Zheng, Qing Y.

2010-01-01

Objective and Background Vestibular evoked myogenic potentials (VEMPs) have been recorded from the neck musculature and the cervical spinal cord in humans and a limited number of laboratory animals in response to loud sound. However, the mouse VEMP has yet to be described. Evaluation of the sacculocollic pathway via VEMPs in mice can set the stage for future evaluations of mutant mice that now play an important role in research regarding human auditory and vestibular dysfunction. Materials and Methods Sound-evoked potentials were recorded from the neck extensor muscles and the cervical spinal cord in normal adult mice and in circling PhexHyp-Duk/y mice with known vestibular abnormalities, including endolymphatic hydrops (ELH). Results Biphasic potentials were recorded from all normal animals. The mean threshold of the VEMP response in normal adult mice was 60 dB normal hearing level with a mean peak latency of 6.25 ± 0.46 and 7.95 ± 0.42 milliseconds for p1 and n1 peaks, respectively. At the maximum sound intensity used (100 dB normal hearing level), 4 of 5 Phex mice did not exhibit VEMP responses, and 1 showed an elevated threshold, but normal response, with regard to peak latency and amplitude. The histologic findings in all of these Phex mice were consistent with distended membranous labyrinth, displaced Reissner membrane, ganglion cell loss, and ELH. Conclusion This is the first report of VEMP recordings in mice and the first report of abnormal VEMPs in a mouse model with ELH. The characteristics of these potentials such as higher response threshold in comparison to auditory brainstem response, myogenic nature of the response, and latency correlation with the cervical recording (accessory nerve nucleus) were similar to those of VEMPs in humans, guinea pigs, cats, and rats, suggesting that the mouse may be used as an animal model in the study of VEMPs. The simplicity and reliability of these recordings make the VEMP a uniquely informative test for assessing vestibular function, and these results suggest that they may be informative in mice with various mutations. However, further investigation is necessary. PMID:19300299

Multimodal nonlinear optical imaging of cartilage development in mouse model

NASA Astrophysics Data System (ADS)

He, Sicong; Xue, Wenqian; Sun, Qiqi; Li, Xuesong; Huang, Jiandong; Qu, Jianan Y.

2017-02-01

Kinesin-1 is a kind of motor protein responsible for intracellular transportation and has been studied in a variety of tissues. However, its roles in cartilage development are not clear. In this study, a kinesin-1 heavy chain (Kif5b) knockout mouse model is used to study the functions of kinesin-1 in the cartilage development. We developed a multimodal nonlinear optical (NLO) microscope system integrating stimulated Raman scattering (SRS), second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) to investigate the morphological and biomedical characteristics of fresh tibial cartilage from normal and mutant mice at different developmental stages. The combined forward and backward SHG imaging resolved the fine structure of collagen fibrils in the extracellular matrix of cartilage. Meanwhile, the chondrocyte morphology in different zones of cartilage was visualized by label-free SRS and TPEF images. The results show that the fibrillar collagen in the superficial zone of cartilage in postnatal day 10 and 15 (P10 and P15) knockout mice was significantly less than that of control mice. Moreover, we observed distorted morphology and disorganization of columnar arrangement of chondrocytes in the growth plate cartilage of mutant mice. This study reveals the significant roles of kinesin-1 in collagen formation and chondrocyte morphogenesis.

Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

PubMed

Nicken, Petra; Empl, Michael T; Gerhard, Daniel; Hausmann, Julia; Steinberg, Pablo

2016-09-01

High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pacsin 2 is required for the maintenance of a normal cardiac function in the developing mouse heart.

PubMed

Semmler, Judith; Kormann, Jan; Srinivasan, Sureshkumar Perumal; Köster, Annette; Sälzer, Daniel; Reppel, Michael; Hescheler, Jürgen; Plomann, Markus; Nguemo, Filomain

2018-02-01

The Pacsin proteins (Pacsin 1, 2 and 3) play an important role in intracellular trafficking and thereby signal transduction in many cells types. This study was designed to examine the role of Pacsin 2 in cardiac development and function. We investigated the development and electrophysiological properties of Pacsin 2 knockout (P2KO) hearts and single cardiomyocytes isolated from 11.5 and 15.5days old fetal mice. Immunofluorescence experiments confirmed the lack of Pacsin 2 protein expression in P2KO cardiac myocytes in comparison to wildtype (WT). Western blotting demonstrates low expression levels of connexin 43 and T-box 3 proteins in P2KO compared to wildtype (WT). Electrophysiology measurements including online Multi-Electrode Array (MEA) based field potential (FP) recordings on isolated whole heart of P2KO mice showed a prolonged AV-conduction time. Patch clamp measurements of P2KO cardiomyocytes revealed differences in action potential (AP) parameters and decreased pacemaker funny channel (I f ), as well as L-type Ca 2+ channel (I CaL ), and sodium channel (I Na ). These findings demonstrate that Pacsin 2 is necessary for cardiac development and function in mouse embryos, which will enhance our knowledge to better understand the genesis of cardiovascular diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in expression of the lysosomal membrane glycoprotein, LAMP-1 in interdigital regions during embryonic mouse limb development, in vivo and in vitro.

PubMed

Stewart, S; Yi, S; Kassabian, G; Mayo, M; Sank, A; Shuler, C

2000-06-01

Syndactyly, a failure of the digits to separate into individual units, affects about 8 to 9 per 1000 newborns and results from an aberration of the normal development of the interdigital tissues. Limb digit separation is the result of programmed cell death (apoptosis). Lysosomes play a role in the process of cell self-destruction. Our experiment was designed to test the hypothesis that the intensity of interdigital lysosomes increases during the separation of digits in vivo and in vitro. The primary mouse monoclonal antibody, 1D4B, detects the presence of lysosomes by identifying the LAMP-1 glycoprotein on the lysosome cell membrane. In our experiment this antibody immunodetected interdigital lysosome proteins in serial sections of limbs from Swiss-Webster mouse embryos, gestational ages E12.5 through E15, key developmental stages for digit separation. Digit separation was associated with an increase in intensity of lysosomal protein staining. In E12.5 limbs, the presence of lysosomes was enriched in the distal aspect of the interdigital tissue. However, the number of lysosomes markedly increased in the E13 and E14 limbs, including the entire length and width of the interdigital tissue in the E14 limbs. This lysosomal protein presence in E14 limbs was significant compared to E12.5, E13, and E15 limbs. By day 12.5, the mouse embryo limb is committed to digit separation. Addition of retinoic acid to the culture medium of limbs earlier in development, such as E12, results in induction of the process of digit separation. Cultured E12 limbs that did not receive an addition of retinoic acid, did not show digit separation. We conclude that in the limb development process, the enrichment in interdigit LAMP-1 proteins, may indicate a relationship between lysosomes, apoptosis, and digit separation. We also conclude that retinoic acid has an important role in digit separation in vivo, as shown in limb development, and demonstrated through the addition of retinoic acid to media of cultured tissues.

MRI Evidence of Endolymphatic Impermeability to the Gadolinium Molecule in the In Vivo Mouse Inner Ear at 9.4 Tesla

PubMed Central

Counter, S Allen; Nikkhou, Sahar; Brené, Stefan; Damberg, Peter; Sierakowiak, Adam; Klason, Tomas; Berglin, Cecilia Engmér; Laurell, Göran

2013-01-01

Objective: Previous in vivo experimental magnetic resonance imaging (MRI) investigations of the mammalian inner ear at 4.7 Tesla have indicated that intravenously injected gadolinium (Gd) penetrates the perilymphatic labyrinth, but not the endolymphatic membranous labyrinth. In the present study, high field MRI at 9.4T was used to visualize the in vivo mouse vestibulo-cochlea system, and to determine whether the endolymphatic system is permeable to a Gd complex. Methods: A 9.4 T Varian magnet equipped with a 12 cm inner diameter gradient system with maximum gradient strength of 600 mT/m, a millipede coil (Varian design) and a Gd contrast agent were used for image acquisition in the normal C57 BL-6 mouse. Results: High-resolution 2D and 3D images of the mouse cochlea were acquired within 80 minutes following intravenous injection of Gd. Gd initially permeated the perilymphatic scala tympani and scala vestibuli, and permitted visualization of both cochlear turns from base to apex. The superior, inferior and lateral semicircular canals were subsequently visualized in 3 planes. The membranous endolymphatic labyrinth was impermeable to intravenously injected Gd, and thus showed no apparent uptake of Gd at 9.4T. Conclusion: The 9.4T field strength MRI permitted acquisition of high resolution images of anatomical and physiological features of the normal, wild type mouse perilymphatic inner ear in vivo, and provided further evidence that the endolymphatic system is impermeable to intravenously injected Gd. PMID:23894262

Deletion of neurturin impairs development of cholinergic nerves and heart rate control in postnatal mouse hearts.

PubMed

Downs, Anthony M; Jalloh, Hawa B; Prater, Kayla J; Fregoso, Santiago P; Bond, Cherie E; Hampton, Thomas G; Hoover, Donald B

2016-05-01

The neurotrophic factor neurturin is required for normal cholinergic innervation of adult mouse heart and bradycardic responses to vagal stimulation. Our goals were to determine effects of neurturin deletion on development of cardiac chronotropic and dromotropic functions, vagal baroreflex response, and cholinergic nerve density in nodal regions of postnatal mice. Experiments were performed on postnatal C57BL/6 wild-type (WT) and neurturin knockout (KO) mice. Serial electrocardiograms were recorded noninvasively from conscious pups using an ECGenie apparatus. Mice were treated with atenolol to evaluate and block sympathetic effects on heart rate (HR) and phenylephrine (PE) to stimulate the baroreflex. Immunohistochemistry was used to label cholinergic nerves in paraffin sections. WT and KO mice showed similar age-dependent increases in HR and decreases in PR interval between postnatal days (P) 2.5 and 21. Treatment with atenolol reduced HR significantly in WT and KO pups at P7.5. PE caused a reflex bradycardia that was significantly smaller in KO pups. Cholinergic nerve density was significantly less in nodal regions of P7.5 KO mice. We conclude that cholinergic nerves have minimal influence on developmental changes in HR and PR, QRS, and QTc intervals in mouse pups. However, cholinergic nerves mediate reflex bradycardia by 1 week postnatally. Deletion of neurturin impairs cholinergic innervation of the heart and the vagal efferent component of the baroreflex early during postnatal development. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function

PubMed Central

Haziza, Sitvanit; Magnani, Roberta; Lan, Dima; Keinan, Omer; Saada, Ann; Hershkovitz, Eli; Yanay, Nurit; Cohen, Yoram; Nevo, Yoram; Houtz, Robert L.; Sheffield, Val C.; Golan, Hava; Parvari, Ruti

2015-01-01

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the CaM KMT gene. PMID:26247364

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

PubMed

Haziza, Sitvanit; Magnani, Roberta; Lan, Dima; Keinan, Omer; Saada, Ann; Hershkovitz, Eli; Yanay, Nurit; Cohen, Yoram; Nevo, Yoram; Houtz, Robert L; Sheffield, Val C; Golan, Hava; Parvari, Ruti

2015-08-01

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the CaM KMT gene.

Accelerated renal disease is associated with the development of metabolic syndrome in a glucolipotoxic mouse model

PubMed Central

Martínez-García, Cristina; Izquierdo, Adriana; Velagapudi, Vidya; Vivas, Yurena; Velasco, Ismael; Campbell, Mark; Burling, Keith; Cava, Fernando; Ros, Manuel; Orešič, Matej; Vidal-Puig, Antonio; Medina-Gomez, Gema

2012-01-01

SUMMARY Individuals with metabolic syndrome are at high risk of developing chronic kidney disease (CKD) through unclear pathogenic mechanisms. Obesity and diabetes are known to induce glucolipotoxic effects in metabolically relevant organs. However, the pathogenic role of glucolipotoxicity in the aetiology of diabetic nephropathy is debated. We generated a murine model, the POKO mouse, obtained by crossing the peroxisome proliferator-activated receptor gamma 2 (PPARγ2) knockout (KO) mouse into a genetically obese ob/ob background. We have previously shown that the POKO mice showed: hyperphagia, insulin resistance, hyperglycaemia and dyslipidaemia as early as 4 weeks of age, and developed a complete loss of normal β-cell function by 16 weeks of age. Metabolic phenotyping of the POKO model has led to investigation of the structural and functional changes in the kidney and changes in blood pressure in these mice. Here we demonstrate that the POKO mouse is a model of renal disease that is accelerated by high levels of glucose and lipid accumulation. Similar to ob/ob mice, at 4 weeks of age these animals exhibited an increased urinary albumin:creatinine ratio and significantly increased blood pressure, but in contrast showed a significant increase in the renal hypertrophy index and an associated increase in p27Kip1 expression compared with their obese littermates. Moreover, at 4 weeks of age POKO mice showed insulin resistance, an alteration of lipid metabolism and glomeruli damage associated with increased transforming growth factor beta (TGFβ) and parathyroid hormone-related protein (PTHrP) expression. At this age, levels of proinflammatory molecules, such as monocyte chemoattractant protein-1 (MCP-1), and fibrotic factors were also increased at the glomerular level compared with levels in ob/ob mice. At 12 weeks of age, renal damage was fully established. These data suggest an accelerated lesion through glucolipotoxic effects in the renal pathogenesis in POKO mice. PMID:22773754

Accelerated renal disease is associated with the development of metabolic syndrome in a glucolipotoxic mouse model.

PubMed

Martínez-García, Cristina; Izquierdo, Adriana; Velagapudi, Vidya; Vivas, Yurena; Velasco, Ismael; Campbell, Mark; Burling, Keith; Cava, Fernando; Ros, Manuel; Oresic, Matej; Vidal-Puig, Antonio; Medina-Gomez, Gema

2012-09-01

Individuals with metabolic syndrome are at high risk of developing chronic kidney disease (CKD) through unclear pathogenic mechanisms. Obesity and diabetes are known to induce glucolipotoxic effects in metabolically relevant organs. However, the pathogenic role of glucolipotoxicity in the aetiology of diabetic nephropathy is debated. We generated a murine model, the POKO mouse, obtained by crossing the peroxisome proliferator-activated receptor gamma 2 (PPARγ2) knockout (KO) mouse into a genetically obese ob/ob background. We have previously shown that the POKO mice showed: hyperphagia, insulin resistance, hyperglycaemia and dyslipidaemia as early as 4 weeks of age, and developed a complete loss of normal β-cell function by 16 weeks of age. Metabolic phenotyping of the POKO model has led to investigation of the structural and functional changes in the kidney and changes in blood pressure in these mice. Here we demonstrate that the POKO mouse is a model of renal disease that is accelerated by high levels of glucose and lipid accumulation. Similar to ob/ob mice, at 4 weeks of age these animals exhibited an increased urinary albumin:creatinine ratio and significantly increased blood pressure, but in contrast showed a significant increase in the renal hypertrophy index and an associated increase in p27(Kip1) expression compared with their obese littermates. Moreover, at 4 weeks of age POKO mice showed insulin resistance, an alteration of lipid metabolism and glomeruli damage associated with increased transforming growth factor beta (TGFβ) and parathyroid hormone-related protein (PTHrP) expression. At this age, levels of proinflammatory molecules, such as monocyte chemoattractant protein-1 (MCP-1), and fibrotic factors were also increased at the glomerular level compared with levels in ob/ob mice. At 12 weeks of age, renal damage was fully established. These data suggest an accelerated lesion through glucolipotoxic effects in the renal pathogenesis in POKO mice.

A modulatory role of the Rax homeobox gene in mature pineal gland function: Investigating the photoneuroendocrine circadian system of a Rax conditional knockout mouse.

PubMed

Rohde, Kristian; Bering, Tenna; Furukawa, Takahisa; Rath, Martin Fredensborg

2017-10-01

The retinal and anterior neural fold homeobox gene (Rax) controls development of the eye and the forebrain. Postnatal expression of Rax in the brain is restricted to the pineal gland, a forebrain structure devoted to melatonin synthesis. The role of Rax in pineal function is unknown. In order to investigate the role of Rax in pineal function while circumventing forebrain abnormalities of the global Rax knockout, we generated an eye and pineal-specific Rax conditional knockout mouse. Deletion of Rax in the pineal gland did not affect morphology of the gland, suggesting that Rax is not essential for pineal gland development. In contrast, deletion of Rax in the eye generated an anophthalmic phenotype. In addition to the loss of central visual pathways, the suprachiasmatic nucleus of the hypothalamus housing the circadian clock was absent, indicating that the retinohypothalamic tract is required for the nucleus to develop. Telemetric analyses confirmed the lack of a functional circadian clock. Arylalkylamine N-acetyltransferase (Aanat) transcripts, encoding the melatonin rhythm-generating enzyme, were undetectable in the pineal gland of the Rax conditional knockout under normal conditions, whereas the paired box 6 homeobox gene, known to regulate pineal development, was up-regulated. By injecting isoproterenol, which mimics a nocturnal situation in the pineal gland, we were able to induce pineal expression of Aanat in the Rax conditional knockout mouse, but Aanat transcript levels were significantly lower than those of Rax-proficient mice. Our data suggest that Rax controls pineal gene expression and via Aanat may modulate melatonin synthesis. © 2017 International Society for Neurochemistry.

Cross-reactive protection against enterohemorrhagic Escherichia coli infection by enteropathogenic E. coli in a mouse model.

PubMed

Calderon Toledo, Carla; Arvidsson, Ida; Karpman, Diana

2011-06-01

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related attaching and effacing (A/E) pathogens. The genes responsible for the A/E pathology are carried on a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Both pathogens share a high degree of homology in the LEE and additional O islands. EHEC prevalence is much lower in areas where EPEC is endemic. This may be due to the development of antibodies against common EPEC and EHEC antigens. This study investigated the hypothesis that EPEC infections may protect against EHEC infections. We used a mouse model to inoculate BALB/c mice intragastrically, first with EPEC and then with EHEC (E. coli O157:H7). Four control groups received either a nonpathogenic E. coli (NPEC) strain followed by EHEC (NPEC/EHEC), phosphate-buffered saline (PBS) followed by EHEC (PBS/EHEC), EPEC/PBS, or PBS/PBS. Mice were monitored for weight loss and symptoms. EPEC colonized the intestine after challenge, and mice developed serum antibodies to intimin and E. coli secreted protein B (encoded in the LEE). Prechallenge with an EPEC strain had a protective effect after EHEC infection, as only a few mice developed mild symptoms, from which they recovered. These mice had an increase in body weight similar to that in control animals, and tissue morphology exhibited mild intestinal changes and normal renal histology. All mice that were not prechallenged with the EPEC strain developed mild to severe symptoms after EHEC infection, with weight loss as well as intestinal and renal histopathological changes. These data suggest that EPEC may protect against EHEC infection in this mouse model.

Antibody-induced albuminuria and accelerated focal glomerulosclerosis in the Thy-1.1 transgenic mouse.

PubMed

Assmann, Karel J M; van Son, Jacco P H F; Dïjkman, Henry B P M; Mentzel, Stef; Wetzels, Jack F M

2002-07-01

Podocytes play an important role in the development of proteinuria and focal glomerulosclerosis. Previously we have demonstrated that a combination of two monoclonal antibodies (mAb) against aminopeptidase A (APA), an enzyme present on podocytes, induces a massive acute albuminuria in mice. The present study examined the relationship between the acute antibody-induced albuminuria and the development of focal glomerulosclerosis in the Thy-1.1 transgenic mouse. This mouse expresses a hybrid human-mouse Thy-1.1 antigen on the podocytes, and slowly but spontaneously develops albuminuria and focal glomerulosclerosis. Five-week-old non-albuminuric Thy-1.1 transgenic and non-transgenic control mice were injected with anti-APA and anti-Thy-1.1 mAb or saline. Albuminuria was measured at days 1, 7, 14 and 21. At day 21 kidneys were processed for light microscopy, immunofluorescence, and electron microscopy. Injection of anti-APA and anti-Thy1.1 mAb in Thy-1.1 transgenic mice induced an albuminuria at day 1 that persisted at day 21. The acute albuminuria after injection of anti-APA mAb was more prominent but transient in non-transgenic mice. In non-trangenic mice no albuminuria could be induced with anti-Thy 1.1 mAb. Light microscopy revealed normal glomeruli at day 1 in all transgenic mice, however, at day 21 advanced glomerulosclerotic lesions were seen in mice injected with either anti-APA mAb (37+/-19% of glomeruli affected) or anti-Thy-1.1 mAb (71+/-5%). Non-transgenic mice did not reveal sclerotic lesions at any time investigated. In the transgenic mice the percentage of focal glomerulosclerosis at day 21 did not correlate with albuminuria at day 21. However, we found a highly significant correlation between percentage of focal glomerulosclerosis and the time-averaged albuminuria over the three-week study period (P < 0.001). Injection of a combination of anti-APA or anti-Thy-1.1 mAb into one mo old, non-albuminuric Thy-1.1 transgenic mice induces an acute albuminuria at day 1 that is accompanied by an accelerated focal glomerulosclerosis at day 21. We suggest that the Thy-1.1 transgenic mouse is an excellent model to study specifically the relation between podocytic injury, albuminuria and the development of focal glomerulosclerosis.

Involvement of aryl hydrocarbon receptor signaling in the development of small cell lung cancer induced by HPV E6/E7 oncoproteins

PubMed Central

2011-01-01

Background Lung cancers consist of four major types that and for clinical-pathological reasons are often divided into two broad categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). All major histological types of lung cancer are associated with smoking, although the association is stronger for SCLC and squamous cell carcinoma than adenocarcinoma. To date, epidemiological studies have identified several environmental, genetic, hormonal and viral factors associated with lung cancer risk. It has been estimated that 15-25% of human cancers may have a viral etiology. The human papillomavirus (HPV) is a proven cause of most human cervical cancers, and might have a role in other malignancies including vulva, skin, oesophagus, head and neck cancer. HPV has also been speculated to have a role in the pathogenesis of lung cancer. To validate the hypothesis of HPV involvement in small cell lung cancer pathogenesis we performed a gene expression profile of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins. Methods Gene expression profile of SCLC has been performed using Agilent whole mouse genome (4 × 44k) representing ~ 41000 genes and mouse transcripts. Samples were obtained from two HPV16-E6/E7 transgenic mouse models and from littermate's normal lung. Data analyses were performed using GeneSpring 10 and the functional classification of deregulated genes was performed using Ingenuity Pathway Analysis (Ingenuity® Systems, http://www.ingenuity.com). Results Analysis of deregulated genes induced by the expression of E6/E7 oncoproteins supports the hypothesis of a linkage between HPV infection and SCLC development. As a matter of fact, comparison of deregulated genes in our system and those in human SCLC showed that many of them are located in the Aryl Hydrocarbon Receptor Signal transduction pathway. Conclusions In this study, the global gene expression of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins led us to identification of several genes involved in SCLC tumor development. Furthermore, our study reveled that the Aryl Hydrocarbon Receptor Signaling is the primarily affected pathway by the E6/E7 oncoproteins expression and that this pathway is also deregulated in human SCLC. Our results provide the basis for the development of new therapeutic approaches against human SCLC. PMID:21205295

Parp-1 genetic ablation in Ela-myc mice unveils novel roles for Parp-1 in pancreatic cancer.

PubMed

Martínez-Bosch, Neus; Iglesias, Mar; Munné-Collado, Jessica; Martínez-Cáceres, Carlos; Moreno, Mireia; Guerra, Carmen; Yélamos, Jose; Navarro, Pilar

2014-10-01

Pancreatic cancer has a dismal prognosis and is currently the fourth leading cause of cancer-related death in developed countries. The inhibition of poly(ADP-ribose) polymerase-1 (Parp-1), the major protein responsible for poly(ADP-ribosy)lation in response to DNA damage, has emerged as a promising treatment for several tumour types. Here we aimed to elucidate the involvement of Parp-1 in pancreatic tumour progression. We assessed Parp-1 protein expression in normal, preneoplastic and pancreatic tumour samples from humans and from K-Ras- and c-myc-driven mouse models of pancreatic cancer. Parp-1 was highly expressed in acinar cells in normal and cancer tissues. In contrast, ductal cells expressed very low or undetectable levels of this protein, both in a normal and in a tumour context. The Parp-1 expression pattern was similar in human and mouse samples, thereby validating the use of animal models for further studies. To determine the in vivo effects of Parp-1 depletion on pancreatic cancer progression, Ela-myc-driven pancreatic tumour development was analysed in a Parp-1 knock-out background. Loss of Parp-1 resulted in increased tumour necrosis and decreased proliferation, apoptosis and angiogenesis. Interestingly, Ela-myc:Parp-1(-/-) mice displayed fewer ductal tumours than their Ela-myc:Parp-1(+/+) counterparts, suggesting that Parp-1 participates in promoting acinar-to-ductal metaplasia, a key event in pancreatic cancer initiation. Moreover, impaired macrophage recruitment can be responsible for the ADM blockade found in the Ela-myc:Parp-1(-/-) mice. Finally, molecular analysis revealed that Parp-1 modulates ADM downstream of the Stat3-MMP7 axis and is also involved in transcriptional up-regulation of the MDM2, VEGFR1 and MMP28 cancer-related genes. In conclusion, the expression pattern of Parp-1 in normal and cancer tissue and the in vivo functional effects of Parp-1 depletion point to a novel role for this protein in pancreatic carcinogenesis and shed light into the clinical use of Parp-1 inhibitors. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Three mouse models of human thalassemia.

PubMed Central

Martinell, J; Whitney, J B; Popp, R A; Russell, L B; Anderson, W F

1981-01-01

Three types of mice with globin gene mutations, called 352HB, 27HB, and Hbath-J, appear to be true animal models of human thalassemia. Expression of the alpha-globin genes in three stocks of mice, each one heterozygous for one of the alpha-globin mutations, was examined at the polypeptide, RNA, and DNA levels. alpha-Globin polypeptide chains, relative to beta-globin chains in heterozygous thalassemic mice, are present at approximately 80% of normal. The ratios of alpha-globin to beta-globin RNA sequences are also 75-80% of normal, exactly reflecting the alpha-globin to beta-globin chain ratios. In the case of mutant 352HB, at least one alpha-globin gene is deleted. Thalassemic mouse erythroid cells appear to compensate partially for the loss of half of their alpha-globin genes. Images PMID:6946454

Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system.

PubMed

Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

2016-03-09

Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production.

Gene Expression Architecture of Mouse Dorsal and Tail Skin Reveals Functional Differences in Inflammation and Cancer.

PubMed

Quigley, David A; Kandyba, Eve; Huang, Phillips; Halliwill, Kyle D; Sjölund, Jonas; Pelorosso, Facundo; Wong, Christine E; Hirst, Gillian L; Wu, Di; Delrosario, Reyno; Kumar, Atul; Balmain, Allan

2016-07-26

Inherited germline polymorphisms can cause gene expression levels in normal tissues to differ substantially between individuals. We present an analysis of the genetic architecture of normal adult skin from 470 genetically unique mice, demonstrating the effect of germline variants, skin tissue location, and perturbation by exogenous inflammation or tumorigenesis on gene signaling pathways. Gene networks related to specific cell types and signaling pathways, including sonic hedgehog (Shh), Wnt, Lgr family stem cell markers, and keratins, differed at these tissue sites, suggesting mechanisms for the differential susceptibility of dorsal and tail skin to development of skin diseases and tumorigenesis. The Pten tumor suppressor gene network is rewired in premalignant tumors compared to normal tissue, but this response to perturbation is lost during malignant progression. We present a software package for expression quantitative trait loci (eQTL) network analysis and demonstrate how network analysis of whole tissues provides insights into interactions between cell compartments and signaling molecules. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Targeted inactivation of the murine Abca3 gene leads to respiratory failure in newborns with defective lamellar bodies

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

Hammel, Markus; Michel, Geert; Hoefer, Christina

2007-08-10

Mutations in the human ABCA3 gene, encoding an ABC-transporter, are associated with respiratory failure in newborns and pediatric interstitial lung disease. In order to study disease mechanisms, a transgenic mouse model with a disrupted Abca3 gene was generated by targeting embryonic stem cells. While heterozygous animals developed normally and were fertile, individuals homozygous for the altered allele (Abca3-/-) died within one hour after birth from respiratory failure, ABCA3 protein being undetectable. Abca3-/- newborns showed atelectasis of the lung in comparison to a normal gas content in unaffected or heterozygous littermates. Electron microscopy demonstrated the absence of normal lamellar bodies inmore » type II pneumocytes. Instead, condensed structures with apparent absence of lipid content were found. We conclude that ABCA3 is required for the formation of lamellar bodies and lung surfactant function. The phenotype of respiratory failure immediately after birth corresponds to the clinical course of severe ABCA3 mutations in human newborns.« less

A High-Calcium and Phosphate Rescue Diet and VDR-Expressing Transgenes Normalize Serum Vitamin D Metabolite Profiles and Renal Cyp27b1 and Cyp24a1 Expression in VDR Null Mice

PubMed Central

Kaufmann, Martin; Lee, Seong Min; Pike, J. Wesley

2015-01-01

Vitamin D receptor (VDR)-mediated 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent gene expression is compromised in the VDR null mouse. The biological consequences include: hypocalcemia, hypophosphatemia, elevated parathyroid hormone (PTH) and 1,25(OH)2D3, and consequential skeletal abnormalities. CYP24A1 is a cytochrome P450 enzyme that is involved in the side chain oxidation and destruction of both 1,25(OH)2D3 and 25-hydroxyvitamin D3 (25-OH-D3). In the current studies, we used liquid chromatography-tandem mass spectrometry technology to compare the metabolic profiles of VDR null mice fed either a normal or a calcium and phosphate-enriched rescue diet and to assess the consequence of transgenic expression of either mouse or human VDR genes in the same background. Serum 1,25(OH)2D3 levels in VDR null mice on normal chow were highly elevated (>3000 pg/mL) coincident with undetectable levels of catabolites such as 24,25-(OH)2D3 and 25-OH-D3-26,23-lactone normally observed in wild-type mice. The rescue diet corrected serum Ca++, PTH, and 1,25(OH)2D3 values and restored basal expression of Cyp24a1 as evidenced by both renal expression of Cyp24a1 and detection of 24,25-(OH)2D3 and the 25-OH-D3-26,23-lactone. Unexpectedly, this diet also resulted in supranormal levels of 3-epi-24,25-(OH)2D3 and 3-epi-25-OH-D3-26,23-lactone. The reappearance of serum 24,25-(OH)2D3 and renal Cyp24a1 expression after rescue suggests that basal levels of Cyp24a1 may be repressed by high PTH. Introduction of transgenes for either mouse or human VDR also normalized vitamin D metabolism in VDR null mice, whereas this metabolic pattern was unaffected by a transgene encoding a ligand binding-deficient mutant (L233S) human VDR. We conclude that liquid chromatography-tandem mass spectrometry-based metabolic profiling is an ideal analytical method to study mouse models with alterations in calcium/phosphate homeostasis. PMID:26441239

PROTECTION OF THE EMBRYO AGAINST THE CONGENITAL AND LETHAL EFFECTS OF X- IRRADIATION. PART I

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

Rugh, R.; Grupp, E.

1960-04-01

The effects of 15 agents, some given before and some after x irradiation to 200 r, have been studied for their effectiveness in protecting the 8.5-day mouse embryo against embryonic or fetal death and the development of the severe cephalic congenital anomaly known as exencephalia (or brain hernia). Some 4979 fetuses were examined. Of the agents studied, only cysteinamine, cystamine, and anoxia proved to be statistically "protective" at all. Cysteinamine and cystamine (both -SH compounds) given I.P. before x irradiation to 200 r allowed 73 to 80% of the 8.5-day embryos to survive to term while the untreated but irradiatedmore » control litters had a survival of only 41%. Funther, there was considerable reduction in bcth uterine death and the congenital anomaly of exencephalia. Anoxia (6% O/sub 2/ + 94% N/sub 2/) aIlowed 71% to come through as ""apparently normal," an increase of 30% over the unprotected irradiated controls. Whether there is long-term damage to the 8.5day embryo from the temporary anoxia alone has not been determined, although the anoxic controls showed 96% " apparently nornnal." Distilled water given I.P. before irradiation, making the milieu of the embryos hypotonic, appeared to be deleterious, causing 2% exencephalia even without x irradiation. When combined with x rays, distilled water reduced the "apparentiy normals" to 31%, or 10% lower than with irradiation alone. Saline in various concentrations was not protective. None of the tissue homogenates (spleen, marrow, liver, or brain of a homologous newborn source) proved to be of any protective value. It is suggested that the protective element in such tissue homogenates may be cellular since the placenta acts as the most efficient filter to allow only the dialyzable substances through to the embryo. However, the fact that the 8.5-day mouse embryo has not yet developed its hematopoietic syatem may explain the failure of homogenates which seem to protect through hematopoietic regeneration. Hypoxia from hypoglycemia following insulin injection was not protective. Insulin or dextrose or the two in combination were -not panticularly harraful to the 8.5-day mouse embryo but when combined with x irradiation were very damaging. "Protection" as used in this study is statistical and relates to the percentage changes in ""apparently normal" fetuses, resorptions, deaths, and congenital anomalies. It does not imply that the surviving mice are without irradiation sequelae. In fact many of the "apparently normals" have eye defects and this might well reveal other and more subtle C. N. 8. damage. On the basis of survival, however, cysteinamine, cystamine, and anoxia did afford some protection. (auth)« less

Development of mice without Cip/Kip CDK inhibitors.

PubMed

Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu; Hara, Eiji; Nakayama, Keiko; Nakayama, Keiichi I

2012-10-19

Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G(0) to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage). Copyright © 2012 Elsevier Inc. All rights reserved.