No description available.

L'organisation en chromatine permet de compacter l'ADN génomique et de réguler finement l'expression du génome. La particule cœur du nucléosome, composée d'un octamère de protéines histones autour desquelles s'enroule l'ADN, peut être modulée par l'incorporation de variants d'histones. Pour l'histone H3, les variants réplicatifs H3.1 et H3.2 permettent une incorporation lors de la réplication de l'ADN, tandis que le variant H3.3 est incorporé tout au long du cycle cellulaire. Les données dans la littérature établissent un lien entre H3.3 et la transcription. L'incorporation d'H3.3 dépend d'une voie d'assemblage faisant intervenir le chaperon HIRA. Mon projet de recherche visait à déterminer si H3.3 et son incorporation via HIRA possédaient un rôle spécifique. Le développement embryonnaire via une régulation fine de l'expression des gènes représentait une situation idéale pour aborder ces questions. L'utilisation du vertébré Xenopus laevis qui ne possède qu'un variant H3 réplicatif : H3.2, m'a permis d'évaluer la fonction de ces variants au cours du développement. J'ai pu montrer que, malgré leur similarité, les variants H3.2 et H3.3 ne sont pas interchangeables. Une altération d'expression d'H3.3 ou l'interférence dans sa voie d'assemblage via son chaperon HIRA conduisent à des défauts majeurs à la gastrulation. Ce phénotype s'accompagne d'un défaut d'expression de gènes mésodermiques, dont le marqueur Xbra. Une désorganisation globale de la chromatine est également observée chez ces embryons. Ces données mettent en lumière l'importance de l'incorporation du variant d'histone H3.3 dans la chromatine au cours d'une étape clé du développement embryonnaire, la gastrulation

Newly established F1 hybrid Embryonic Stem cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell based genetics feasible. An F1 hybrid ES cell line, G4 was used to generate transgenic over-expressing cell lines. The consequence of the expression of a panel of transgenes was assessed directly from ES cell-derived embryos produced by the tetraploid complementation assay. The generation of ES cell-derived embryos/animals was very efficient. A sufficient number of mutants for initial phenotypic analyses was derived only a few weeks after the establishment of the cell lines. The genes used in the study had either angiogenic/vasculogenic, anti-angiogenic or unknown properties. Of these transgenic mouse lines VEGF-A and Flt-Fc were used to further elucidate the effects of altered VEGF signaling on cell fate decisions in embryonic development and ES differentiation in two experimental systems. A. Early but transient Flk-1 activation led to enhanced generation of blood progenitors, whereas continuous activation of Flk-1 abolished this effect and enhanced endothelial cell generation. Ex vivo analysis of cells derived from E7.5 embryos demonstrated that sFlt-1-mediated control of Flk-1 activity also impacted the fate of hematopoietic and endothelial cells. The Flt-1-Fc transgenic mouse model was used to alter Flk-1 activation in vivo and show the relevance of the in vitro observations. These results demonstrate that sFlt-1 regulates Flk-1 activation in an oxygen responsive manner. Inhibition of Flk-1 activation by sFlt-1 increases the specification of hemangioblasts to blood cells consistent with a VEGF-independent default mechanism. B. Ubiquitous over-expression of VEGF164 isoform led to E8.75 embryonic lethality. The primary cause of lethality was the failure to form an organized cardiovascular system, which was manifested in three ways: the absence of yolk sac blood vessels, the lack of embryonic-maternal circulation due to the failure of allantochorionic fusion and improper cardiac function. The described phenotypes suggest that VEGF does not inhibit embryonic or extra-embryonic mesoderm formation at gastrulation but perturbs the balance amongst the mesodermal components.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Newly established F1 hybrid Embryonic Stem cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell based genetics feasible. An F1 hybrid ES cell line, G4 was used to generate transgenic over-expressing cell lines. The consequence of the expression of a panel of transgenes was assessed directly from ES cell-derived embryos produced by the tetraploid complementation assay. The generation of ES cell-derived embryos/animals was very efficient. A sufficient number of mutants for initial phenotypic analyses was derived only a few weeks after the establishment of the cell lines. The genes used in the study had either angiogenic/vasculogenic, anti-angiogenic or unknown properties. Of these transgenic mouse lines VEGF-A and Flt-Fc were used to further elucidate the effects of altered VEGF signaling on cell fate decisions in embryonic development and ES differentiation in two experimental systems. A. Early but transient Flk-1 activation led to enhanced generation of blood progenitors, whereas continuous activation of Flk-1 abolished this effect and enhanced endothelial cell generation. Ex vivo analysis of cells derived from E7.5 embryos demonstrated that sFlt-1-mediated control of Flk-1 activity also impacted the fate of hematopoietic and endothelial cells. The Flt-1-Fc transgenic mouse model was used to alter Flk-1 activation in vivo and show the relevance of the in vitro observations. These results demonstrate that sFlt-1 regulates Flk-1 activation in an oxygen responsive manner. Inhibition of Flk-1 activation by sFlt-1 increases the specification of hemangioblasts to blood cells consistent with a VEGF-independent default mechanism. B. Ubiquitous over-expression of VEGF164 isoform led to E8.75 embryonic lethality. The primary cause of lethality was the failure to form an organized cardiovascular system, which was manifested in three ways: the absence of yolk sac blood vessels, the lack of embryonic-maternal circulation due to the failure of allantochorionic fusion and improper cardiac function. The described phenotypes suggest that VEGF does not inhibit embryonic or extra-embryonic mesoderm formation at gastrulation but perturbs the balance amongst the mesodermal components.

The objective of this work is to investigate the elastic modulus of gastrula-stage avian embryos and the effect of substrate stiffness on presumptive precardiac cell fate. Our overall hypothesis is that the mechanical microenvironment, specifically, tissue modulus and substrate stiffness, influences gastrulation and cardiac induction. Large-scale morphogenetic movements during early embryo development are driven by complex changes in biochemical and biophysical factors. Current models for amniote primitive streak morphogenesis and gastrulation take into account numerous genetic pathways but largely ignore the role of mechanical forces. Here, we used atomic force microscopy (AFM) to obtain for the first time precise biomechanical properties of the early avian embryo. Our data reveal that the primitive streak is significantly stiffer than neighboring regions of the epiblast, and that it is stiffer than the pre-primitive streak epiblast. To test our hypothesis that these changes in mechanical properties are due to a localized increase of actomyosin contractility, we inhibited actomyosin contractility via the Rho kinase (ROCK) pathway using the small-molecule inhibitor Y-27632. Our results using several different assays show the following: 1) primitive streak formation was blocked; 2) the time-dependent increase in primitive streak stiffness was abolished; and 3) convergence of epiblast cells to the midline was inhibited. Taken together, our data suggest that actomyosin contractility is necessary for primitive streak morphogenesis, and specifically, ROCK plays a critical role. To better understand the underlying mechanisms of this fundamental process, future models should account for the findings presented in this study. As presumptive cardiac cells traverse the course of differentiation into cardiac myocytes during cardiogenesis, the sequence, magnitude, and spatiotemporal map of biomechanical and biochemical signals has not been fully explored. There have been many studies detailing the induction of cardiogenesis on a variety of substrates and extracellular matrix (ECM) proteins, but none have completed a rigorous study of the effects of substrate stiffness on the induction of precardiac cells prior to the onset of cardiac gene expression (smooth muscle alpha actin [SMAA] at stage 5.) We investigate the effects of the mechanical environment on precardiac cell behaviors in an in vitro setting to elucidate the effect of substrate stiffness and inducing factors on precardiac tissue and the potential connection between them. The cells in the anterior portion of the primitive streak are fated to form the heart, and we show differing levels of SMAA expression on substrates of differing moduli, which suggests that substrate stiffness may play a role in cardiac differentiation. We cannot determine the physical mechanisms during morphogenesis without understanding the response of precardiac cells to changes in their mechanical environment.

GTPases are integral components of virtually every known signal transduction pathway, and mutations in GTPases frequently cause disease. A genomic analysis identified and annotated 174 GTPases in the sea urchin genome (with 90% expressed in the embryo), covering five classes of GTP-binding proteins: the Ras superfamily, the heterotrimeric G proteins, the dynamin superfamily, the SRP/SR GTPases, and the translational GTPases. The sea urchin genome was found to contain large lineage-specific expansions within the Ras superfamily. For the Rho, Rab, Arf and Ras subfamilies, the number of sea urchin genes relative to vertebrate orthologs suggests reduced genomic complexity in the sea urchin. However, gene duplications in the sea urchin increased overall numbers, such that total sea urchin gene numbers of these GTPase families approximate vertebrate gene numbers. This suggests lineage-specific expansions as an important component of genomic evolution in signal transduction. A focused analysis on RhoA, a monomeric GTPase, shows it contributes to multiple signal transduction pathways during sea urchin development. The data reveal that RhoA inhibition in the sea urchin results in a failure to invaginate during gastrulation. Conversely, activated RhoA induces precocious archenteron invagination, complete with the associated actin rearrangements and extracellular matrix secretion. Although RhoA regulates convergent extension movements in vertebrates, our experiments show RhoA activity does not regulate convergent extension in the sea urchin. Instead, the results suggest RhoA serves as a trigger to initiate invagination, and once initiation occurs RhoA activity is no longer involved in subsequent gastrulation movements. RhoA signaling was also observed during endomesodermal specification in the sea urchin. Data show that LvRhoA activity is required, downstream of a partially characterized Early Signal, for SoxB1 clearance from endomesodermal nuclei (and subsequent expression of GataE and Endo16 genes). Investigations also suggest that within the endomesoderm, RhoA clears SoxB1 as part of Wnt8 signaling, as activated RhoA is sufficient to rescue Wnt8-inhibited embryos. These data provide evidence of the first molecular components involved in SoxB1 clearance, as well as highlight a previously unrecognized role for RhoA during endomesodermal specification. These analyses suggest RhoA signaling is integral to the proper specification and morphogenesis of the sea urchin endomesoderm. / dissertation

Debate continues on the merits of strictly limiting alcohol consumption during all of pregnancy, and whether “safe” consumption levels and/or times exist. Only a relatively few experimental studies have been conducted that limit the timing of exposure to specific events during development and the exposure level to one that might model sporadic, incidental drinking during pregnancy. In the present study, the effects of two acute gavage exposures to low and moderate levels of ethanol (peak blood ethanol concentrations (BEC) of 104 and 177 mg/dl, respectively) either during gastrulation on gestational day (GD) 7 (at GD7:0 h and GD7:4 h) or during neurulation on GD8 (at GD8:6 h and GD8:10 h) on the spatial learning and memory abilities of adult mice in the radial arm maze (RAM) were examined. Mice were selected from a prenatal ethanol exposure (PAE) cohort that had been tested as neonates for their sensorimotor development (Schambra et al., 2015) and as juveniles and young adults for open field activity levels and emotionality (Schambra et al., 2016). Mice exposed on either of the two gestational days to acute, low or moderate levels of ethanol were deficient in overall performance in the RAM in adulthood. Importantly, mice in ethanol exposed groups took longer to reach criterion in the RAM, and many mice in these groups failed to do so after 48 trials when testing was terminated. Exposure to a low level of ethanol on either GD7 or GD8, or a moderate level on GD7, resulted in significant impairment in spatial reference (long-term) memory, while only mice exposed on GD7 to the low level of ethanol were significantly impaired in spatial working (short-term) memory. Mice exposed to the low ethanol level on either day had significantly shorter response latencies, which may reflect impairment of processes related to response inhibition or executive attention in these mice. For all measures, distributions of individual scores revealed a relatively small subset of mice in each PAE group who scored well outside the range of the control group, which skewed the population distributions to varying degrees in the direction of worse performance for the PAE groups. Overall the data suggest that after acute, low level ethanol exposure early in gestation, the likelihood that an individual mouse embryo experienced measureable ill-effects due to the exposure was rather low, but in a few of the embryos, damage occurred that resulted in significant deficits in later performance. The overall characteristics of our cohort of PAE mice, including delayed sensorimotor development, mild hypoactivity and increased emotionality, as shown in previous studies, together with deficits in spatial learning and memory as shown here, resemble those in a subset of human Fetal Alcohol Spectrum Disorder (FASD) diagnoses, specifically ADHD-Inattentive type (ADHD-I) and/or Sluggish Cognitive Tempo (SCT). Although possible correspondences between mechanisms underlying PAE-induced deficits in mice and those operating in humans remain undefined, further study with this mouse PAE model may ultimately help advance understanding of the causes of these conditions in affected children. This study highlights the possibility of risk associated with low to moderate sporadic alcohol consumption during the first month of human pregnancy.

The formation of the anterior-posterior axis requires a symmetry-breaking event that starts gastrulation. Ultimately, the morphogenetic movements of gastrulation reshape the embryo to its final tri-dimensional form. In mouse embryos, the identity of the molecule that breaks the bilateral symmetry and sets in motion gastrulation remains elusive. The Wnt signaling pathway plays a pivotal role during axial specification and gastrulation in metazoans. Loss-of-function experiments have demonstrated a requirement of Wnt3 for gastrulation in mice. But because Wnt3 is expressed sequentially in two tissues, the visceral endoderm and the epiblast, its tissue specific requirements remain uncertain. Here, we report that embryos lacking Wnt3 specifically in the visceral endoderm do not form a primitive streak, mesoderm, endoderm or any derivatives. Visceral endoderm-specific Wnt3 mutants also lack primordial germ cells. Moreover, we provide data demonstrating that Wnt3 carries out its actions in the epiblast via the canonical Wnt pathway. Together, these data suggest that the posterior visceral endoderm via Wnt3, regulates the development of mouse embryos in a similar fashion to the amphibian Nieuwkoop center. Next, we conditionally ablated Wnt3 locus in the epiblast to investigate whether Wnt3 expression is also required in that tissue. Embryos lacking Wnt3 expression in the epiblast, but retaining its expression in the visceral endoderm, show delayed but not absent gastrulation. We conclude that the expression of Wnt3 in the epiblast is required for maintenance but not initiation of gastrulation in mouse embryos. Furthermore, we used in vitro and in vivo approaches to demonstrate that the Wnt3-mediated activation of the canonical Wnt pathway leads to β-catenin occupancy followed by transcription of key loci, including the Wnt3 locus itself, during gastrulation in mice. Our data indicate the presence of an autoregulatory loop in which Wnt3 controls its own expression and orchestrates the process of gastrulation in the mouse embryo.