Project description:Formation of epiblast (EPI) - the founder line of all embryonic lineages - and extra-embryonic supportive tissues is one of the key events in mammalian development. The prevailing model of early mammalian development is based almost exclusively on the mouse. Here, we provide a comprehensive, stage-by-stage analysis of EPI and extra-embryonic primitive endoderm (PrE) formation during preimplantation development of the rabbit. Although we observed that rabbit embryos have several features in common with mouse embryos, including a stage-related initiation of lineage specification, our results demonstrate the existence of some key differences in lineage specification among mammals. Contrary to the current view, our data suggest that reciprocal repression of GATA6 and NANOG is not fundamental for the initial stages of PrE versus EPI specification in mammals. Furthermore, our results provide insight into the observed discrepancies relating to the role of FGF/ERK signalling in PrE versus EPI specification between mouse and other mammals.
Project description:The classical cell sorting experiments undertaken by Townes and Holtfreter described the intrinsic propensity of dissociated embryonic cells to self-organize and reconcile into their original embryonic germ layers with characteristic histotypic positioning. Steinberg presented the differential adhesion hypothesis to explain these patterning phenomena. Here, we have reappraised these issues by implementing embryoid bodies to model the patterning of epiblast and primitive endoderm layers. We have used combinations of embryonic stem (ES) cells and their derivatives differentiated by retinoic acid treatment to model epiblast and endoderm cells, and wild-type or E-cadherin null cells to represent strongly or weakly adherent cells, respectively. One cell type was fluorescently labeled and reconstituted with another heterotypically to generate chimeric embryoid bodies, and cell sorting was tracked by time-lapse video microscopy and confirmed by immunostaining. When undifferentiated wild-type and E-cadherin null ES cells were mixed, the resulting cell aggregates consisted of a core of wild-type cells surrounded by loosely associated E-cadherin null cells, consistent with the differential adhesion hypothesis. However, when mixed with undifferentiated ES cells, the differentiated primitive endoderm-like cells sorted to the surface to form a primitive endoderm layer irrespective of cell-adhesive strength, contradicting the differential adhesion hypothesis. We propose that the primitive endoderm cells reach the surface by random movement, and subsequently the cells generate an apical/basal polarity that prevents reentry. Thus, the ability to generate epithelial polarity, rather than adhesive affinity, determines the surface positioning of the primitive endoderm cells.
Project description:Anterior-posterior axis specification in the mouse requires signalling from a specialised extra-embryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the embryo and move to the prospective anterior. Embryological and genetic analysis has demonstrated that the AVE is required for anterior patterning and for correctly positioning the site of primitive streak formation by inhibiting Nodal activity. We have carried out a genetic ablation of the Hex-expressing cells of the AVE (Hex-AVE) by knocking the Diphtheria toxin subunit A into the Hex locus in an inducible manner. Using this model we have identified that, in addition to its requirement in the anterior of the embryo, the Hex-AVE sub-population has a novel role between 5.5 and 6.5dpc in patterning the primitive streak. Embryos lacking the Hex-AVE display delayed initiation of primitive streak formation and miss-patterning of the anterior primitive streak. We demonstrate that in the absence of the Hex-AVE the restriction of Bmp2 expression to the proximal visceral endoderm is also defective and expression of Wnt3 and Nodal is not correctly restricted to the posterior epiblast. These results, coupled with the observation that reducing Nodal signalling in Hex-AVE ablated embryos increases the frequency of phenotypes observed, suggests that these primitive streak patterning defects are due to defective Nodal signalling. Together, our experiments demonstrate that the AVE is not only required for anterior patterning, but also that specific sub-populations of this tissue are required to pattern the posterior of the embryo.
Project description:The Zonula Occludens proteins ZO-1 and ZO-2 are cell-cell junction-associated adaptor proteins that are essential for the structural and regulatory functions of tight junctions in epithelial cells and their absence leads to early embryonic lethality in mouse models. Here, we use the embryoid body, an in vitro peri-implantation mouse embryogenesis model, to elucidate and dissect the roles ZO-1 and ZO-2 play in epithelial morphogenesis and de novo tight junction assembly. Through the generation of individual or combined ZO-1 and ZO-2 null embryoid bodies, we show that their dual deletion prevents tight junction formation, resulting in the disorganization and compromised barrier function of embryoid body epithelial layers. The disorganization is associated with poor microvilli development, fragmented basement membrane deposition and impaired cavity formation, all of which are key epithelial tissue morphogenetic processes. Expression of Podocalyxin, which positively regulates the formation of microvilli and the apical membrane, is repressed in embryoid bodies lacking both ZO-1 and ZO-2 and this correlates with an aberrant submembranous localization of Ezrin. The null embryoid bodies thus give an insight into how the two ZO proteins influence early mouse embryogenesis and possible mechanisms underlying the embryonic lethal phenotype.
Project description:Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in embryoid body cultures, representing the inductive key step of further differentiation events in this system. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells.
Project description:Interactions between the endoderm and mesoderm that mediate myocardial induction are difficult to study in vivo because of the small size of mammalian embryos at relevant stages. However, we and others have demonstrated that signals from endodermal cell lines can influence myocardial differentiation from both mouse and human embryoid bodies (EBs), and because of this, assays that utilize embryonic stem (ES) cells and endodermal cell lines provide excellent in vitro models to study early cardiac differentiation. Extraembryonic endoderm (XEN) stem cells have a particular advantage over other heart-inducing cell lines in that they can easily be derived from both wild type and mutant mouse blastocysts. Here we describe the first isolation of a Nodal mutant XEN stem cell line. Nodal(-/-) XEN cell lines were not isolated at expected Mendelian ratios, and those that were successfully established, showed an increase in markers for the anterior visceral endoderm (AVE). Since AVE represents the heart-inducing endoderm in the mouse, cardiac differentiation was compared in EBs treated with conditioned medium (CM) collected from wild type or Nodal(-/-) XEN cells. EBs treated with CM from Nodal(-/-) cells began beating earlier and showed early activation of myocardial genes, but this early cardiac differentiation did not cause an overall increase in cardiomyocyte yield. By comparison, CM from wild type XEN cells both delayed cardiac differentiation and caused a concomitant increase in overall cardiomyocyte formation. Detailed marker analysis suggested that early activation of cardiac differentiation by Nodal(-/-) XEN CM caused premature differentiation and subsequent depletion of cardiac progenitors.
Project description:Independent mouse knockouts of Etv2 and Flk1 are embryonic lethal and lack hematopoietic and endothelial lineages. We previously reported that Flk1 activates Etv2 in the initiation of hematopoiesis and vasculogenesis. However, Flk1 and its ligand VEGF are expressed throughout development, from E7.0 to adulthood, whereas Etv2 is expressed only transiently during embryogenesis. These observations suggest a complex regulatory interaction between Flk1 and Etv2. To further examine the Flk1 and Etv2 regulatory interaction, we transduced Etv2 and Flk1 mutant ES cells with viral integrants that inducibly overexpress Flk1 or Etv2. We demonstrated that forced expression of Etv2 rescued the hematopoietic and endothelial potential of differentiating Flk1 and Etv2 mutant cells. We further discovered that forced expression of Flk1 can rescue that of the Flk1, but not Etv2 mutant cells. Therefore, we conclude that the requirement for Flk1 can be bypassed by expressing Etv2, supporting the notion that disruption of Etv2 expression is responsible for the early phenotypes of the Etv2 and Flk1 mutant embryos.
Project description:Centrioles comprise the heart of centrosomes, microtubule-organizing centers. To study the function of centrioles in lung and gut development, we genetically disrupted centrioles throughout the mouse endoderm. Surprisingly, removing centrioles from the endoderm did not disrupt intestinal growth or development but blocked lung branching. In the lung, acentriolar SOX2-expressing airway epithelial cells apoptosed. Loss of centrioles activated p53, and removing p53 restored survival of SOX2-expressing cells, lung branching, and mouse viability. To investigate how endodermal p53 activation specifically killed acentriolar SOX2-expressing cells, we assessed ERK, a prosurvival cue. ERK was active throughout the intestine and in the distal lung buds, correlating with tolerance to centriole loss. Pharmacologically inhibiting ERK activated apoptosis in acentriolar cells, revealing that ERK activity protects acentriolar cells from apoptosis. Therefore, centrioles are largely dispensable for endodermal growth and the spatial distribution of ERK activity in the endoderm shapes the developmental consequences of centriolar defects and p53 activation.
Project description:The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signalling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation, and therefore represent a valuable tool for investigating PrE lineage differentiation.
Project description:Embryoid body (EB) formation forms an important step in embryonic stem cell differentiation invivo. In murine embryonic stem cell (mESC) cultures EB formation is inhibited by the inclusion of leukaemic inhibitory factor (LIF) in the medium. Assembly of mESCs into aggregates by positive dielectrophoresis (DEP) in high field regions between interdigitated oppositely castellated electrodes was found to initiate EB formation. Embryoid body formation in aggregates formed with DEP occurred at a more rapid rate-in fact faster compared to conventional methods-in medium without LIF. However, EB formation also occurred in medium in which LIF was present when the cells were aggregated with DEP. The optimum characteristic size for the electrodes for EB formation with DEP was found to be 75-100 microns; aggregates smaller than this tended to merge, whilst aggregates larger than this tended to split to form multiple EBs. Experiments with ESCs in which green fluorescent protein (GFP) production was targeted to the mesodermal gene brachyury indicated that differentiation within embryoid bodies of this size may preferentially occur along the mesoderm lineage. As hematopoietic lineages during normal development derive from mesoderm, the finding points to a possible application of DEP formed EBs in the production of blood-based products from ESCs.