Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro

Cardiomyogenesis proceeds in the presence of signals emanating from extra-embryonic lineages emerging before and during early eutherian gastrulation. In embryonic stem cell derived embryoid bodies, primitive endoderm gives rise to visceral and parietal endoderm. Parietal endoderm undergoes an epithelial to mesenchymal transition shortly before first cardiomyocytes start to contract rhythmically. Here, we demonstrate that Secreted Protein, Acidic, Rich in Cysteine, SPARC, predominantly secreted by mesenchymal parietal endoderm specifically promotes early myocardial cell differentiation in embryoid bodies. SPARC enhanced the expression of bmp2 and nkx2.5 in embryoid bodies and fetal cardiomyocytes. Inhibition of either SPARC or Bmp2 attenuated in both cases cardiomyogenesis and downregulated nkx2.5 expression. Thus, SPARC directly affects cardiomyogenesis, modulates Bmp2 signaling, and contributes to a positive autoregulatory loop of Bmp2 and Nkx2.5 in cardiomyocytes.     

Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm

The definitive endoderm forms during gastrulation and is rapidly transformed into the gut tube which is divided along the anterior-posterior axis into the foregut, midgut, and hindgut. Lineage tracing and genetic analysis have examined the origin of the definitive endoderm during gastrulation and demonstrated that the majority of definitive endoderm arises at the anterior end of the primitive streak (APS). Foxh1 and Foxa2 have been shown to play a role in specification of the APS and definitive endoderm. However, prior studies have focused on the role of these factors in specification of foregut definitive endoderm, while their role in the specification of midgut and hindgut definitive endoderm is less understood. Furthermore, previous analyses of these mutants have utilized definitive endoderm markers that are restricted to the anterior endoderm, expressed in extraembryonic endoderm, or present in other germ layers. Here, we characterized the expression of several novel definitive and visceral endoderm markers in Foxh1 and Foxa2 null embryos. In accordance with previous studies, we observed a deficiency of foregut definitive endoderm resulting in incorporation of visceral endoderm into the foregut. Interestingly, this analysis revealed that formation of midgut and hindgut definitive endoderm is unaffected by loss of Foxh1 or Foxa2. This finding represents a significant insight into specification and regionalization of mouse definitive endoderm.     

Paracrine promotion of cardiomyogenesis in embryoid bodies by LIF modulated endoderm

In the vertebrate embryo the heart is the first organ to form. Embryonic and extra-embryonic tissues are supposed to contribute to cardiac lineage commitment before and during gastrulation in a paracrine fashion. Evidence has accumulated that factors secreted by the anterior lateral endoderm and extra-embryonic endoderm contribute to cardiomyogenesis. Here we exploit in vitro differentiation of embryonic stem cells in embryoid bodies to study differentiation of the extraembryonic endodermal lineage, gastrulation-like processes, and the influence of endoderm on cardiomyogenesis. We demonstrate that in embryoid bodies primitive endoderm differentiates to visceral and parietal endoderm and that parietal endoderm influences onset of cardiomyogenesis in a concentration-dependent manner. Both increased concentrations of leukemia inhibitory factor and its absence in lif-/- embryoid bodies hampered parietal endoderm formation. Reduced differentiation of parietal endoderm correlated with an attenuation of cardiomyogenesis even in the presence of LIE These and previous results suggest that leukemia inhibitory factor is directly and indirectly, via endoderm formation, involved in the regulation of cardiomyogenesis. Increased proliferation of parietal endoderm in lifr -/- embryoid bodies and addition of conditioned lif -/- cell culture supernatant promoted cardiomyogenesis, demonstrating for the first time that parietal endoderm also contributes to cardiomyogenesis in embryoid bodies in a paracrine and leukemia inhibitory factor and its receptor independent pathway. New factors signaling independently of the leukemia inhibitory-factor receptor pathway may sustain cardiomyocyte cell proliferation and thus be a future target for gene therapy of cardiomyopathies and cell therapy of the myocardium.     

Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis

During mouse gastrulation, primordial germ cells (PGCs) become clustered at the base of the allantois and move caudally into the hindgut endoderm before entering the genital ridges. The precise roles of endoderm tissues in PGC migration, however, remain unclear. By using Sox17 mutants with a specific endoderm deficiency, we provide direct evidence for the crucial role of hindgut expansion in directing proper PGC migration. In Sox17-null embryos, PGCs normally colonize in the allantois and then a small front-row population of PGCs moves properly into the most posterior gut endoderm. Defective hindgut expansion, however, causes the failure of further lateral PGC movement, resulting in the immobilization of PGCs in the hindgut entrance at the later stages. In contrast, the majority of the remaining PGCs moves into the visceral endoderm layer, but relocate outside of the embryonic gut domain. This leads to a scattering of PGCs in the extraembryonic yolk sac endoderm. This aberrant migration of Sox17-null PGCs can be rescued by the supply of wildtype hindgut cells in chimeric embryos. Therefore, these data indicate that hindgut morphogenic movement is crucial for directing PGC movement toward the embryonic gut side, but not for their relocation from the mesoderm into the endoderm.     

Localization of Brachyury (T) in embryonic and extraembryonic tissues during mouse gastrulation

T-box gene family members have important roles during murine embryogenesis, gastrulation, and organogenesis. Although relatively little is known about how T-box genes are regulated, published gene expression studies have revealed dynamic and specific patterns in both embryonic and extraembryonic tissues of the mouse conceptus. Mutant alleles of the T-box gene Brachyury (T) have identified roles in formation of mesoderm and its derivatives, such as somites and the allantois. However, given the cell autonomous nature of T gene activity and conflicting results of gene expression studies, it has been difficult to attribute a primary function to T in normal allantoic development. We report localization of T protein by sectional immunohistochemistry in both embryonic and extraembryonic tissues during mouse gastrulation, emphasizing T localization within the allantois. T was detected in all previously reported sites within the conceptus, including the primitive streak and its derivatives, nascent embryonic mesoderm, the node and notochord, as well as notochord-associated endoderm and posterior neurectoderm. In addition, we have clarified T within the allantois, where it was first detected in the proximal midline of the late allantoic bud (approximately 7.5 days postcoitum, dpc) and persisted within an expanded midline domain until 6-somite pairs (s; approximately 8.5 dpc). Lastly, we have discovered several novel T sites, including the developing heart, visceral endoderm, extraembryonic ectoderm, and its derivative, chorionic ectoderm. Together, these data provide a unified picture of T in the mammalian conceptus, and demonstrate T's presence in unrelated cell types and tissues in highly dynamic spatiotemporal patterns in both embryonic and extraembryonic tissues.     

The endoderm gene regulatory network in sea urchin embryos up to mid-blastula stage

As the result of early specification processes, sea urchin embryos eventually form various mesodermal cell lineages and a gut consisting of fore-, mid- and hindgut. The progression of specification as well as the overall spatial organization of the organism is encoded in its gene regulatory networks (GRNs). We have analyzed the GRN driving endoderm specification up to the onset of gastrulation and present in this paper the mechanisms which determine this process up to mid-blastula stage. At this stage, the embryo consists of two separate lineages of endoderm precursor cells with distinct regulatory states. One of these lineages, the veg2 cell lineage, gives rise to endoderm and mesoderm cell types. The separation of these cell fates is initiated by the spatially confined activation of the mesoderm GRN superimposed on a generally activated endoderm GRN within veg2 descendants. Here we integrate the architecture of regulatory interactions with the spatial restriction of regulatory gene expression to model the logic control of endoderm development.     

Signals from primary mesenchyme cells regulate endoderm differentiation in the sea urchin embryo

Primary mesenchyme cells (PMC), the skeletogenic cells derived from the micromeres of the sea urchin embryo, are involved in the differentiation of the gut. When PMC were deleted from the mesenchyme blastula, both formation of the constrictions in the gut and expression of endoderm-specific alkaline phosphatase were significantly delayed. Therefore, the correct timing of gut differentiation depends on the existence of PMC, probably via a type of promotive signal. To date, the only role of PMC in other tissue differentiation has been a suppressive signal for the conversion of secondary mesenchyme cells (SMC) into skeletogenic cells. The present experiments using PMC ablation and transplantation showed that both signaling processes occurred in the same short period during gastrulation, but the embryos kept their competence for gut differentiation until a later stage. Further investigations indicated that conversion of SMC did not cause delay in gut differentiation and that SMC did not mediate the PMC signal to the endoderm. Therefore, the effect of PMC on gut differentiation could be a new role that is independent of the suppressive effect for SMC conversion.     

Embryonic fates for extraembryonic lineages: New perspectives

The prevailing view of the functions of the extraembryonic lineages of the mammalian embryo has been that they serve solely to support its intrauterine development. In recent years, a number of studies have suggested that the extraembryonic mesoderm and visceral endoderm in fact contribute cells to tissues of the developing animal. In this mini‐review, we discuss evidence that the yolk sac is an early source of hematopoietic stem and progenitor cells and that the cells of the visceral endoderm, once thought to be segregated solely to the yolk sac, constitute a subpopulation of cells within the developing gut tube and perhaps other endodermal structures. Fascinating questions remain to be addressed and are likely to establish a new paradigm for studying early mammalian development. Understanding the processes that give rise to stem cell populations in development may lead to advances in stem cell therapies and regenerative medicine. J. Cell. Biochem. 107: 586–591, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular approaches to the study of mesoderm formation in amphibians

The mesoderm is the region of the embryo that gives rise to muscle, blood and connective tissues; it becomes segregated from the ectoderm and endoderm at gastrulation. Embryological studies have revealed, however, that the potential for certain embryonic cells to become part of the mesoderm is established well before gastrulation, most likely through an extracellular signalling process termed ‘induction’. The recent characterization of mesoderm-specific mRNAs and proteins now permits an analysis of the very earliest events involved in the specification of the mesoderm at the molecular level. Such experiments should contribute to our understanding of the mechanisms involved in controlling cell differentiation.     

Human parthenogenetic stem cells produce enriched populations of definitive endoderm cells after trichostatin A pretreatment

Human parthenogenetic stem cells (hpSC) hold great promise as a source of pluripotent stem cells for cell-based transplantation therapy due to their ethical method of derivation as well as the enhanced capacity for immunomatching with significant segments of the human population. We report here the directed differentiation of hpSC to produce enriched populations of definitive endoderm. Moreover, we find that treatment of undifferentiated hpSC by trichostatin A (TSA) before applying the directed differentiation protocol significantly increases the proportion of definitive endoderm cells in the final population. TSA-pretreated as well as non-TSA-treated hpSC undergoing differentiation toward definitive endoderm demonstrate a similar temporal sequence of gene expression to that which occurs in the course of definitive endoderm differentiation during vertebrate gastrulation and for differentiation of hESCs to definitive endoderm. Creation of the definitive endoderm lineages from hpSC represents the critical first step toward the development of hpSC-based cellular therapies for diseases of the liver or pancreas.     

FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo

At the end of gastrulation in avians and mammals, the endoderm germ layer is an undetermined sheet of cells. Over the next 24-48 h, endoderm forms a primitive tube and becomes regionally specified along the anterior-posterior axis. Fgf4 is expressed in gastrulation and somite stage embryos in the vicinity of posterior endoderm that gives rise to the posterior gut. Moreover, the posterior endoderm adjacent to Fgf4-expressing mesoderm expresses the FGF-target genes Sprouty1 and 2 suggesting that endoderm respond to an FGF signal in vivo. Here, we report the first evidence suggesting that FGF4-mediated signaling is required for establishing gut tube domains along the A-P axis in vivo. At the gastrula stage, exposing endoderm to recombinant FGF4 protein results in an anterior shift in the Pdx1 and CdxB expression domains. These expression domains remain sensitive to FGF4 levels throughout early somite stages. Additionally, FGF4 represses the anterior endoderm markers Hex1 and Nkx2.1 and disrupts foregut morphogenesis. FGF signaling directly patterns endoderm and not via a secondary induction from another germ layer, as shown by expression of dominant-active FGFR1 specifically in endoderm, which results in ectopic anterior expression of Pdx1. Loss-of-function studies using the FGF receptor antagonist SU5402 demonstrate that FGF signaling is necessary for establishing midgut gene expression and for maintaining gene expression boundaries between the midgut and hindgut from gastrulation through somitogenesis. Moreover, FGF signaling in the primitive streak is necessary to restrict Hex1 expression to anterior endoderm. These data show that FGF signaling is critical for patterning the gut tube by promoting posterior and inhibiting anterior endoderm cell fate.     

Endoderm and mesoderm reciprocal signaling mediated by CXCL12 and CXCR4 regulates the migration of angioblasts and establishes the pancreatic fate

We have discovered that angioblasts trigger an early inductive event in pancreatic differentiation. This event occurs soon after gastrulation, before the formation of blood vessels. Morphological studies revealed that Lmo2-expressing angioblasts reside in proximity to the somitic mesoderm and the gut endoderm from which pancreatic progenitors arise. The chemokine ligand CXCL12 expressed in the gut endoderm functions to attract the angioblasts that express its receptor CXCR4. Angioblasts then signal back to the gut endoderm to induce Pdx1 expression. Gain-of-function and loss-of-function experiments for CXCL12 and CXCR4 were performed to test their function in blood vessel formation and pancreatic differentiation. The ectopic expression of Cxcl12 in the endoderm attracted the angioblasts and induced ectopic Pdx1 expression, resulting in an expanded pancreatic bud and an increased area of insulin-expressing cells. By contrast, in chick embryos treated with beads soaked in AMD3100, an inhibitor of CXCR4, the migration of angioblasts towards the Cxcl12-expressing gut endoderm was arrested, causing a malformation of blood vessels. This led to the generation of a smaller pancreatic bud and a reduced area of insulin-expressing cells. Taken together, these results indicate that the gut endoderm and angioblasts attract each other through reciprocal CXCL12 and CXCR4 signaling. This has a pivotal role in the fate establishment of the pancreatic progenitor cells and in the potentiation of further differentiation into endocrine β-cells.     

Intestinal lineage commitment of embryonic stem cells

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.     

The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages

The cell movements underlying the morphogenesis of the embryonic endoderm, the tissue that will give rise to the respiratory and digestive tracts, are complex and not well understood. Using live imaging combined with genetic labeling, we investigated the cell behaviors and fate of the visceral endoderm during gut endoderm formation in the mouse gastrula. Contrary to the prevailing view, our data reveal no mass displacement of visceral endoderm to extraembryonic regions concomitant with the emergence of epiblast-derived definitive endoderm. Instead, we observed dispersal of the visceral endoderm epithelium and extensive mixing between cells of visceral endoderm and epiblast origin. Visceral endoderm cells remained associated with the epiblast and were incorporated into the early gut tube. Our findings suggest that the segregation of extraembryonic and embryonic tissues within the mammalian embryo is not as strict as believed and that a lineage previously defined as exclusively extraembryonic contributes cells to the embryo.     

文昌鱼sfy1基因的克隆及其在早期发育中的表达

文昌鱼是公认现存最接近于脊椎动物的一种头索动物,具有与脊椎动物相似但简单得多的身体图式[1],因而是研究脊椎动物发育机制起源和进化的宝贵材料,也是发育生物学的经典实验模型之一.近年来,人们在对果蝇和脊椎动物发育分子机制的研究取得了一系列重大突破之后,利用发育调控基     

Early development of the gut: new light on an old hypothesis

This review deals with the early development of the gut. It draws largely on information provided from the study of avian embryos. Evidence that concerns the early determination of the regional fate of the endoderm and mesoderm of the gut is reviewed. Gut endoderm can undergo a limited degree of differentiation from a remarkably early age when cultured in the absence of mesoderm and there is evidence that points to the establishment of a pre-pattern in the early mesoderm before the genes responsible for patterning in gut are active. Initially, at least at cranial levels, those parts of the mesoderm and endoderm that are in contact are not those parts that will ultimately be in apposition; the consequence of this for any signalling between these layers is considered. In the light of the above information, the probable role of mesenchyme in gut development is re-examined.