Early endocardial formation originates from precardiac mesoderm as revealed by QH-1 antibody staining

The formation of endocardial endothelium in quail embryos was investigated using in vivo and in vitro systems. At stage 7+ (2 somite), the initial emergence of endothelial cells within the bilateral heart forming region (HFR) was detected in quail embryos by immunohistochemistry with QH-1 (an anti-quail endothelial cell marker) and confocal microscopy. We consistently observed more QH-1 positive cells in the right HFR than the left. At stage 8 (4 somite), the HFR, including QH-1 positive cells, were located in the splanchnic mesoderm after formation of the coelom. During stage 8, the HFR migrated along the margin of anterior intestinal portal in association with the endoderm. By stage 8+ (5 somite), the two HFR had fused at the midline and formed a plexus of QH-1 positive endothelial precursor cells. The definitive endocardium developed as a single, hollow, tube within this plexus. Posteriorly, QH-1 positive cells of the HFR established vascular-like connections with QH-1 positive cells that had formed outside (peripheral to) the HFR. During migration and subsequent determination, the precardiac mesoderm is continuously associated with the basement membrane of the anterior endoderm. To determine the role of endoderm on endocardial endothelial cell formation and development, precardiac mesoderm from stage 5 embryos, which does not express QH-1 antigen, was explanted onto the surface of collagen gels. When co-cultured with endoderm, the outgrowth of free cells from the mesoderm was much more extensive, many of which invaded the gel and expressed the QH-1 antigen; mesoderm cultured without endoderm did not seed nor express QH-1 antigen. These findings suggest that the segregation of endothelial and myocardial lineages may occur by an endoderm-mediated, mesenchymal formation.     

Chordin affects pronephros development in Xenopus embryos by anteriorizing presomitic mesoderm.

Spemann's organizer emits signals that pattern the mesodermal germ layer during Xenopus embryogenesis. In a previous study, we demonstrated that FGFR1 activity within the organizer is required for the production of both the somitic muscle- and pronephros-patterning signals by the organizer and the expression of chordin, an organizer-specific secreted protein (Mitchell and Sheets [2001] Dev. Biol. 237:295-305). Studies from others in both chicken and Xenopus embryos provide compelling evidence that pronephros forms by means of secondary induction signals emitted from anterior somites (Seufert et al. [1999] Dev. Biol. 215:233-242; Mauch et al. [2000] Dev. Biol. 220:62-75). Here we provide several lines of evidence in support of the hypothesis that chordin influences pronephros development by directing the formation of anterior somites. Chordin mRNA was absent in ultraviolet (UV) -irradiated embryos lacking pronepheros (average DAI<2) but was always found in UV-irradiated embryos that retain pronepheros (average DAI>2). Furthermore, ectopic expression of chordin in embryos and in tissue explants leads to the formation of anterior somites and pronephros. In these experiments, pronephros was only observed in association with muscle. Chordin diverted somatic muscle cells to more anterior positions within the somite file in chordin-induced secondary trunks and induced the expression of the anterior myogenic gene myf5. Finally, depletion of chordin mRNA with DEED antisense oligonucleotides substantially reduced somitic muscle and pronephric tubule and duct formation in whole embryos. These data and previous studies on ectoderm and endoderm (Sasai et al. [1995] Nature 377:757) support the idea that chordin functions as an anteriorizing signal in patterning the germ layers during vertebrate embryogenesis. Our data support the hypothesis that chordin directs the formation of anterior somites that in turn are necessary for pronephros development.     

Properties of surface and junctional membranes of embryonic cells isolated from blastula stages of Xenopus laevis.

1. Some membrane properties of endoderm and mesoderm cells isolated from late blastula stages of Xenopus laevis have been examined using electrophysiological techniques. 2. Cells were isolated by treatment of whole embryos with Ca-free EDTA containing media, or mechanically by micro-dissection, and cultured in Ca-containing Holtfreter solution (60 mM-NaCl) or Ringer solution (120 mM-NaCl). 3. Membrane potentials lay between -6 and -84 mV; specific membrane resistances ranged from 500 to 29,000 omega cm2; there was no difference between EDTA isolated and mechanically isolated cells. 4. Relative and absolute cation and anion conductances varied from cell to cell. Some cells were anion impermeable; the cation conductance ranged from 35 to 300 mumho/cm2. 5. The resting potential of some cells was largely determined by the concentration gradient and membrane permeability of K ions. In other cells the potential was maintained either by some other ion or by an electrogenic pump. [K]i came to approximately 130 mM in Ringer solution (the value pertaining in the intact embryo) and similar to 60 mM in Holtfreter solution. 6. In most pairs and small clumps of cells ionic current spread from one cell to the next; some single cells and groups of cells were uncoupled from their neighbours. 7. The junctional resistance lay between 10(5) and 10(8) omega; it behaved as a linear resistor in most cell pairs studied. In three pairs the intercellular junction showed rectifying properites. 8. By the late blastula stage of development presumptive endoderm and mesoderm cells form a heterogeneous population with widely varying passive membrane properties. The significance of these findings is discussed in relation to current hypotheses for the formation of spatial patterns during differentiation.     

Differentiation of gut endoderm in dependence of the notochord

Summary Presumptive intraembryonic endoderm, either isolated or together with adhering mesoderm, from 19-h chick embryos, was grafted to the coelom of 50-h host embryos. The viability of such grafts was low and endodermal differentiation was poor. In a second series the endoderm (with or without adhering mesoderm) was combined with a fragment of notochordal tissue from 48–60-h donor embryos. Then the recovery was much higher, notably after longer periods of in vivo culture. After 10 days of cultivation well-developed entero-endocrine (argyrophilic) cells were found among the regular enterocytes in both series.     

Is extraembryonic angiogenesis in the chick embryo controlled by the endoderm?

Summary The area vasculosa of the chick embryo is subdivided into two concentric zones: the inner transparent area pellucida vasculosa (AVP) and the less transparent surrounding area opaca vasculosa (AOV). The different optical properties of these zones are caused by the different morphology of the endoderm, which consists of flat cells in the APV and of high-prismatic cells containing large yolk vacuoles in the AOV. The present study describes how this endodermal subdivision of the area vasculosa is related to the development of the extraembryonic vascular pattern. By injection of ink into the vascular system of chick embryos at stages 12 to 20 (Hamburger and Hamilton 1951 HH), it has been demonstrated that the vascular net of the area vasculosa from stage 14 (HH) onwards develops into different patterns in APV and AOV. The small loops of uniform capillary vessels of stage 13 (HH) are widened due to the rapid expansion of the extraembryonic mesoderm. In the AOV from stage 14 (HH) onwards numerous small blood vessels sprout into the enlarged intervascular spaces. This process is maximal at stage 17 (HH). In contrast, the blood vessels of the APV remain largely unbranched. These findings suggest that the development of the extraembryonic vascular pattern is controlled by the endodermal pattern. To test this hypothesis, both zones (APV and AOV) were examined by light microscopy, transmission and scanning electron microscopy, in vivo observations and by treatment with bromodeoxyuridine (BrdU). TEM examinations show that the ultrastructural organization of the APV mesoderm is different from that of the AOV: The splanchnopleuric cells of the APV form a continuous cover around the endothelial cells connected by numerous desmosomes, whereas the splanchnopleuric cells of the AOV are frequently separated by gaps. The largest gaps are seen in the small blood vessels at stage 17 (HH). These results should be considered in relation to the dynamic changes in the vascular pattern of the AOV. The endodermal cells of APV and AOV are two different populations. In vivo observation of the endodermal transition from APV to AOV detected no transformations of APV cells into AOV cells or vice versa. The borderline between the zones is stable.The AOV endoderm, having been overgrown by the expanding mesoderm, stops proliferating almost completely, whereas the proliferation of the APV endoderm is unaffected by contact with the mesoderm. The rate of its proliferation is approximately as high as that of the AOV prior to contact with the expanding mesoderm (results after treatment with BrdU). The contact of the basal side of the AOV endoerm with mesoderm is closer than that of the APV. In the AOV the basal compartments of endodermal cells show numerous small coated vesicles, probably exocytotic in nature.The transition between zones in the endoderm was found to be formed by small vaulted cells bearing microvilli on their surface. These cells probably are daughter cells of the primary hypoblast cells, which have been withdrawn to the margin of the APV by the invading endoblast.     

Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.

Wilms' tumor 1-associating protein (WTAP) was previously identified as a protein associated with Wilms' tumor-1 (WT-1) protein that is essential for the development of the genitourinary system. Although WTAP has been suggested to function in alternative splicing, stabilization of mRNA, and cell growth, its in vivo function is still unclear. We generated Wtap mutant mice using a novel gene-trap approach and showed that Wtap mutant embryos exhibited defective egg-cylinder formation at the gastrulation stage and died by embryonic day 10.5. Although they could form extraembryonic tissues and anterior visceral endoderm, Wtap mutant embryos and embryonic stem cells failed to differentiate into endoderm and mesoderm. The chimera analysis showed that Wtap in extraembryonic tissues was required for the formation of mesoderm and endoderm in embryonic tissues. Taken together, our findings indicate that Wtap is indispensable for differentiation of mesoderm and endoderm in the mouse embryo.     

An endothelial cell niche induces hepatic specification through dual repression of Wnt and Notch signaling

Complex cross-talk between endoderm and the microenvironment is an absolute requirement to orchestrate hepatic specification and expansion. In the mouse, the septum transversum and cardiac mesoderm, through secreted bone morphogenetic proteins (BMP) and fibroblast growth factors (FGF), respectively, instruct the adjacent ventral endoderm to become hepatic endoderm. Consecutively, endothelial cells promote expansion of the specified hepatic endoderm. By using a mouse reporter embryonic stem cell line, in which hCD4 and hCD25 were targeted to the Foxa2 and Foxa3 loci, we reconstituted an in vitro culture system in which committed endoderm cells coexpressing hCD4-Foxa2 and hCD25-Foxa3 were isolated and cocultured with endothelial cells in the presence of BMP4 and bFGF. In this culture setting, we provide mechanistic evidence that endothelial cells function not only to promote hepatic endoderm expansion but are also required at an earlier step for hepatic specification, at least in part through regulation of the Wnt and Notch pathways. Activation of Wnt and Notch by chemical or genetic approaches increases endoderm cell numbers but inhibits hepatic specification, and conversely, chemical inhibition of both pathways enhances hepatic specification and reduces proliferation. By using identical coculture conditions, we defined a similar dependence of endoderm harvested from embryos on endothelial cells to support their growth and hepatic specification. Our findings (1) confirm a conserved role of Wnt repression for mouse hepatic specification, (2) uncover a novel role for Notch repression in the hepatic fate decision, and (3) demonstrate that repression of Wnt and Notch signaling in hepatic endoderm is controlled by the endothelial cell niche.     

Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development.

Studies on nonmammalian vertebrate embryos have indicated that retinoic acid (RA) is required for pancreas development. We have analyzed mouse embryos carrying a null mutation of the gene encoding retinaldehyde dehydrogenase 2 (Raldh2), which controls RA synthesis. Raldh2-/- embryos specifically lack expression of Pdx1 (a homeobox gene required for pancreas development) and Prox1 in dorsal endodermal but not ventral endodermal pancreatic precursor tissues. Ventral endodermal expression of Hex is not affected in Raldh2-/- embryos, indicating that liver specification is not dependent upon RA. Also, expression of Foxa2 across the dorsoventral axis of the endoderm is not affected in Raldh2-/- embryos, indicating that a lack of RA does not cause a general defect in foregut endoderm development. Comparison of wild-type and Raldh2-/- embryos carrying an RA-reporter transgene demonstrates that RA activity is normally present throughout the endoderm except in the ventral-most region but is totally missing in endoderm of Raldh2-/- embryos. Thus, Raldh2 expressed in adjacent splanchnic lateral plate mesoderm provides an RA signal to dorsal endoderm. Dorsal Pdx1 expression is rescued in Raldh2-/- embryos by low-dose maternal administration of RA, which preferentially restores RA-reporter expression in the dorsal endoderm. Our findings demonstrate a specific role for RA in mouse embryos as a mesodermally synthesized signal needed for dorsal endodermal expression of Pdx1 during development of the dorsal pancreatic lineage.     

Development of the chick embryo endoderm studied by S.E.M

Summary The endoderm of a series of chick embryos from the unincubated egg to Hamburger and Hamilton stage 5 was examined by scanning electron microscopy (SEM). During this period the endoderm develops from a few scattered cells to a complete epithelial layer. Prior to the formation of the primitive streak endoderm cells can be observed delaminating from the ectoderm. These cells are round and have few processes except where they contact each other. At stage 2 cells appear in the endoderm over the primitive streak which have broad flat processes. This suggests that the cells originate directly from the streak. Away from the streak the endoderm cells are either smooth or have short microvilli. In later streak stages a mixture of smooth and some microvillous cells form a hexagonal pattern. This pattern is occasionally modified and holes are found in the endoderm with cell processes protruding from below the endoderm level. Sometimes whole cells, smaller and rounder than the majority of the endoderm cells are associated with this disturbance of the pattern. These cells are connected to the mesoderm by a long cytoplasmic process and it is suggested that they could be cells entering the endoderm from the middle layer, having accompanied the mesoderm cells through the primitive streak.     

Xenopus hairy2b specifies anterior prechordal mesoderm identity within Spemann's organizer.

Spemann's organizer is a region of the gastrula stage embryo that contains future anterior endodermal and dorsal mesodermal tissues. During gastrulation, the dorsal mesoderm is divided into the prechordal mesoderm and the chordamesoderm. However, little is known regarding how this division is established. We analyzed the role of the anterior prechordal mesoderm-specific gene Xhairy2b in the regionalization of the organizer. We found that mesoderm-inducing transforming growth factor-beta signaling induced Xhairy2b expression. On the other hand, the ectopic expression of Xhairy2b induced the expression of organizer-specific genes and resulted in the formation of a secondary dorsal axis lacking head and notochord structures. We also showed that Xhairy2b down-regulated the expression of ventral mesodermal, anterior endodermal, and chordamesodermal genes. In Xhairy2b-depleted embryos, defects in the specification of anterior prechordal mesoderm identity were observed as the border between the prechordal mesoderm and the chordamesoderm was anteriorly shifted. These results suggest that Xhairy2b establishes the identity of the anterior prechordal mesoderm within Spemann's organizer by inhibiting the formation of neighboring tissues.     

Sebox regulates mesoderm formation in early amphibian embryos

Background: Mix/Bix genes are important regulators of mesendoderm formation during vertebrate embryogenesis. Sebox, an additional member of this gene family, has been implicated in endoderm formation during early embryogenesis in zebrafish. However, it remains unclear whether Sebox plays a unique role in early Xenopus embryos. Results: In this study, we provide evidence that Sebox is uniquely required for the formation of mesoderm during early Xenopus embryogenesis. Sebox is dynamically expressed in the involuted mesoderm during gastrulation. It is activated by Nodal/Activin signaling and modulated by zygotic Wnt/β‐catenin signaling. Overexpression of Sebox perturbs movements during convergent extension and inhibits the expression of mesodermal, but not endodermal, genes induced by Nodal/Activin signaling. Depletion of Sebox using a specific morpholino increases the expression of noncanonical wnt5a, wnt5b, and wnt11b. Depletion of Sebox also up‐regulates the expression of pcdh8.2, a paraxial mesoderm‐specific protocadherin, in a Wnt11B‐dependent manner. Sebox morphants display reduced development of the head and notochord. Conclusions: Our findings illustrate that Sebox, a unique member of the Mix/Bix gene family, functions downstream of Nodal/Activin signaling and is required for the proper expression of noncanonical Wnt ligands and the normal development of mesoderm in Xenopus. Developmental Dynamics 244:1415–1426, 2015. © 2015 Wiley Periodicals, Inc.