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.     

Is chemotaxis a factor in the migration of precardiac mesoderm in the chick?

Summary The chick heart is formed from bilateral patches of presumptive cardiac mesoderm cells which migrate over the endoderm and fuse in the midline. We have tested the possibility that this migration is controlled, at least in part, by a chemotactic substance exuded by the anterior end of the endoderm. We have used chick/quail combinations to follow naturally marked cells during the course of their migration. Chimaeric embryos were formed by fusing together parts of chick and quail embryos of stage 5–6. Each embryo possessed two pairs of precardiac regions, the quail pair lying immediately anterior to that of the chick. These chimaeras were then explanted in embryo culture. In the event of chemotaxis, cells from the posterior end of the quail precardiac mesoderm might be expected to invade the chick area. Samples of explants and chimaeras were examined at intervals from 2 to 24 h, but in no case were cells found to have changed their direction of migration as a result of the proximity of anterior endoderm. It is concluded that this work does not provide evidence for a chemotactic attraction by the anterior end of the endoderm. Supported by the following grants: NIH HD 21048, HD 06819, and AHA 880696 (JWL); the British Heart Foundation, and Action Research (R.B.); and an SERC postgraduate studentship (HSE).     

Conserved and divergent expression patterns of markers of axial development in eutherian mammals

Background : Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior–posterior (A‐P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A‐P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. Results : All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2‐positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp‐positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A‐P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. Conclusions : Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents. Developmental Dynamics 245:67–86, 2016. © 2015 Wiley Periodicals, Inc.     

Early mesoderm differentiation in the chick embryo

Summary Two groups of experiments were carried out. In the first group, grafts of quail mesoderm whose presumptive fate was to form somites or heart tissues, were taken from quail embryos (stage 4–5 of Hamburger and Hamilton 1951) and inserted beneath the ectoderm of chick embryos of stage 3–4 immediately lateral to the primitive streak. Whilst most grafts contributed to the somites and/or the heart, 22 out of a total of 46 were found to have contributed also to the pharyngeal endoderm. Although three of these grafts were known to have included some quail endoderm cells, the remainder were considered to consist of mesoderm alone. It is concluded that mesoderm at the primitive streak stages is still capable of forming endoderm.In the second group of experiments, grafts of quail somites (stage 10–14) were inserted beneath the ectoderm of chick embryos of stage 3–4. In 18 out of 23 cases the graft cells were found in somitic tissue, but they were also found in the endoderm (4 specimens), lateral plate (3 specimens) and endothelium (4 specimens). It is concluded that even at stages 10–14, the somite-derived cells are still not completely determined to form somite derivatives. In those cases where the grafted somites differentiated further, sclerotome cells which migrated from them did not necessarily move towards the host notochord.     

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.     

Lineage and fate of each blastomere of the eight-cell sea urchin embryo

A fluoresceinated lineage tracer was injected into individual blastomeres of eight-cell sea urchin (Strongylocentrotus purpuratus) embryos, and the location of the progeny of each blastomere was determined in the fully developed pluteus. Each blastomere gives rise to a unique portion of the advanced embryo. We confirm many of the classical assignments of cell fate along the animal-vegetal axis of the cleavage-stage embryo, and demonstrate that one blastomere of the animal quartet at the eight-cell stage lies nearest the future oral pole and the opposite one nearest the future aboral pole of the embryo. Clones of cells deriving from ectodermal founder cells always remain contiguous, while clones of cells descendant from the vegetal plate (i.e., gut, secondary mesenchyme) do not. The locations of ectodermal clones contributed by specific blastomeres require that the larval plane of bilateral symmetry lie approximately equidistant (i.e., at a 45 degree angle) from each of the first two cleavage planes. These results underscore the conclusion that many of the early spatial patterns of differential gene expression observed at the molecular level are specified in a clonal manner early in embryonic sea urchin development, and are each confined to cell lineages established during cleavage.     

Development of mouse embryos in hanging drop culture

Mouse blastocysts were cultured in hanging drops for up to 6 days in order to study development under conditions that avoid the distortion of embryos typically seen when they are allowed to attach to a glass or plastic surface. The survival rate of embryos in hanging drops was equal to that of embryos attached to culture dishes and superior to that of embryos suspended in gyrating flasks. Development of the embryonic portion was similar to that in vivo and on culture dishes but slower than in vivo; the egg cylinder stage was reached after 8-10 equivalent gestation days (4 to 6 days in culture), while that stage is reached at 5.5 to 6 days in vivo. The trophectoderm, however, developed in a unique manner. The cells migrated away from the inner cell mass (ICM), similar to embryos on a culture dish, but without a surface on which to spread they clustered distal to the ICM. In vivo, trophectoderm remained covering the ICM. By 5 days in hanging drop culture the embryos had developed a segmented appearance with trophoblast giant cells at the abembryonic pole, extraembryonic cells not covered by vacuolated endoderm in the central region, and embryonic endoderm surrounding a developing proamniotic cavity in embryonic ectoderm at the embryonic pole. These observations suggest that the trophectoderm is able to follow a developmental program independent of that in the embryonic portion and that its behavior is dominated by the different adhesive properties of the trophoblastic and embryonic cells.     

The rostro-caudal position of cardiac myocytes affect their fate.

During chick embryogenesis, cells destined to form cardiac myocytes are located within the primitive streak at stage 3 in the same relative anterior-posterior distribution as in the prelooped heart. The most rostral cells contribute to the extreme anterior pole of the heart, the bulbus cordis, and the most caudal to the extreme posterior end, the sinoatrial region. After gastrulation, these cells commit to the myocyte lineage and, retaining their relative positions, migrate to the anterior lateral plate. From stages 5 to 10 they diversify into atrial and ventricular myocytes, with the former located posteriorly and the latter, anteriorly. To determine the effect of a change in the rostro-caudal position of these cells on their diversification, anterior lateral plate mesoderm and the underlying endoderm were cut and rotated 180 degrees along the longitudinal axis, at stages 4-8. The subsequent diversification of these precursor cells into atrial and ventricular myocytes was examined using lineage-specific markers. Our results showed that altering location along the longitudinal axis through stage 6 changed the normal fate of a precursor cell. The orientation of the overlying ectoderm did not alter normal morphogenesis or determination of fate.     

The tyrosine hydroxylase gene is expressed in endoderm and pancreas of early quail embryos

The initial expression of the gene encoding tyrosine hydroxylase (TH) was studied in the trunk of quail embryos by in situ hybridization. We detected the presence of quail TH mRNA on embryonic day 3.5 (E3.5) in the sympathetic ganglia and aortic plexus, both neural crest derived structures. In contrast, the TH gene was expressed much earlier in the endodermal layer of E2 embryos, i. e. from the 8-somite stage onwards. TH mRNA was found also in the pancreatic bud, an endoderm-derived structure. The TH protein and catecholamines were subsequently looked for in these structures. TH immunoreactivity was found in cells of E2 explanted endoderm, but no catecholamine histofluorescence was observed before or after a few days in culture. TH-positive cells were also detected in cultures of pancreatic rudiments, explanted from E3 to E6 quail embryos. We suggest that the TH-positive cells of the endoderm are the progenitors of the catecholaminergic cells of the pancreas and of the enterochromaffin cells of the gut. The hypothesis that the TH-positive cells of the endoderm are involved in the expression of the catecholaminergic phenotype by neural crest cells is discussed.     

Fate diversity of primitive streak cells during heart field formation in ovo.

In amniote embryos, cells from a rostral portion of the primitive streak migrate anterolaterally and establish the heart field mesoderm, from which two cardiac cell lineages, cardiomyocytes and endocardial endothelial cells, differentiate. The endoderm underlying the heart field has been postulated as the source of several paracrine factors that may serve to induce each of these cell types. However, it has been unclear how these signal molecules, which are expressed broadly in the endoderm, instruct individual cells of the heart field mesoderm to enter either the cardiomyocyte lineage or the endocardial cell lineage. To clarify lineage relationships of these two cardiac cell types, the fate of chicken primitive streak cells was traced for the first time in ovo. By using replication-defective retroviral-mediated gene transfer, we demonstrate that cells in the rostral half of Hamburger and Hamilton (HH) stage 3 primitive streak generate a daughter population that proliferates and migrates into the heart field, differentiating into either endocardial or myocardial cells, but not both cell types. The results suggest that the rostral portion of the primitive streak at HH stage 3 consists of at least two distinct subpopulations, entering either the cardiomyocyte lineage or the endocardial cell lineage. Thus, in ovo these two cell lineages of the heart are already segregated within the primitive streak, significantly before their migration to the heart field. When the precardiomyocytes and pre-endocardial cells arrive at the heart field, each mesodermal cell subpopulation may be permissive to paracrine signal(s) from underlying endoderm to initiate their terminal differentiation into either muscle or endothelial cells.     

Ability of neural crest cells from the embryonic chick to differentiate into cartilage before their migration away from the neural tube.

Whether neural crest cells from the avian embryo are determined for chondrogenesis before they begin their migration away from the neural tube (i.e., before H. H. stages 8.5--9) was investigated by establishing neural folds from embryos of H. H. stages 5--11 either in organ culture, or as grafts to the chorioallantoic membranes of host embryos. Cartilage differentiated from neural folds taken from embryos of H. H. stages 5--7 but not from those taken from older embryos. This stage specific pattern was reversed when the tissue adjacent to the neural tube was grafted to the chorioallantoic membrane. Cartilage only formed from tissues isolated later than H. H. stage 8; i.e., when these adjacent tissues contain neural crest cells. We concluded that neural crest cells are determined for chondrogenesis while still in the neural tube and before their migration to the face and head. This is in contrast to the situation in the only other group which has been examined, the urodele amphibians.     

Ability of neural crest cells from the embryonic chick to differentiate into cartilage before their migration away from the neural tube

Whether neural crest cells from the avian embryo are determined for chondrogenesis before they begin their migration away from the neural tube (i.e., before H. H. stages 8.5-9) was investigated by establishing neural folds from embryos of H. H. stages 5-11 either in organ culture, or as grafts to the chorioallantoic membranes of host embryos. Cartilage differentiated from neural folds taken from embryos of H. H. stages 5-7 but not from those taken from older embryos. This stage specific pattern was reversed when the tissue adjacent to the neural tube was grafted to the chorioallantoic membrane. Cartilage only formed from tissues isolated later than H. H. stage 8; i.e., when these adjacent tissues contain neural crest cells. We concluded that neural crest cells are determined for chondrogenesis while still in the neural tube and before their migration to the face and head. This is in contrast to the situation in the only other group which has been examined, the urodele amphibians.     

Origin and early migration of primordial germ cells in the chick embryo: A study of the stages definitive primitive streak through eight somites

The germinal crescent in the chick embryo is characterized by small, PAS-positive, nonglycogen granules from 1.5 to 5 μ in diameter. The primordial germ cells (PGCs) were found to originate in and separate from the germinal crescent endoderm through stage 7 (2 somites). Shortly after separation most of the granules in the PGCs lost their organization and the PAS-positive material was distributed irregularly throughout the cytoplasm. A few of these granules remained within the cells indefinitely. Glycogen of an agranular nature which had shifted to one pole of the cell was observed at stage four. Granular glycogen which was distributed throughout the cytoplasm was not observed prior to stage 7 or 8. Cell counts on individual embryos showed noticeable variations as to the number of germ cells between embryos of the same stage. For example, in stage 4 embryos the minimum number of cells counted, including attached and free, was 78 and the maimum 169, while in stage 9 the minimum was 83 and the maximum 469 cells. After separation the germ cells were observed almost anywhere between the ectoderm and the endoderm although the majority remained in the area where they originated.     

The DVE changes distal epiblast fate from definitive endoderm to neurectoderm by antagonizing nodal signaling.

To assess the function of the distal visceral endoderm (DVE) of embryonic day 5.5 (E5.5) embryos, we established a system to directly ablate the DVE and observe the consequences after culture. When the DVE was successfully ablated, such embryos (DVE-ablated embryos) showed deregulated expression of Nodal and Wnt3 and ectopically formed the primitive streak at the proximal portion of the embryo. The DVE and anterior visceral endoderm (AVE) are implicated in the development of neurectoderm. We found that the distal epiblast of E5.5 embryo rotates anteriorly by the beginning of gastrulation. These cells remained to be anteriorly located during gastrulation and contributed to the ectoderm in the anterior side of the embryo. This indicates that the distal epiblast of E5.5 embryo becomes neurectoderm in normal development. In DVE-ablated embryos, the distal epiblast did not show any movement during culture and was abnormally fated to early definitive endoderm lineage. The data suggest that down-regulation of Nodal signaling in the distal epiblast of E5.5 embryo may be an initial step of neural development.     

Can gastric endoderm change the regionally specific inducing ability of presumptive small intestinal mesoderm?

This study was designed to establish the source of gut mesoderm's ability to induce regional pattern in the endoderm. The most obvious possibility is induction by the endoderm through epithelial-mesenchymal interaction. To test this experimentally, reciprocal quail/chick combinations were prepared of early proventricular endoderm (that is already known to be regionally determined) and presumptive small intestinal mesoderm. The combinations were cultured for 7 days to allow for 'programming' of the mesoderm by the endoderm. After removal of the proventricular endoderm the mesoderm was combined with young gizzard endoderm. It is known that gizzard endoderm can be provoked to develop in either a proventricular or a small intestinal direction by association with the appropriate mesoderm. Thus, by combining intestinal mesoderm 'programmed' by association with proventricular endoderm with gizzard endoderm, the subsequent differentiation of the gizzard endoderm would indicate whether or not the inducing ability of the intestinal mesenchyme had been altered. In addition to such experimental grafts, three types of control graft were prepared. The results of the experiment, based on the morphology of the grafts and the immunocytochemical analysis of selected endocrine cell types, showed that in the majority of cases the gizzard endoderm developed the features of small intestine, not those of proventriculus. This indicates that at the stages studied, endoderm does not act to program mesoderm with which it is associated. If this does occur, it must take place at an earlier stage, i.e., before the time of explantation of the presumptive small intestinal mesoderm (1.25 days of incubation).     

Notch signaling can regulate endoderm formation in zebrafish.

Early in vertebrate development, the processes of gastrulation lead to the formation of the three germ layers: ectoderm, mesoderm, and endoderm. The mechanisms leading to the segregation of the endoderm and mesoderm are not well understood. In mid-blastula stage zebrafish embryos, single marginal cells can give rise to both endoderm and mesoderm (reviewed by Warga and Stainier [2002] The guts of endoderm formation. In: Solnica-Krezel L, editor. Pattern formation in zebrafish. Berlin: Springer-Verlag. p 28-47). By the late blastula stage, however, single marginal cells generally give rise to either endoderm or mesoderm. To investigate this segregation of the blastoderm into cells with either endodermal or mesodermal fates, we analyzed the role of Notch signaling in this process. We show that deltaC, deltaD, and notch1 are expressed in the marginal domain of blastula stage embryos and that this expression is dependent on Nodal signaling. Activation of Notch signaling from an early stage leads to a reduction of endodermal cells, as assessed by sox17 and foxA2 expression. We further find that this reduction in endoderm formation by the activation of Notch signaling is preceded by a reduction in the expression of bonnie and clyde (bon) and faust/gata5, two genes necessary for endoderm formation (Reiter et al. [1999] Genes Dev 13:2983-2995; Reiter et al. [2001] Development 128:125-135; Kikuchi et al. [2001] Genes Dev 14:1279-1289). However, activation of Notch signaling in bon mutant embryos leads to a further reduction in endodermal cells, also arguing for a bon-independent role for Notch signaling in endoderm formation. Altogether, these results suggest that Notch signaling plays a role in the formation of the endoderm, possibly in its segregation from the mesoderm.     

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.     

Localization of Mg++-dependent adenosine triphosphatase and alkaline phosphatase activities in the postimplantation mouse embryos in day 5 and 6

Summary Mg⁺⁺-dependent adenosine triphosphatase (Mg-ATPase) and alkaline phosphatase (ALPase) activities were histo- and cytochemically investigated in postimplantation mouse embryos from day 5 to day 6. In day 5 postimplantation embryos, Mg-ATPase activity was detected in the embryonic ectoderm and weakly in the visceral endoderm. Weak ALPase activity was found in the embryonic ectoderm and visceral endoderm. Parietal endoderm, both in day 5 and in day 6 embryos, had very weak or no Mg-ATPase and ALPase activities. Mg-ATPase activity in day 6 embryos was found with the same localization as that in day 5 embryos. No ALPase activity was observed in their embryonic ectoderm. Extraembryonic ectodermal cell mass had the strongest Mg-ATPase activity in these stage embryos.These results suggest that the localization of both enzyme activities in postimplantation mouse embryos is closely related to the morphogenesis. As regards the proamniotic cavity formation, the fact that Mg-ATPase activity was still observed in the embryonic ectoderm in these stages suggests the involvement of active transport system on the production of nascent proamniotic cavity fluid.