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.     

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.     

Determination of anterior endoderm in Xenopus embryos.

Grafts of anterior endoderm from embryos at stage 28 or later developed according to their fate (i.e., anterior) when transplanted posteriorly. Conversely, grafts from earlier embryos developed according to their new location (i.e., posterior). However, endoderm grafted along with its mesodermal and ectodermal sheath retained its fate regardless of the stage of the donor. We conclude that anterior endoderm in Xenopus embryos is determined at about stage 25 under the influence of mesoderm.     

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.     

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.     

Endoderm/mesoderm multiplication rates in stage 5–12 chick embryos

Multiplication rates for the endoderm/mesoderm layer of the head-process to 17-somite-stage chick embryo were studied by implanting essentially identical transplants labeled with tritiated thymidine into paired recipient embryos. One recipient was fixed as soon as the transplant had healed (after 30 min) and the other was reincubated an additional 3.5 to 22.5 hr; the ratios of labeled cells in the paired embryos provided points on a graph that indicated that doubling of endoderm/mesoderm cells in head-process-stage chick embryos occurs at approximately 4.0 and 17.2 hr of reincubation.     

The effect of cytochalasin B on chick mesoderm cells as studied by scanning electron microscopy

Summary The effects of cytochalasin B (CCB) on chick mesoderm cells in vivo was examined by scanning electron microscopy (SEM). The embryos were mounted for New Culture and the mesoderm exposed by dissecting off the endoderm. Cytochalasin B was suspended in saline and the embryos flooded with the suspension. Control embryos were treated with saline alone. The embryos were reincubated for varying times at 37°C.In the treated embryos the mesoderm cells were rounded and separated from each other. Many had long branched processes and rough surfaces. These changes became more pronounced as treatment time was increased. They were also reversible on reincubating treated embryos in the absence of cytochalasin B. The morphological changes produced by CCB are thought to be due to an effect on the cytoskeleton, either a direct disruptive effect or detachment of skeletal microfilaments from the cell membrane. There may also be a direct removal of cell surface materials leading to the observed surface roughening of treated cells.     

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.     

Edge cell migration in the extraembryonic mesoderm of the chick embryo

Summary The expansion of the extraembryonic mesoderm was investigated in chick embryos of 2 and 3 days incubation with special regard to the mesodermal edge cells. These cells are lying immediately distal to the sinus terminalis and have the shape of migrating cells. By SEM examination they appear to be linked together to form a uniform edge which extends numerous spike-like filopodia. The shape of these filopodia corresponds to their microtubule pattern, as shown by immunofluorescence staining. Filopodia contain strong bundles of microtubules. By in vivo observation at high magnification, the migration of edge cells was demonstrated, and the results of SEM and immunofluorescence studies could be confirmed. By local application of cytochalasin D, distal to the region of the sinus terminalis, the migration of edge cells was inhibited selectively. Subsequent to the inhibition of migration, the expansion of the mesoderm stopped although the interstitial growth of the mesoderm in drug-treated regions remained unaffected. Thus the edge cells have a promotor function in the expansive growth of the extraembryonic mesoderm. The proliferating mesoderm, located proximally to the edge cells, has no expansive tendency of its own. The selectivity of the cytochalasin effect was checked by examination of the phalloidin stained actin pattern. Furthermore, by in vivo observations at low magnification and by transplantation of endoderm from quail to chick it could be confirmed that the extraembryonic mesoderm spreads out invasively between ectoderm and endoderm separating the two sheets. The promotion of this invasion can be regarded as an additional function of the edge cells. An expansion of the mesoderm can also be observed after endoderm removal. In regions freed from endoderm the mesoderm expands faster than in adjacent regions still covered by endoderm. There is no promoting influence of endoderm on mesodermal expansion. On the contrary, expansion itself is facilitated, when the conditions for invasion are abolished by removing the endoderm.     

Scanning electron microscopy of the development of the mesoderm layer in chick embryos

Summary The distribution of mesoderm, the structure of mesoderm cells and relationship between mesoderm and ectoderm were examined by SEM in embryos at stages 3 to 5. The mesoderm was displayed by removal of the endoderm and by fracturing the embryos through mesoderm containing regions. Within the mesoderm layer four zones could be distinguished by their cell shape and arrangement — the primitive streak, a multilayered compact area around the margins of the area pellucida, multilayered and loosely arranged cells near the primitive streak and a flattened monolayer of cells around the advancing lateral and anterior edges of the mesoderm sheet. Secretion of basement membrane by the ectoderm was seen to precede the arrival of mesoderm cells. This suggests that ectoderm alone can synthesize basement membrane without mesodermal contribution.     

Emergence of endothelial and hemopoietic cells in the avian embryo

Summary During organogenesis, endothelial cells develop through two different mechanisms: differentiation of intrinsic precursors in organ rudiments constituted of mesoderm associated with endoderm, and colonization by extrinsic precursors in organs constituted of mesoderm associated with ectoderm (Pardanaud et al. 1989). On the other hand, both types of rudiment are colonized by extrinsic hemopoietic stem cells. In the present work we extend our former study by investigating the hemangioblastic (i.e. hemopoietic and angioblastic) potentialities of primordial germ layers in the area pellucida during the morphogenetic period. By means of interspecific grafts between quail and chick embryos, we show that splanchnopleural mesoderm gives rise to abundant endothelial cells, and to numerous hemopoietic cells in a permissive microenvironment, while somatopleural mesoderm produces very few cells belonging to these lineages, or none. Thus we confirm that the angioblastic capacities of the mesoderm differ radically, depending on its association with ectoderm or endoderm. Furthermore, at this embryonic period, both endothelial and hemopoietic potentialities are displayed by splanchnopleural mesoderm. However the site of emergence of intraembryonic hemopoietic stem cells appears spatially restricted by comparison to more widespread angioblastic capacities.     

The establishment of a somitomeric pattern in the mesoderm of the gastrulating mouse embryo

Mesoderm formation in the mouse embryo begins at 6.5–6.75 days p.c. (postcoitum) when a primitive streak is formed along the posterior side of the egg cylinder. Epiblast cells in a localized region separate from one another and spread laterally between the primitive endoderm and the rest of the epiblast. The newly formed mesoderm contributes to both embryonic and extraembryonic regions. When the endoderm is removed, a definitive somitomeric pattern is first observed in the lateral wings of mesoderm of the mid-primitive-streak-stage embryo. The sequential appearance and the placement of somitomeres in the gastrulating mouse embryo are closely related to the general changes in physical dimensions and to the pattern of tissue growth which occur during the maturation of the egg cylinder. By the late-primitive-streak stage, about four somitomeres are present in the paraxial mesoderm on either side of the embryonic axis. These somitomeres will undergo morphogenesis and give rise to the cranial segments and head mesenchyme of neurulating embryos (Meier and Tam, 1982). The midline or axial mesoderm, consisting of prechordal plate and notochord, is derived from the head process mesoderm originating from the anterior end of the primitive streak. Cells of the head process are compact and adherent to the endoderm. The early presence of a somitomeric pattern which persists and is added to throughout subsequent phases of mesoderm formation suggests that spreading mesodermal cells have relatively stable neighbor relationships. This morphological evidence supports the idea that the expansion of the mesoderm during gastrulation results from tissue growth and progressive deposition of cells from the primitive streak. Cell migration may be limited principally to nonsomitomeric mesodermal cells found in the leading edge of the spreading lateral wings.     

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).     

The developmental fate of androgenetic, parthenogenetic, and gynogenetic cells in chimeric gastrulating mouse embryos

Both a maternal and a paternal genomic contribution are necessary for completion of embryonic development in the mouse. Parthenogenetic embryos, with only a maternally inherited genome, and androgenetic embryos, with only a paternally inherited genome, fail to develop to term, and these two types of isoparental embryos fail in development in characteristic ways. In this paper we describe the construction of chimeras between single androgenetic, parthenogenetic, and gynogenetic blastomeres and normal eight-cell embryos. We allow the development of the chimeras to reach the late-gastrulating-stage embryo and then analyze the tissue distributions of the isoparental component. The isoparental embryos are derived from a transgenic mouse line carrying plasmid and mouse beta-globin sequences. The isoparental cells are detected in histological sections of chimeras by DNA-DNA in situ hybridization to the transgene, using a biotinylated DNA probe with an enzymatic detection system. We found strong tissue preferences for the androgenetic, parthenogenetic, and gynogenetic cells in chimeras. Androgenetic cells contributed strongly to all trophectoderm-derived tissue, with only a rare contribution to any tissues of the embryo proper, extraembryonic mesoderm, or extraembryonic endoderm. Parthenogenetic cells shared a developmental fate similar to gynogenetic cells, contributing to all tissues of the embryo proper and to the extraembryonic mesoderm, but only rarely to the extraembryonic endoderm or to any trophectoderm-derived tissues.     

Germ layer differentiation during early hindgut and cloaca formation in rabbit and pig embryos

Relative to recent advances in understanding molecular requirements for endoderm differentiation, the dynamics of germ layer morphology and the topographical distribution of molecular factors involved in endoderm formation at the caudal pole of the embryonic disc are still poorly defined. To discover common principles of mammalian germ layer development, pig and rabbit embryos at late gastrulation and early neurulation stages were analysed as species with a human‐like embryonic disc morphology, using correlative light and electron microscopy. Close intercellular contact but no direct structural evidence of endoderm formation such as mesenchymal–epithelial transition between posterior primitive streak mesoderm and the emerging posterior endoderm were found. However, a two‐step process closely related to posterior germ layer differentiation emerged for the formation of the cloacal membrane: (i) a continuous mesoderm layer and numerous patches of electron‐dense flocculent extracellular matrix mark the prospective region of cloacal membrane formation; and (ii) mesoderm cells and all extracellular matrix including the basement membrane are lost locally and close intercellular contact between the endoderm and ectoderm is established. The latter process involves single cells at first and then gradually spreads to form a longitudinally oriented seam‐like cloacal membrane. These gradual changes were found from gastrulation to early somite stages in the pig, whereas they were found from early somite to mid‐somite stages in the rabbit; in both species cloacal membrane formation is complete prior to secondary neurulation. The results highlight the structural requirements for endoderm formation during development of the hindgut and suggest new mechanisms for the pathogenesis of common urogenital and anorectal malformations.     

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).     

Formation and differentiation of extraembryonic mesoderm in the rhesus monkey

Differentiation of extraembryonic mesoderm in the rhesus monkey was studied from the epithelial penetration stage of implantation (stage 4) through the first week of postimplantation development (to stage 6). It was found that the first cells that appeared between the primitive endoderm (hypoblast) and trophoblast were separated from the latter by a basal lamina but appeared to be either loosely attached to the endoderm or to have been detached from it. Cells in this intermediate position differentiated cytologically into mesenchymal cells, which, by stage 5, had a distinctive intraendoplasmic reticulum marker. This differentiation occurred prior to the time at which the primitive streak could be recognized. By the time the primitive streak was readily discernible (stage 6), the extraembryonic mesoderm had already produced substantial extracellular matrix. The sequence of differentiation was repeated, with a 1- to 2-day lag, in the secondary implantation site. No evidence of a contribution from cytotrophoblast or primitive streak to the extraembryonic mesoderm was found. It is concluded that the origin of the first extraembryonic mesoderm in the rhesus monkey is probably a two-step process, with formation of a reticulum from primitive endoderm followed by differentiation in situ into mesenchymal cells. The first blood vessels formed also differentiated in situ from the extraembryonic mesenchymal cells. Primitive capillaries were identifiable as early as the 13th day of pregnancy.