Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of prestreak and early primitive streak stages of the mouse embryo was studied in vitro by microinjecting horseradish peroxidase into single axial endoderm cells of 6.7-day-old embryos and tracing the labelled descendants either through gastrulation (1 day of culture) or to early somite stages (2 days of culture). Descendants of endoderm cells from the anterior half of the axis were found at the extreme cranial end of the embryo after 1 day and in the visceral yolk sac endoderm after 2 days, i.e. they were displaced anteriorly and anterolaterally. Descendants of cells originating over and near the anterior end of the early primitive streak, i.e. posterior to the distal tip of the egg cylinder, were found after 1 day over the entire embryonic axis and after 2 days in the embryonic endoderm at the anterior intestinal portal, in the foregut, along the trunk and postnodally, as well as anteriorly and posteriorly in the visceral yolk sac. Endoderm covering the posterior half of the early primitive streak contributed to postnodal endoderm after 1 day (at the late streak stage) and mainly to posterior visceral yolk sac endoderm after 2 days. Clonal descendants of axial endoderm were located after 2 days either over the embryo or in the yolk sac; the few exceptions spanned the caudal end of the embryo and the posterior yolk sac. The clonal analysis also showed that the endoderm layer along the posterior half of the axis of prestreak- and early-streak-stage embryos is heterogeneous in its germ layer fate. Whereas the germ layer location of descendants from anterior sites did not differ after 1 day from that expected from the initial controls (approx. 90% exclusively in endoderm), only 62% of the successfully injected posterior sites resulted in labelled cells exclusively in endoderm; the remainder contributed partially or entirely to ectoderm and mesoderm. This loss from the endoderm layer was compensated by posterior-derived cells that remained in endoderm having more surviving descendants (8.4 h population doubling time) than did anterior-derived cells (10.5 h population doubling time). There was no indication of cell death at the prestreak and early streak stages; at least 93% of the cells were proliferating and more than half of the total axial population were in, or had completed, a third cell cycle after 22 h culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Origin and differentiation of the yolk sac and extraembryonic mesoderm in presomite human and rhesus monkey embryos.

Reexamination of presomite human and rhesus monkey embryos in the Carnegie Collection provides no evidence to corroborate the hypothesis that the trophoblast is the source of all extraembryonic tissues in these embryos. Instead, the present study indicates that the developmental pattern of the yolk sac and extraembryonic mesoderm is homologous to that in other eutharian mammals. The primary yolk sac of 10- to 11-day human blastocysts is partially filled with a meshwork of extraembryonic endoderm, whereas such a meshwork is absent in the rhesus monkey. It is suggested that this endodermal meshwork develops as the result of interstitial implantation in the human embryo. A small secondary yolk sac develops in 12- to 13-day human and macaque embryos as the result of pinching off of a portion of the larger primary yolk sac. Development of a secondary yolk sac in higher primates appears to be related causally to differential rates of expansion of the blastocyst and primary yolk sac within the simplex uterus. The caudal margin of the primitive streak develops precociously in 12- to 14-day human and macaque embryos, and this appears to be the source of all the extraembryonic mesoderm of the chorion, chorionic villi, and body stalk. It is suggested that the peripheral spread of extraembryonic mesoderm plays in inductive role in the development of chorionic villi, similar to other types of epithelial-mesenchymal inductive interactions. In contrast to previous hypotheses, the human and macaque trophoblasts appear to give rise only to additional trophoblast.     

The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo.

The prospective fate of cells in the primitive streak was examined at early, mid and late stages of mouse gastrula development to determine the order of allocation of primitive streak cells to the mesoderm of the extraembryonic membranes and to the fetal tissues. At the early-streak stage, primitive streak cells contribute predominantly to tissues of the extraembryonic mesoderm as previously found. However, a surprising observation is that the erythropoietic precursors of the yolk sac emerge earlier than the bulk of the vitelline endothelium, which is formed continuously throughout gastrula development. This may suggest that the erythropoietic and the endothelial cell lineages may arise independently of one another. Furthermore, the extraembryonic mesoderm that is localized to the anterior and chorionic side of the yolk sac is recruited ahead of that destined for the posterior and amnionic side. For the mesodermal derivatives in the embryo, those destined for the rostral structures such as heart and forebrain mesoderm ingress through the primitive streak early during a narrow window of development. They are then followed by those for the rest of the cranial mesoderm and lastly the paraxial and lateral mesoderm of the trunk. Results of this study, which represent snapshots of the types of precursor cells in the primitive streak, have provided a better delineation of the timing of allocation of the various mesodermal lineages to specific compartments in the extraembryonic membranes and different locations in the embryonic anteroposterior axis.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Differential gene expression during early murine yolk sac development

The visceral yolk sac (VYS), composed of extraembryonic mesoderm and visceral endoderm, is the initial site of blood cell development and serves important nutritive and absorptive functions. In the mouse, the visceral endoderm becomes a morphologically distinct tissue at the time of implantation (E4.5), while the extraembryonic mesoderm arises during gastrulation (E6.5–8.5). To isolate genes differentially expressed in the developing yolk sac, polymerase chain reaction (PCR) methods were used to construct cDNA from late primitive streak to neural plate stage (E7.5) murine VYS mesoderm and VYS endoderm tissues. Differential screening led to the identification of six VYS mesoderm-enriched clones: ribosomal protein L13a, the heat shock proteins hsc 70 and hsp 86, guanine-nucleotide binding protein-related gene, cellular nucleic acid binding protein, and ã-enolase. One VYS endoderm-specific cDNA was identified as apolipoprotein C2. In situ hybridization studies confirmed the differential expression of these genes in E7.5 yolk sac tissues. These results indicate that representative cDNA populations can be obtained from small numbers of cells and that PCR methodologies permit the study of gene expression during early mammalian postimplantation development. While all of the mesoderm-enriched genes were ubiquitously expressed in the embryo proper, apolipoprotein C2 expression was confined to the visceral endoderm. These results are consistent with the hypothesis that at E7.5, the yolk sac endoderm provides differentiated liver-like functions, while the newly developing extraembryonic mesoderm is still a largely undifferentiated tissue. © 1995 wiley-Liss, Inc.     

Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of midstreak to neural plate stages of the mouse embryo was studied by microinjecting horseradish peroxidase (HRP) into single axial endoderm cells in situ, and tracing the labeled descendants to early somite stages in vitro. Axial endoderm cells along the anterior fifth of the late streak/neural plate stage embryo contributed descendants either to the yolk sac endoderm or to the anterior intestinal portal. Cells of the exposed head process contributed to the trunk endoderm and notochord; neighboring endoderm cells contributed to the dorsal foregut. Contributions to the ventral foregut came from endoderm at, and anterior to, the distal tip of the younger, midstreak embryo (in which the head process was not yet exposed). Endoderm over the primitive streak contributed to the postsomite endoderm. We argue from these results and those in the literature that during gastrulation the axial embryonic endoderm is of mixed lineage: (1) an anterior population of cells is derived from primitive endoderm and contributes to the yolk sac endoderm; (2) a population at, and anterior to, the distal tip of the midstreak embryo, extending more anteriorly at late streak/neural plate stages, is presumed to emerge from primitive ectoderm at the beginning of gastrulation and contributes to the foregut and anterior intestinal portal; (3) the axial portion of the head process that begins to incorporate into the ventral surface at the late streak stage contributes to notochord and trunk endoderm. Cells or their descendants that were destined to die within 24 hr were evident at the midstreak stage. There was a linear trend in the incidence of cell death among labeled cells at the late streak/neural plate stages, ranging from 27% caudal to the node to 57% in the anterior fifth of the embryo. The surviving axial endoderm cells divided sufficiently fast to double the population in 24 hr.     

Scanning Electron Microscopy (SEM) of the Chick Embryo Primitive Streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2–5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the primitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arranged cells with long, thin processes.
Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.     

Placentation in the lizard Gerrhonotus coeruleus with a comparison to the extraembryonic membranes of the oviparous Gerrhonotus multicarinatus (Sauria,Anguidae)

Paraffin sections of an ontogenetic series of embryos of the viviparous lizard Gerrhonotus coeruleus and the oviparous congener G. multicarinatus reveal that although general features of the development of the chorioallantoic and yolk sac membranes are similar, differences are evident in the distribution of the chorioallantoic membrane in late stage embryos. An acellular shell membrane surrounds the egg throughout gestation in both species although the thickness of this structure is much reduced in G. coeruleus over that of G. multicarinatus. The initial vascular membrane to contact the shell membrane in both species is a trilaminar omphalopleure (choriovitelline membrane) composed of ectoderm, mesoderm of the area vasculosa, and endoderm. This transitory membrane is replaced by the vascularized chorioallantois as the allantois expands to contact the inner surface of the chorion. Prior to the establishment of the chorioallantois at the embryonic pole, a membrane begins to form within the yolk ventral to the sinus terminalis. This membrane, which becomes vascularized, extends across the entire width of the abembryonic region and isolates a mass of yolk ventral to the yolk mass proper. The outer membrane of the yolk pole is a nonvascular bilaminar omphalopleure (chorionic ectoderm and yolk endoderm). In G. multicarinatus the bilaminar omphalopleure is supported internally by the vascularized allantoic membrane, whereas in G. coeruleus the allantois does not extend beyond the margin of the isolated yolk mass and the bilaminar omphalopleure is supported by the vascularized intravitelline membrane. Both the chorioallantoic placenta (uterine epithelium, chorionic ectoderm and mesoderm, and allantoic mesoderm and endoderm) and the yolk sac placenta at the abembryonic pole (uterine epithelium, chorionic ectoderm, and yolk sac endoderm) persist to the end of gestation in G. coeruleus.     

Developmentally regulated expression of the cell-cell adhesion glycoprotein cell-CAM 120/80 in peri-implantation mouse embryos and extraembryonic membranes

Peri-implantation mouse embryos and extraembryonic membranes were examined immunohistochemically for the expression of the cell-cell adhesion molecule (cell-CAM) 120/80. Cell-CAM 120/80 was seen along the lateral borders of all cells in the blastocyst but became undetectable on trophoblastic giant cells, some mononuclear trophoblastic cells and parietal yolk sac cells when blastocysts were cultured in vitro. In postimplantation embryos in vivo, all parts of the early egg-cylinder reacted with the antibody to cell-CAM 120/80 except for the cells of the parietal endoderm and the primary trophoblastic giant cells. In the late stage egg-cylinder, no cell-CAM 120/80 was seen on the cells of the primitive mesoderm or on the primordial germ cells. The germ cells in genital ridges and fetal gonads remained cell-CAM 120/80-negative throughout the fetal stages of development. In the extraembryonic membranes, the visceral yolk sac, amnion, and the cells of the placental labyrinth were cell-CAM 120/80-positive, whereas, the parietal yolk sac cells and the spongiotrophoblast cells were negative. These data show that cell-CAM 120/80 is found on cells arranged into epithelial layers in the early embryo and extraembryonic tissues, but is not expressed in the dissociated cells differentiating from these epithelia. Thus, the expression of cell-CAM 120/80 appears to be developmentally regulated.     

Visceral Endoderm Expression of Yin-Yang1 (YY1) Is Required for VEGFA Maintenance and Yolk Sac Development

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.     

Endodermal origin of yolk-sac-derived teratomas

Mutant mice deficient in glucose-6-phosphate dehydrogenase were used to induce teratomas. This enzyme is linked to the X chromosome, which can be inactivated in female embryo. The differences in the enzyme activity between yolk sac mesoderm and embryo versus yolk sac endoderm can be detected in female concepti by using appropriate crosses of wild-type and G6PD-deficient mice. Histochemical study showed that the dual cell population was observed in heterozygous embryos and in the embryomas derived from them. The teratomas derived from the corresponding yolk sac, however, were G6PD-positive from wild-type and G6PD-negative from homozygous enzyme-deficient mothers. We conclude that yolk-sac-derived teratomas are of endodermal origin because of the fact that the paternal X chromosome is inactivated in the yolk sac endoderm, whereas in the yolk sac mesoderm, as in the embryo, the inactivation is at random.     

Scanning electron microscopic observations on the 9-day opossum (Didelphis virginiana) embryo

All three germ layers are present in the opossum embryo by the 9th prenatal day. The embryo proper is part of, and continuous with, the remainder of the chorionic wall. The wall of the yolk sac-chorion away from the embryo consists only of an outer covering of ectoderm and an inner layer of endoderm. Ectodermal cells covering the neural folds have dome-shaped apices and often show large, bleb-like expansions. Microvilli are short and few in number. The apical surfaces of ectodermal cells that overlie the parietal mesoderm are relatively smooth and show scattered, short microvilli that tend to be concentrated at cell junctions. The apices of ectodermal cells that cover the extraembryonic region are more rounded, and the cells balloon from the surface. Each cell shows abundant elongate microvilli and occasional cytoplasmic blebs. Endodermal cells that line the chorion and form the third (innermost) layer of the embryo are similar in their surface morphology.     

Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

Ultrastructure of the pre-implantation shark yolk sac placenta

During ontogeny, the yolk sac of viviparous sharks differentiates into a yolk sac placenta which functions in gas exchange and hematrophic nutrient transport. The pre-implantation yolk sac functions in respiration and yolk absorption. In a 10.0 cm embryo, the yolk sac consists of six layers, viz. (1) somatic ectoderm; (2) somatic mesoderm; (3) extraembryonic coelom; (4) capillaries; (5) endoderm; and (6) yolk syncytium. The epithelial ectoderm is a simple cuboidal epithelium possessing the normal complement of cytoplasmic organelles. The endoplasmic cisternae are dilated and vesicular. The epithelium rests upon a basal lamina below which is a collagenous stroma that contains dense bodies of varying diameter. They have a dense marginal zone, a less dense core, and a dense center. The squamous mesoderm has many pinocytotic caveolae. The capillary endothelium is adjacent to the mesoderm and is delimited by a basal lamina. The endoderm contains yolk degradation vesicles whose contents range from pale to dense. The yolk syncytium contains many morphologically diverse yolk granules in all phases of degradation. Concentric membrane lamellae form around yolk bodies as the main yolk granules begin to be degraded. During degradation, yolk platelets exhibit a vesicular configuration.     

Pathological axes of wound repair: Gastrulation revisited

Post-traumatic inflammation is formed by molecular and cellular complex mechanisms whose final goal seems to be injured tissue regeneration. In the skin -an exterior organ of the body- mechanical or thermal injury induces the expression of different inflammatory phenotypes that resemble similar phenotypes expressed during embryo development. Particularly, molecular and cellular mechanisms involved in gastrulation return. This is a developmental phase that delineates the three embryonic germ layers: ectoderm, endoderm and mesoderm. Consequently, in the post-natal wounded skin, primitive functions related with the embryonic mesoderm, i.e. amniotic and yolk sac-derived, are expressed. Neurogenesis and hematogenesis stand out among the primitive function mechanisms involved. Interestingly, in these phases of the inflammatory response, whose molecular and cellular mechanisms are considered as traces of the early phases of the embryonic development, the mast cell, a cell that is supposedly inflammatory, plays a key role. The correlation that can be established between the embryonic and the inflammatory events suggests that the results obtained from the research regarding both great fields of knowledge must be interchangeable to obtain the maximum advantage.     

Differentiation of the embryonic disc, amnion, and yolk sac in the rhesus monkey

The inner cell mass of the blastocyst has differentiated into epiblast and hypoblast (primitive endoderm) prior to implantation. Since endoderm cells extend beyond the epiblast, it can be considered that both parietal and visceral endoderm are present. At implantation, epiblast cells begin to show marked evidence of polarity. They form a spherical aggregate with their basal ends toward the basal lamina and apical ends toward the interior. The potential for an internal space is formed by this change in polarity of the cells. No cytological evidence of separation of those cells that will form amniotic epithelium from the rest of the epiblast is seen until a cavity begins to form. The amniotic epithelium is originally contiguous with overlying cytotrophoblast, and a diverticulum remains in this position during early development. Epiblast forms a pseudostratified columnar epithelium, but dividing cells are situated toward the amniotic cavity rather than basally. The first evidence of a trilaminar disc occurs when a strand of cells contiguous with epiblast is found extending toward visceral endoderm. These presumptive mesoderm cells are undifferentiated, whereas extraembryonic mesoderm cells are already a distinct population forming extracellular materials. After implantation, visceral endoderm cells proliferate forming an irregular layer one to three cells thick. Visceral endoderm cells have smooth apical surfaces, but very irregular basal surfaces, and no basal lamina. At the margins of the disc, visceral endoderm is continuous with parietal endoderm and reflects back over the apices of the marginal visceral endoderm cells. This sacculation by visceral endoderm cells precedes pinching off of the secondary yolk sac from the remaining primary yolk sac.