The Developing Chicken Yolk Sac Acquires Nutrient Transport Competence by an Orchestrated Differentiation Process of Its Endodermal Epithelial Cells

During chicken yolk sac (YS) growth, mesodermal cells in the area vasculosa follow the migrating endodermal epithelial cell (EEC) layer in the area vitellina. Ultimately, these cells form the vascularized YS that functions in nutrient transfer to the embryo. How and when EECs, with their apical aspect directly contacting the oocytic yolk, acquire the ability to take up yolk macromolecules during the vitellina-to-vasculosa transition has not been investigated. In addressing these questions, we found that with progressive vascularization, the expression level in EECs of the nutrient receptor triad, LRP2-cubilin-amnionless, changes significantly. The receptor complex, competent for uptake of yolk proteins, is produced by EECs in the area vasculosa but not in the area vitellina. Yolk components endocytosed by LRP2-cubilin-amnionless, preformed and newly formed lipid droplets, and yolk-derived very low density lipoprotein, shown to be efficiently endocytosed and lysosomally processed by EECs, probably provide substrates for resynthesis and secretion of nutrients, such as lipoproteins. In fact, as directly demonstrated by pulse-chase experiments, EECs in the vascularized, but not in the avascular, region efficiently produce and secrete lipoproteins containing apolipoprotein A-I (apoA-I), apoB, and/or apoA-V. In contrast, perilipin 2, a lipid droplet-stabilizing protein, is produced exclusively by the EECs of the area vitellina. These data suggest a differentiation process that orchestrates the vascularization of the developing YS with the induction of yolk uptake and lipoprotein secretion by EECs to ensure embryo nutrition.     

Primitive lymphohematopoietic precursor cell lines generated in culture from day 7 early-mid-primitive streak stage mouse embryo.

During mouse development, the first lymphohematopoietic precursor cells and myeloid or erythroid cell lineage-determined cells can be detected in the yolk sac at days 8-8.5 of gestation. The characteristics of the cells that give rise to these yolk sac primitive lymphohematopoietic cells and the molecular events controlling this process remain poorly defined. We show here that cell suspensions from day 7 early-mid-primitive streak stage embryo proper generated early immature PgP-1+ Joro 177+ Lin- hematopoietic cells and some Mac-1+ myeloid and TER 119+ erythroid cells after co-culture with the yolk sac-derived stromal cell line YS6 without addition of exogenous cytokines. Purified Lin- hematopoietic cells generated in these cultures did not express genes known to be transcribed at early stages of lymphoid, myeloid or erythroid cell differentiation and were able to give rise to T and B lymphocytes, myeloid cells and erythroid cells after appropriate further induction in vitro. Several cell lines were established in culture with a mixture of four cytokines from the PgP-1+ Joro 177+ Lin- cell population. The cell lines shared phenotypic and genotypic characteristics with the PgP-1+ Joro 177+ Lin- cell population generated in culture from day 7 embryo proper and they were able to reconstitute the lymphohematopoietic system of irradiated mice. Taken together these results support a model of lymphohematopoiesis in which cells from day 7 early-mid-primitive streak mouse embryo proper migrate and colonize the visceral yolk sac. There they generate primitive lymphohematopoietic precursor cells and the first erythroid and myeloid hematopoietic cells under the influence of yolk sac stromal cells like the YS6 cells described here.     

Insufficient VEGFA activity in yolk sac endoderm compromises haematopoietic and endothelial differentiation

Vascular endothelial growth factor A (VEGFA) plays a pivotal role in the first steps of endothelial and haematopoietic development in the yolk sac, as well as in the establishment of the cardiovascular system of the embryo. At the onset of gastrulation, VEGFA is primarily expressed in the yolk sac visceral endoderm and in the yolk sac mesothelium. We report the generation and analysis of a Vegf hypomorphic allele, Vegf(lo). Animals heterozygous for the targeted mutation are viable. Homozygous embryos, however, die at 9.0 dpc because of severe abnormalities in the yolk sac vasculature and deficiencies in the development of the dorsal aortae. We find that providing 'Vegf wild-type' visceral endoderm to the hypomorphic embryos restores normal blood and endothelial differentiation in the yolk sac, but does not rescue the phenotype in the embryo proper. In the opposite situation, however, when Vegf hypomorphic visceral endoderm is provided to a wild-type embryo, the 'Vegf wild-type' yolk sac mesoderm is not sufficient to support proper vessel formation and haematopoietic differentiation in this extra-embryonic membrane. These findings demonstrate that VEGFA expression in the visceral endoderm is absolutely required for the normal expansion and organisation of both the endothelial and haematopoietic lineages in the early sites of vessel and blood formation. However, normal VEGFA expression in the yolk sac mesoderm alone is not sufficient for supporting the proper development of the early vascular and haematopoietic system.     

The effects of simulated microgravity on avian embryonic development.

Based on the few reports available, microgravity (MG) can have adverse effects on the early development of vascularised extra-embryonic membranes in avian eggs. Whether gravity or oxygen availability is the stimulus for development of the blood vessels in the chorioallantoic membranes (CAM) remains unclear. Under gravity the blastoderm forms on top of the yolk sac, closest to the oxygen rich region beneath the shell membranes, and from there the CAM buds from an abdominal extension subsequently to form a close contact with shell membranes. Then as the embryo develops it spreads beneath the eggshell surface to maximise the surface area of the CAM vascular bed available for O2 uptake. To investigate how simulated MG influences development of the CAM and embryo we conducted experiments on chicken embryos during incubation in a 3D-clinostat (control or continuous MG treatment at 5 rpm). Further, to determine if CAM angiogenesis is directed towards regions of high O2 tension or gravity we investigated the effects of wax treatment (50% shell surface area) on development in MG. We found that clinostat MG caused embryonic failure between day 0-5 by preventing normal development of CAM-shell membrane complex. Thereafter acute MG promoted increases in CAM mass, but did not affect embryo mass. Preliminary findings suggest that combined acute MG and wax treatment did not significantly affect embryonic growth in either MG or control groups, but retarded CAM growth in control embryos only. Finally, we will present evidence to show that acute and prolonged exposure to MG does not prevent normal growth and hatching, but might have more subtle effects on hatchling physiology, including reduced heart mass.     

Embryonic growth and mobilization of energy and material during incubation in the checkered keelback snake, Xenochrophis piscator

We collected 20 checkered keelback snakes (Xenochrophis piscator) to study embryonic growth and mobilization of energy and material during incubation. Females laid eggs between late May and late June. The eggs were incubated at 27 degrees C (+/-0.3). One egg from each clutch was dissected at five-day intervals starting at oviposition. The mean incubation length at 27 degrees C was 48.9 days. We identified three phases of embryonic growth or yolk depletion in X. piscator. Phase 1, between oviposition and Day 20, was one of minimal transfer of energy and material from yolk to embryo. Phase 2, between Day 20 and Day 39-40, was characterized by increasingly rapid embryonic growth or yolk depletion. Phase 3, between Day 39-40 and hatching, was characterized by reduced embryonic growth or yolk depletion. Approximately 71% of dry mass, 53% of non-polar lipids and 66% of energy were transferred from the egg contents to the hatchling during incubation. Our data confirm that oviposition is not timed to coincide with the onset of rapid embryonic growth in oviparous squamate reptiles. The greater conversion efficiencies of energy and material from egg to hatchling in snakes can be attributed to their lower energetic costs of embryonic development and greater residual yolk sizes.     

半滑舌鳎胚胎发育中后期卵黄囊超微结构观察

在胚胎发育中期,半滑舌鳎胚胎由胚体、卵黄囊和卵周液构成.对半滑舌鳎胚胎发育中后期的卵黄囊进行超微结构观察.结果表明,卵黄囊是由卵黄囊膜和包裹其内的卵黄物质组成.在半滑舌鳎胚胎发育过程中,卵黄囊内的卵黄物质逐渐消耗产生低分子量的卵黄磷蛋白分裂小泡.分裂小泡转移到卵黄囊内部消黄细胞中,在消黄细胞的作用下分裂小泡转化成卵黄颗粒.随后卵黄颗粒在卵黄囊内表面聚集成囊状结构并转移运输到卵黄囊膜内部,最后把卵黄物质从卵黄囊膜转移并释放到卵周液中,为胚胎发育提供营养.     

Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.     

Ontogeny of T cells: development of pre-T cells from fetal liver and yolk sac in the thymus microenvironment

The patterns of development of T cells from the very early stem cells that settle in the embryonic thymus have been studied. For this purpose, mouse embryonic thymuses (14 days) depleted of thymocytes were reconstituted with hemopoietic stem cells from fetal liver (FL) and yolk sac (YS) and T-cell development was followed in vitro in organ culture. It was found that cells derived from FL and YS of 10- to 14-day-old embryos were capable of reconstituting depleted thymic explants and exhibiting membrane markers in a pattern similar to that of thymocytes developing in intact thymic explants. Furthermore, these cells responded to concanavalin A in proliferative and cytotoxic assays as measured by limiting-dilution analysis. Thus, lymphohemopoietic stem cells emerging in the embryo prior to thymus lymphoid development are capable of differentiation in the thymus microenvironment into T cells, identified by phenotypic markers and functions that are characteristic of cells developing in the intact embryonic thymus.     

Rhythmic contractions in chick amnio-yolk sac and snake amnion during embryogenesis

The rhythmic movements of fetal membranes in chick and reptile embryos were studied to explore the developmental role of the extra-embryonic motor activity. In the snakes Lamprophis fuliginosus and Elaphe radiata, rhythmic contractions of amnion inside the developing egg were recorded from the 11th incubation day until pre-hatching stages (ca. day 60-72). The duration of these contractions averaged 2.02+/-0.27 min. The frequency ranged from 2 to 6 per 10 min and averaged 4.61+/-0.57 per 10 min. A tendency of frequency to increase toward the end of embryogenesis was observed. Lowering the temperature from 28 to 20 degrees C significantly decreased the frequency of amnion contractions to 2.85+/-0.91 per 10 min. The isolated snake amnion retained its capacity for spontaneous contraction. Noradrenaline inhibited, acetylcholine stimulated and serotonin did not affect the rhythmic activity of the isolated snake amnion. Similar effects were found when these agents were applied into the snake amniotic cavity. In the chick, yolk sac rhythmic contractions were recorded from the fifth until the 12th incubation days. The duration of these contractions ranged from 15 to 60 s, their frequency averaged 11.8+/-3.18 per 10 min and depended on temperature. The low temperature threshold was approximately 30 degrees C. After surgical removal of the amnion and embryo, the yolk sac continued contracting inside the egg. The yolk sac rhythmic contractions likely participate in the space movement of the embryo inside the egg during embryogenesis.     

Ontogeny of the mouse hematopoietic system

In vertebrates the extraembryonic mesoderm of the yolk sac (YS) is the first site during embryogenesis where morphologically discernible hematopoiesis may be found. Later hematopoiesis shifts into the embryo proper, first to the liver, the major fetal hematopoietic site, then to definitive hematopoietic territories, the spleen and bone marrow. It is widely accepted that in the mouse this picture reflects the migration of pluripotent hematopoietic stem cells (HSC) from the YS accompanied by subsequent colonization of the hematopoietic tissues during embryogenesis. However, there is no conclusive evidence showing unequivocally the initiating role of the YS in murine adult hematopoiesis. Recently, we have demonstrated the important role of embryo body tissues in the development of CFU-S before the establishment of definitive hematopoiesis in the fetal liver. This finding suggests that the early development of the hematopoietic system in the mouse is more complex than has been previously proposed and we consider here the early hematopoietic events in the developing mouse embryo.     

B cell potential can be obtained from pre-circulatory yolk sac, but with low frequency

The ontogenic source of definitive hematopoietic system has been identified in non-mammalian vertebrates such as birds and amphibians by orthotopic embryo grafting, but remains unclear for mammals because of technical difficulties. Here, we successfully generated mouse chimeras by grafting yolk sac (YS) on YS of the host embryos before establishing circulation between YS and embryo proper and cultured the whole embryo for 66 h. Donor YS were isolated from C57BL/6 Ly-5.1 and EGFP-transgenic mouse embryos, and recipient embryos from C57BL/6 Ly-5.2 mouse. Almost one-half of the grafts in YS-YS chimeras survived and had obvious blood flow; graft-derived cells comprised 12.7+/-0.9% of the blood cells in the circulation. These graft-derived blood cells consisted mainly of erythroid cells, some myeloid cells and a few blastic cells. In addition, CD19(+) B cells were generated from the graft-derived cells isolated from aorta-gonad-mesonephros (AGM) regions of the YS-YS chimeras; however, the frequency of the YS-derived B cell was low (1.0+/-0.6%) when co-cultured with OP9 stromal cells. These results demonstrate that B cell potential exists in YS before the circulation. Although the major source for B cell is intra-embryonic AGM region, YS may contribute to definitive lymphopoiesis in vivo in mice.     

Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis

Our investigations concerning the importance of cell surface macromolecules during embryonic development led us to the discovery in 1961 that heterologous anti-rat kidney serum produced teratogenesis, growth retardation and embryonic death when injected into the pregnant rat during early organogenesis. It was established that IgG was the teratogenic agent, primarily directed against the visceral yolk sac (VYS) but not the embryo. Heterologous anti-rat VYS serum was prepared which was teratogenic localized in the VYS and served as a model for producing VYS dysfunction and embryonic malnutrition. The role of the yolk sac placenta in histiotrophic nutrition is now recognized to be critical for normal embryonic development during early organogenesis in the rodent. VYS antiserum affects embryonic development primarily by inhibiting endocytosis of proteins by the VYS endoderm, resulting in a reduction in the amino acids supplied to the embryo. Our laboratory has recently developed teratogenic monoclonal yolk sac antibodies (MCA) which can be utilized; to study VYS plasma membrane synthesis and recycling, to compare yolk sac function among different species, and to identify components of the plasma membrane involved in pinocytosis. MCA prepared against certain VYS antigens provide an opportunity to study embryonic nutrition with minimal interference with the nutritional state of the mother. Recent developments in the study of the human yolk sac along with our laboratory's ability to isolate a spectrum of yolk sac antigens, prepare monoclonal antibodies, and perform functional studies, should provide information that will increase our understanding of yolk sac function and dysfunction in the human and determine the relative importance of various amino acids to normal development during mammalian organogenesis.     

In vitro analysis of glucose metabolism and embryonic growth in postimplantation rat embryos

The glucose metabolism and embryonic development of rat embryos during organogenesis was studied using embryo culture. Glucose uptake and embryonic growth and differentiation of 10.5-day explants (embryos + membranes) were limited by the decreasing glucose concentration, but not the increasing concentration of metabolites, in the culture media during the second 24 h of a 48 h culture. No such limitations were found on the embryonic development of 9.5-day explants during a 48 h culture although glucose uptake was slightly reduced at very low concentrations of glucose. From the head-fold stage to the 25-somite stage of development, glucose uptake was characteristic of the stage of development of the embryo and not the time it had been in culture. Embryonic growth of 9.5-day explants was similar to that previously observed in vivo. Glucose uptake by 9.5-day explants was dependent on the surface area of the yolk sac and was independent of the glucose concentration in the culture media (within the range of 9.4 to 2.5 mM). The proportion of glucose converted to lactate was 100% during the first 42h of culture then fell to about 50% during the final 6h. The protein contents of both the extraembryonic membranes and the embryo were dependent on the glucose uptake.     

Gastrulation and angiogenesis,not endothelial specification,is sensitive to partial deficiency in vascular endothelial growth factor-a in mice

Mouse embryogenesis is dose sensitive to vascular endothelial growth factor-A (VEGF-A), and mouse embryos partially deficient in VEGF-A die in utero because of severe vascular defects. In this study, we investigate the possible causes that underlie this phenomenon. Although the development of vascular defects in VEGF-A-deficient embryos seems to suggest that endothelial differentiation depends on the presence of a sufficient level of VEGF-A, we were surprised to find that endothelial differentiation per se is insensitive to a significant loss of VEGF-A activity. Instead, the development of the multipotent mesenchymal cells, from which endothelial progenitors arise in the yolk sac, is most highly dependent on VEGF-A. As a result of VEGF-A deficiency, dramatically fewer multipotent mesenchymal cells are generated in the prospective yolk sac. However, among the small number of mesenchymal cells that do enter the prospective yolk sac, endothelial differentiation occurs at a normal frequency. In the embryo proper, vasculogenesis is initiated actively in spite of a significant VEGF-A deficiency, but the subsequent steps of vascular development are defective. We conclude that a full-level VEGF-A activity is not critical for endothelial specification but is important for two distinct processes before and after endothelial specification: the development of the yolk sac mesenchyme and angiogenic sprouting of blood vessels.     

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.     

Cultured chick yolk sac epithelium: structure and IgG surface binding

The chick yolk sac endoderm transports maternal immunoglobulin G (IgG) from the yolk into the embryo during development, providing the newly hatched chick with passive immunity until it becomes immunocompetent. To study this transport process, chick yolk sac endodermal cells isolated from embryos of 6 to 18 days of incubation were grown in vitro on a collagen substrate. The cultured cells possessed a remarkable structural similarity to the in vivo tissue and reformed a polarized confluent epithelium with tight junctions and desmosomes joining the cells at their apical margins. In addition, the cells exhibited apical microvilli, numerous phagolysosomes in the cytoplasm and retained the expression of the yolk sac endoderm-specific enzyme marker, cysteine lyase. Importantly, the cultured cells retained the ability to specifically bind IgG as demonstrated by indirect immunofluorescence. Chicken IgG bound to the cultured cells at 4 degrees C in a diffuse pattern that clustered into a punctate pattern when a second antibody was used. Cultures from yolk sacs of day 6 through day 18 of development all demonstrated this immunofluorescent labeling for at least 14 days in culture. These results demonstrate that cultured yolk sac endoderm maintains its differentiated morphology and ability to bind IgG.     

Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplantation embryos

The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early and slightly later stage cell differentiation in the developing bovine embryo. In vitro–produced Day 6 embryos were transferred into a recipient heifer and after 7 days of further in vivo culture, ovoid and elongated Day 13 embryos were recovered by flushing both uterine horns after slaughter. The primitive YS fluid and cellular components were isolated from 12 ovoid and three elongated embryos and using nano-high-performance liquid chromatography, tandem mass spectrometry, and isobaric tag for relative and absolute quantitation proteomic analysis, a total of 9652 unique proteins were identified. We performed GO term and keyword analyses of differentially expressed proteins in the fluid and the cells of the two embryonic stages, along with a discussion of the biological perspectives of our data with relation to morphogenesis and embryo-maternal communication. Our study thereby provides a considerable contribution to the current knowledge of bovine preimplantation development.