Hemopoiesis in the human yolk sac

The endodermal layer of the human yolk sac was examined three-dimensionally with light microscopy on serial sections using scanning electron microscopy and transmission electron microscopy to find the origin of hemopoiesis in the yolk sac. Cell-labelling techniques were also employed using the monoclonal anti-transferrin receptor antibody. Orifices of the endodermal and intracellular tubules facing the yolk-sac cavity were demonstrated on the endodermal surface. Various-sized blood cells in various stages of differentiation and maturation were distributed in the yolk-sac cavity and tubules and were observed also at the orifices of the tubules. The morphological and the immunological findings suggest that blood cells with large nuclei in the endodermal layer are the most immature. The present results suggest that blood cells originate from the endodermal layer and are carried to the embryo through the yolk sac cavity and the vitelline duct. It is probable that the endodermal and intracellular systems of tubules have an important role in the transport of blood cells, including stem cells.     

Ultrastructure of the full-term shark yolk sac placenta. I. Morphology and cellular transport at the fetal attachment site

During ontogeny, the yolk sac of some viviparous sharks differentiates into a yolk sac placenta that persists to term. The placenta is non-invasive and non-deciduate. Hematrophic transport is the major route of nutrient transfer from mother to fetus. The placental unit consists of: (1) an umbilical stalk; (2) the smooth, proximal portion of the placenta; (3) the distal, rugose portion; (4) the egg envelope; and (5) the maternal uterine tissues. Exchange of metabolites is effected through the intervening egg envelope. The distal rugose portion of the placenta is the fetal attachment site. It consists of: (1) surface epithelial cells; (2) a collagenous stroma with vitelline capillaries; and (3) an innermost boundary cell layer. The columnar surface epithelial cells are closely apposed to the inner surface of the egg envelope. Wide spaces occur between the lateral margins of adjacent cells. Surface epithelial cells contain an extensive apical canalicular-tubular system and many whorl-like inclusions in their basal cytoplasm. Capillaries of the vitelline circulation are closely situated to these cells. A well-developed collagenous stroma separates the surface epithelium from an innermost boundary cell layer. In vitro exposure of full-term placentae to solutions of trypan blue and horseradish peroxidase (HRP) reveals little uptake by the smooth portion of the placenta but rapid absorption by the surface epithelial cells of the distal, rugose portion. HRP enters these cells by an extensive apical system of smooth-walled membranous anastomosing canaliculi and tubules. Prominent whorl-like inclusions that occupy the basal cytoplasm of the surface cells, adjacent to the pinocytotically active endothelium of the vitelline capillaries, are hypothesized to be yolk proteins that are transferred from the mother to embryo throughout gestation.     

Structure of the endodermal epithelium of the chick yolk sac during early stages of development.

The structure of the areas pellucida and vasculosa of the early chick embryo (stages 11-29) was examined by light, transmission and scanning electron microscopy. The most striking feature of the endodermal cells of these areas is the presence of large intracellular yolk drops which are characteristic of the regions in which they are found; lipid-like homogeneous drops in the area pellucida, heterogeneously composed pleomorphic drops in the mid-region of the area vasculosa and granular drops at the periphery of the area vasculosa in the region of the sinus terminalis. On morphological criteria it is postulated that granular drops may arise by direct engulfment of extracellular yolk, but this does not appear to be true for pleomorphic or homogeneous drops. Since the apical junctions between endodermal cells across the yolk sac are tight, they seal off the extraembryonic compartment from the vitelline circulation and presumably prevent intercellular passage of the yolk constituents. Thus the endodermal epithelium must mediate the transport of nutrients from the yolk mass to the developing embryo. Endodermal cells exhibit a variation across the yolk sac in the presence and number of structures associated with uptake of extracellular materials. The mid-region of the area vasculosa appears to be the most endocytotically active region with an abundance of microvilli, bristle-coated pits and vesicles and apical canaliculi and vacuoles. There is a close association between the endoderm and vitelline blood vessels and this association is maintained, as the yolk sac develops, by the formation of small vessels juxtaposed between the vascular surface of the endoderm and the walls of the large vitelline vessels.     

Yolk sac cholesteryl ester secretion rates can be manipulated in the Golden Syrian hamster: effect of yolk sac cholesterol concentrations

The yolk sac is one of two extra-embryonic fetal tissues that separates the fetal and maternal circulations. The yolk sac can secrete lipoprotein particles to the vitelline vessels, which supply yolk sac-derived nutrients to the embryo. The amount and composition of lipoproteins secreted from the rat yolk sac can be manipulated by fatty acid content and gestational age. The goals of the current studies were to determine, first, if tissue cholesterol concentration could mediate cholesterol secretion rate from the yolk sac and, second, if some of the secreted cholesterol could be derived from the maternal circulation. Golden Syrian hamsters were fed 2% added cholesterol to increase the yolk sac cholesterol concentration. Yolk sac explants secreted similar amounts of triglyceride and apolipoproteins B and E into the media regardless of yolk sac cholesterol concentration. In contrast, yolk sacs with greater cholesterol concentrations secreted 2.3-fold more cholesterol into the media as compared to control yolk sacs; the increase was found mostly as cholesteryl ester. At least part of the secreted cholesterol was maternally derived. These data demonstrate that yolk sac cholesterol concentration influences cholesterol secretion rates, and that at least some of the cholesterol secreted originates from the maternal circulation.     

Atrial natriuretic peptide binding in rat placenta,yolk sac,decidua, and materal placental vessels

Using in vitro autoradiography, binding sites of ¹²⁵I-ANP (atrial natriuretic peptide) were localized in the rat placenta, visceral yolk sac, and decidua at 16, 18, and 20 days of gestation. There was diffuse binding over the labyrinthine region of the placenta and an intense binding over the decidual gland and visceral yolk sac. In the yolk sac, ANP localized over the cores of the villi where it may be involved with the regulation of transport across the membranes or the flow of blood through the vitelline vessels. Of particular interest was binding over the maternal blood vessels supplying the decidual region and placenta. Receptors were located on the endothelial cells and smooth muscle cells of the arteries and veins, indicating that ANP may be involved with regional regulation of blood flow to the placenta.     

Hedgehog is required for murine yolk sac angiogenesis.

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh(-/-) mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and alpha-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh(-/-) lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or SMO: Whereas Ihh(-/-) yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo(-/-) yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh(-/-) yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.     

Immunohistochemical characterization of extracellular matrix components of yolk sac tumors

Proteoglycans (PGs) were isolated from yolk sac tumor and chondroitin sulfate large PG (core molecule with a molecular weight congruent to 200,000) and small PG (core molecule with a molecular weight congruent to 50,000) were detected. Immunohistochemical localization of PGs in three yolk sac tumors was investigated using monoclonal antibodies raised against both small and large PGs, which were purified from human ovarian fibroma capsule and a yolk sac tumor, respectively. The localization of large PG was observed to be distinct from that of small PG. A markedly positive reaction for antibody against large PG was observed in myxomatous areas, perivascular and perivesicular portions; hyaline globules were the most intensely reactive. In the areas showing a polyvesicular vitelline tumor pattern, the compact connective tissue stroma consisted of small PGs. It is conceivable that large PGs are synthesized by immature mesenchymal cells and also by epithelial-like cells as a basement membrane component, whereas small PGs are synthesized by mature fibroblastic cells synthesizing collagen. Immunohistochemical localization of other extracellular matrix components (laminin, fibronectin, type I-IV collagen) was also studied in relation to PG localization.     

The visceral yolk sac--an important site of synthesis and secretion of apolipoprotein B containing lipoproteins in the feto-placental unit of the rat.

Rat fetuses exhibit a high serum LDL concentration at term. Delivery caused a marked decrease of the LDL apolipoprotein (apo) B concentration independent of whether this occurred on days 21, 22 or 23 of gestation. The interruption of the yolk sac circulation by a ligature in situ for 6 h led to the same alterations of the LDL-apo B concentration as Caesarean section. Immunoelectronmicroscopic studies provided evidence that the epithelial cells of the visceral yolk sac exhibited electron dense LDL-sized and apo B containing particles which were localized over the compartments of the Golgi complexes, endoplasmatic reticulum, secretory vesicles and intercellular spaces, but not over the cell nuclei, mitochondria or lysosomes. ApoB containing LDL-sized particles could be obtained by ultracentrifugation from the disrupted material of the microsomal fraction of yolk sac homogenates. Isolated segments of the yolk sac membranes were capable to secrete apoB containing lipoproteins floating in the d less than 1.020 g/ml as well as in the d = 1.020-1.064 g/ml fraction with a 10-fold higher amount of apoB in the higher density class. Incorporation experiments with [35S] methionine gave evidence that these lipoproteins were at least partially provided with newly synthesized apoB predominantly found in the LDL fraction. The size of the negatively stained particles in the d = 1.020-1.064 g/ml fraction secreted from yolk sac segments corresponded to that of LDL from fetal rat serum. In contrast their acylglycerol content was significantly higher, whereas the percentage contribution of total cholesterol and protein was markedly reduced in comparison with serum LDL of the fetus. In summary, biochemical and ultrastructural studies provide clear cut evidence that the rat yolk sac is able to synthesize and to deliver apo B containing lipoproteins in the density ranges of VLDL, IDL and particular of LDL thus contributing to the supply of serum lipoproteins in the rat fetus. By recalculation of recent tracer kinetic data (Plonné et al. (1990) J. Lipid Res. 31, 747) using a mathematical step function model it was possible to assess the contribution of the rat yolk sac to the LDL influx into the fetal serum.     

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.     

In vitro activation of the in vivo colony-forming units of the mouse yolk sac.

Experiments were performed to investigate the presence of colony-forming units (CFU) in the mouse embryonic yolk sac during the developmental period in which the yolk sac is the sole hemopoietic organ. Injection of yolk sac cell suspensions from normal embryos into syngeneic, lethally irradiated adult recipients evoked a very low number of spleen colonies. However, prior cultivation of yolk sacs in vitro caused a dramatic increase in the spleen colony-forming capacity--as high as 84-fold--following 48 hours in culture. The yolk sac origin of the spleen colonies was confirmed by: (a) Chromosomal marker analysis; (b) dose-response analysis; (c) demonstrating that the above colonies were not of endogenous origin induced by the mere injection of grafted cells. We conclude that the yolk sac contains many precursors of colony-forming cells which though undetectable by immediate grafting apparently become activated in culture by an as yet unknown induction process.     

The human foetal yolk sac

Summary Specimens of human foetal yolk sac from conceptuses of 8 and 10 weeks menstrual age were studied with the electron microscope. At 8 weeks columns of endodermal cells projected into the underlying mesenchyme. Several types of endodermal cell were identified; some contained much granular endoplasmic reticulum and abundant glycogen; others resembled the haemocytoblasts present in the mesenchyme and yet others contained membrane-bounded channels similar to those seen in megakaryocytes. It was suggested that the endoderm is the site of origin of the blood cells but that, while the platelets may be formed within the endoderm, the normal development of the red cells is conditional upon their early release into the mesenchyme and possibly the attainment of an intravascular position. Intravascular macrophages were identified and their role in determining the nature of the blood picture during the period of functional acitvity of the sac discussed. The morphology of the epithelium on the external surface of the sac was discussed in relation to the possibility of its playing a part in the exchange of materials between the yolk sac and the chorionic cavity.Supported in part by grant no. 5-T01-GM-00582-08 from the U.S. Public Health Service.     

An electron microscopic study of absorption of peroxidase-conjugated immunoglobulin G by guinea pig visceral yolk sac in vitro.

In the guinea pig and some other animals, passive immunity is conferred on the developing fetus by passage of immunoglobulin from mother to fetus across the yolk sac. In order to examine the cytological pathway involved in immunoglobulin transport, guinea pig visceral yolk sacs from late in gestation were exposed in vitro to peroxidase-conjugated guinea pig immunoglobulin G (IgG-HRP). Tissue was then fixed, incubated to show the site of localization of peroxidase reaction product and prepared for electron microscopy. The results suggested that the first step in the uptake of IgG-HRP by yolk sac is attachment of the protein to the surface coats of endocytic invaginations at the apical surfaces of the endodermal cells. The endocytic vesicles then appear to pinch off from the surface and move deeper into the cytoplasm. Some of the small endocytic vesicles fuse with large apical vacuoles, which often contain large amounts of reaction product. Other small endocytic vesicles pinch off from the surface, move deeper into the cytoplasm and fuse with the lateral plasmalemma; their protein content is emptied into the intercellular space by exocytosis. From the intercellular spaces the protein presumably diffuses across the basement membrane and connective tissue spaces and enters the vitelline capillary bed. It is postulated that the latter cellular pathway, involving small vesicles and the intercellular spaces, is utilized by those immunoglobulins which are transferred intact across the yolk sac endoderm.     

Embryonic macrophages of early rat yolk sac: Immunohistochemistry and ultrastructure with reference to endodermal cell layer

Macrophages are multifunctional cells that participate in numerous biological processes; they actively phagocytose foreign particles and cell debris. Embryonic tissue macrophages are present at early stages of mammalian development; their ontogeny and function is still under investigation. Our study used immunohistochemistry and electron microscopy to investigate early rat yolk sac macrophages using mouse antirat macrophage monoclonal antibodies (mAb) Mar 1 and Mar 3 produced by our laboratory. Mar 3 mAb revealed the first emergence of immature macrophages in the rat yolk sac at fetal day nine coinciding with the beginning of yolk sac haemopoiesis that consisted mainly of erythropoiesis, while Mar 1 mAb detected specifically rat yolk sac macrophages at about the 13th to 14th day of gestation. Immunoreactivity against Mar mAbs was mainly located in the yolk sac endodermal cell layer, which may signify endodermal origin of the yolk sac macrophages. Ultrastructurally mature yolk sac macrophages contained numerous endocytic vesicles or vacuoles, well-developed Golgi saccules and many electron dense granules in their cytoplasm and a number of microvillous projections from the cell surface. After establishment of the circulation between yolk sac and embryo, Mar 3 positive cells were also demonstrated inside fetal undifferentiated mesenchymal tissue at fetal day 12. The study demonstrated the first emergence of immature yolk sac macrophages being among the earliest haemopoietic cells formed in mammalian development. Thus, Mar mAbs managed to detect macrophage differentiation antigens through their development early in the rat yolk sac.     

Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis

Wnts are secreted signaling molecules implicated in various developmental processes and frizzled proteins are the receptors for these Wnt ligands. To investigate the physiological roles of frizzled proteins, we isolated and characterized a novel mouse frizzled gene Fzd5. Fzd5 mRNA was expressed in the yolk sac, eye and lung bud at 9.5 days post coitum. Fzd5 specifically synergized with Wnt2, Wnt5a and Wnt10b in ectopic axis induction assays in Xenopus embryos. Using homologous recombination in embryonic stem cells, we have generated Fzd5 knockout mice. While the heterozygotes were viable, fertile and appeared normal, the homozygous embryos died in utero around 10.75 days post coitum, owing to defects in yolk sac angiogenesis. At 10.25 days post coitum, prior to any morphological changes, endothelial cell proliferation was markedly reduced in homozygous mutant yolk sacs, as measured by BrdU labeling. By 10.75 days post coitum, large vitelline vessels were poorly developed, and the capillary plexus was disorganized. At this stage, vasculogenesis in the placenta was also defective, although that in the embryo proper was normal. Because Wnt5a and Wnt10b co-localized with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for the Fzd5-dependent signaling for endothelial growth in the yolk sac.     

Yolk sac failure in embryopathy due to hyperglycemia: ultrastructural analysis of yolk sac differentiation associated with embryopathy in rat conceptuses under hyperglycemic conditions

Diabetes mellitus in pregnancy is associated with an increased incidence of various congenital anomalies that occur during organogenesis. Because a well functioning yolk sac is crucial to embryonic growth and development during this period, we performed an ultrastructural study of the effects of excess glucose (total glucose 750 mg/dl, osmolality 305 mOsm/kg) on pregnancy day 10 (Witschi stage 13) rat conceptuses cultured for 48 hr in heat-inactivated male rat serum with and without added d- or l-glucose. Embryos exposed to excess d-glucose demonstrated decreased conceptus size (P less than 0.001), and gross malformations in a dose-related fashion. The visceral yolk sac capillaries and vitelline vessels of conceptuses in excess d-glucose were sparse, patchy, and nonuniformly located. Ultrastructurally, the visceral yolk sac endodermal cells had reduced numbers of rough endoplasmic reticulum, ribosomes, and mitochondria. These obvious defects in yolk sac structure suggest that hyperglycemia during organogenesis has a primary deleterious effect on yolk sac function with resultant embryopathy.     

Characterization of a rat yolk sac carcinoma cell line

A continuous cell line was established from an experimentally induced rat yolk sac carcinoma. In the early passages both visceral and parietal yolk sac carcinoma were present (designated L1). When the cell line was reestablished in culture after serial transplantations in rats, only parietal yolk sac carcinoma could be identified (designated L2). This cell line expresses parietal yolk sac endoderm characteristics in that it synthesizes basement membrane components, in particular, laminin, but also entactin, collagen IV, and heparan sulfate proteoglycan. In addition, a noncartilage chondrotin sulfate proteoglycan is synthesized. This rat yolk sac carcinoma cell line L2 will be a valuable model for the study of basement membrane components.     

Ontogeny of vitamin D action on the morphology and calcium transport properties of the chick embryonic yolk sac

The ontogeny of the calcium transport properties and hormonal modulation of the yolk sac membrane in amniote embryos is presently poorly understood. We investigated the role of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on plasma calcium values, yolk sac morphology and the ability of the yolk sac membrane to transport 45Ca from yolk to embryo. 1,25-(OH)2D3 treatment caused significant hypercalcaemia in 9-, 12- and 15-day embryos. Additionally, this hormone caused a hypertrophy of the endodermal cell layer that comprises the bulk of the yolk sac membrane. Both of these effects were the most dramatic in the 15-day embryo, the oldest age tested. 45Ca added to the yolk was transported into the blood rapidly across the yolk sac membrane. 1,25-(OH)2D3 significantly enhanced this transport in all age groups. [14C]Inulin was also taken across the yolk sac membrane, but at a slower rate than 45Ca; this transport was unaffected by 1,25-(OH)2D3. Thus, the yolk sac responds to 1,25-(OH)2D3 treatment both morphologically and functionally. The mechanism for transport appears to be a specific one, rather than a simple enhancement of non-specific endocytosis.     

In vitro differentiation of mouse embryonic yolk sac cells

The embryonic yolk sac is the first site in the mammalian embryo in which cells are found that can carry out cell-mediated immune functions, yet the relation of cells of this primitive hematopoietic organ to the development of the mature immune system has not been established. We have initiated a series of experiments to determine the potential of cells of the mouse yolk sac to differentiate in vitro, in order to get an insight into the development of immunocompetence in this primary population of hematopoietic stem cells. The present paper describes the conditions promoting stem-cell differentiation and provides an initial characterization of cell surface phenotypes of the cell lineages established in vitro. Yolk sac cells obtained from 10- to 13-day mouse embryos were maintained in culture for more than 18 months, giving rise to a variety of cell types belonging to the hematopoietic lineages and culminating in the establishment of long-term cell lines. Supernatants of secondary mixed leukocyte cultures were found to be an effective source of growth factors promoting the initial differentiation as well as the maintenance of these cells. Flow-cytometric analysis showed that, in contrast to freshly obtained yolk sac cells, which had no detectable Thy 1 antigen, cells expressing significant levels of Thy 1 were obtained after 1 week or more of culture. Ly1 and Lyt 2 antigens were detected only rarely and the L3T4 (GK 1.5) antigen was never expressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular remodeling of the mouse yolk sac requires hemodynamic force

The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a(-/-)). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.