Yolk Sac Development Research Articles

The mammalian yolk sac placenta

There are three fetal membranes in mammals, namely the yolk sac, allantois and amnion, but only the first two form a placenta. In Monotremata (monotremes), Marsupialia (marsupials) and Placentalia (eutherians), the yolk sac transfers nutrients originating from uterine glandular secretion or the maternal blood. Ontogenetically, the yolk sac of most eutherians loses contact with the peripheral chorion and forms a free splanchnopleuric yolk sac that transfers substances from the exocoelomic cavity, not directly from the endometrium as in marsupials and rodents. This free yolk sac has been preserved in humans and substances originating from glandular secretions are transferred from the exocoelomic cavity to the embryo via this route. The therian yolk sac expresses numerous growth and transcription factors, associated binding proteins and receptors that control its differentiation and function, including hematopoiesis and angiogenesis. Errors in yolk sac development and function could contribute to embryonic malformation, miscarriage and growth diseases. Factors regulating transfer and metabolism, notably insulin and IGF2, are imprinted in the human yolk sac, as in all therians so far studied. This suggests persistence of a strong selective pressure for parentally controlled allocation of resources to the growing embryo via this fetal membrane. The metabolic and biosynthetic functions of the yolk sac of the ancestral therian stem species, as well as hematopoiesis in the eutherian ancestor, appear to have been retained by the human yolk sac. Thus, the yolk sac of humans, like that of all viviparous mammals, is a true placenta crucial for early embryonic development and survival.

Molecular and Morphological Changes in Placenta and Embryo Development Associated with the Inhibition of Polyamine Synthesis during Midpregnancy in Mice

Polyamines play an essential role in murine development, as demonstrated by both gene ablation in ornithine decarboxylase (ODC)-deficient embryos and pharmacological treatments of pregnant mice. However, the molecular and cellular mechanisms by which ODC inhibition affects embryonic development during critical periods of pregnancy are mostly unknown. Our present results demonstrate that the contragestational effect of alpha-difluoromethylornithine (DFMO), a suicide inhibitor of ODC, when given at d 7-9 of pregnancy, is associated with embryo growth arrest and marked alterations in the development of yolk sac and placenta. Blood island formation as well as the transcript levels of embryonary globins alpha-like x chain and beta-like y-chain was markedly decreased in the yolk sac. At the placental level, abnormal chorioallantoic attachment, absence of the spongiotrophoblast layer and a deficient development of the labyrinthine zone were evident. Real-time RT-PCR analysis showed that transcript levels of the steroidogenic genes steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase VI, and 17alpha-hydroxylase were markedly decreased by DFMO treatment in the developing placenta at d 9 and 10 of pregnancy. Plasma values of progesterone and androstenedione were also decreased by DFMO treatment. Transcriptomic analysis also detected changes in the expression of several genes involved in placentation and the differentiation of trophoblastic lineages. In conclusion, our results indicate that ODC inhibition at d 8 of pregnancy is related to alterations in yolk sac formation and trophoblast differentiation, affecting processes such as vasculogenesis and steroidogenesis.

The patatin-like lipase family in Gallus gallus

BackgroundIn oviparous species, genes encoding proteins with functions in lipid remodeling, such as specialized lipases, may have evolved to facilitate the assembly and utilization of yolk lipids by the embryo. The mammalian gene family of patatin-like phospholipases (PNPLAs) has received significant attention, but studies in other vertebrates are lacking; thus, we have begun investigations of PNPLA genes in the chicken (Gallus gallus).ResultsWe scanned the draft chicken genome using human PNPLA sequences, and performed PCR to amplify and sequence orthologous cDNAs. Full-length cDNA sequences of galline PNPLA2/ATGL, PNPLA4, -7, -8, -9, and the activator protein CGI-58, as well as partial cDNA sequences of avian PNPLA1, -3, and -6 were obtained. The high degree of sequence identities (~50 to 80%) between the avian and human orthologs suggests conservation of important enzymatic functions. Quantitation by qPCR of the transcript levels of PNPLAs and CGI-58 in 21 tissues indicates that expression patterns and levels diverge greatly between species. A particularly interesting tissue in which certain PNPLAs may contribute to physiological specialization is the extraembryonic yolk sac.ConclusionKnowledge about the exact in-vivo functions of PNPLAs in any system is still sparse. Thus, studies about the temporal expression patterns and functions of the enzymes identified here, and of other already known extracellular lipases and co-factors, in the yolk sac and embryonic tissues during embryogenesis are called for. Based on the information obtained, further studies are anticipated to provide important insights of the roles of PNPLAs in the yolk sac and embryo development.

Early Ontogeny of <i>Tropheus Moorii</i> Boulenger 1898 (Pisces, <i>Cichlidae</i>, Lake Tanganyika) in Laboratory Conditions

In this work were shown some crucial moments in early Troheus moorii development (for example: begin of exogenous feeding period, end of endogenous feeding period, time of finish fins development and others). Fertilized eggs, obtained after natural spawn from females of Tropheus species kept in tank, were incubated in laboratory conditions. Hatched embryos were reared in incubation tank, in constance temperature, until resorption of the yolk sac and finish fins development. Embryos were fed ad libitum with live Artemia nauplii. In Tropheus, the fins became fully developed and the yolk sac supplies depleted on the same day, 25 th since the mating. Since this species keeps its offspring in the mouth for 30–35 days (YANAGISAWA ,S ATO 1990), the young stay in they shelter for another one to two weeks. When they are let out for the first time, they are in an advanced stage of their development, mature enough to live like the adults of their species. Since their yolk sack supplies are depleted before the termination of parental care period, it is obvious that they must be intrabucally fed in this time. It is confirmed by the fact that in aquarium conditions incubating females pick the food particles as the non-incubating females do. The food had been found on a regular basis in incubating females mouths when acquiring eggs and embryos for observations. Since the young Tropheus already can ingest exogenous food while being still in mothers mouth, it is on purpose to feed crushed food to the incubating females in aquarium conditions.

Blockade of c-Jun N-terminal kinase activation abrogates hyperglycemia-induced yolk sac vasculopathy in vitro

Maternal hyperglycemia has an impact on both the function and morphology of the rodent visceral yolk sac; the objective of the present study was to determine whether hyperglycemia in vitro affects yolk sac vasculogenesis. Because maternal hyperglycemia triggers c-Jun N-terminal kinase (JNK) 1 and 2 activation in the yolk sac, we tested whether the inhibition of JNK activation would ameliorate hyperglycemia-induced yolk sac vasculopathy. In rodents, the yolk sac serves as the primitive placenta after implantation and before the formation of the chorioallantoic placenta. Furthermore, during this early stage, the nutrition from mother to embryo is considered to be facilitated by a tissue-to-tissue form of nutrition, referred to as histiotropic nutrition, and subsequently via yolk sac facilitation (hemotropic nutrition). In addition, during embryopathy, teratogen such as hyperglycemia is associated with concomitant injury to the yolk sac and embryo. Rat embryos at embryonic day 9 were cultured under euglycemic (150 mg/dL glucose) and hyperglycemic (500 mg /dL glucose) conditions. JNK activation was inhibited using a JNK1/2-specific inhibitor SP60025 at concentrations of 40, 400, and 800 nM. After 48 hours, the development of yolk sac vasculatures was evaluated by assigning to arbitrative scores on the basis of yolk sac vasculature morphology. The correlation between yolk sac vasculature and embryonic malformation rates was assessed. Levels of phosphorylated JNK1/2 and Bcl-2-associated X protein (Bax) in the yolk sacs from conceptuses of the euglycemic and hyperglycemic groups were determined by Western blotting with densitometric quantification. Under hyperglycemic conditions, yolk sac development was morphologically impaired. The yolk sac vasculature score of the hyperglycemic group was significantly lower than that of the euglycemic group. Yolk sac vasculature morphologic scores were inversely correlated with embryonic malformation rates. Levels of phosphorylated JNK1/2 and Bax in yolk sacs of the hyperglycemic group were significantly higher than those in yolk sacs of the euglycemic group. JNK1/2-specific inhibitor, SP600125, ameliorated the adverse effect of hyperglycemia on yolk sac vasculature development. Whereas the vasculature morphologic score of yolk sacs in the hyperglycemic group was 54% lower than that of euglycemic group, the vasculature morphologic score of yolk sacs in hyperglycemic plus 800 nM SP600125 group was as same as that in the euglycemic group. Thus, SP600125 at 800 nM completely reversed hyperglycemia-induced vasculopathy as well as embryopathy. Hyperglycemia in vitro induces yolk sac vasculopathy. Embryonic malformation is inversely correlated with the yolk sac vasculature development, suggesting that hyperglycemia-induced yolk sac vasculopathy may be one of the causative factors in hyperglycemia-induced embryonic malformation. Blockade of JNK1/2 activation restores hyperglycemia-induced vasculopathy and reduces the malformation rates. These findings indicate that JNK1/2 activation mediates the deleterious effect of hyperglycemia on yolk sac vasculature and embryonic development.

Transient Over-Expression of HOX-Regulating Genes CDX2 and MLL during Human Embryoid Body Differentiation Greatly Augments the Generation of Multipotent Hematopoietic Progenitors.

Homeobox (HOX) genes play critical roles in normal anterior-posterior patterning of embryonic development, and in hematopoietic stem cell (HSC) development. Conversely, dysregulated expressions of HOX-regulating factors such as CDX2 (Caudal) and MLL (Mixed Lineage Leukemia) are directly linked to development of acute leukemia. Although CDX family (e.g. cdx4) and mll factors play important roles in murine HSC development, their role in normal human embryonic blood development is obscure. The role of CDX genes (e.g., CDX1, CDX2, CDX4), expressed exclusively during embryonic development, is difficult to evaluate in human hematopoietic development, since fetal tissue is difficult to obtain. Our group has developed a human embryonic stem cell (hESC) differentiation system that recapitulates the 2nd–6th gestational weeks of human yolk sac (YS) development, and initiates from an embryonic hemangioblastic progenitor of primitive and definitive hematopoiesis. The role of HOX-regulating genes (and also HOX-regulating microRNAs (miRNAs), e.g., mir196) that regulate the earliest stages of human hematopoietic development can therefore be studied directly in vitro using our hESC model. We tested the effects of pulsatile, transient over-expression of HOX-regulating factors and miRNAs on the generation of primitive and definitive hematopoeitic progenitors during human embryoid body (hEB) differentiation. Since expression of HOX-regulating genes and miRNAs follow temporal, transient expression patterns during normal embryonic, and also hEB development, we developed a methodology that allows similar transient expression of DNA and RNA molecules at multiple time points of advancing hEB differentiation. This method, termed whole embryoid body (WEB) nucleofection was optimized using GFP-expressing DNA constructs, GFP-silencing siRNA, and also miRNA molecules within intact, whole hEB. WEB nucleofection allowed expression in 15–90% of day 4–9 hEB cells without disrupting their three-dimensional structural integrity, and with minimal toxicity. GFP-nucleofected day 5–13 hEB demonstrated peak expression levels at 48 hrs post-nucleofection, and expression was sustained for approximately one week. A FITC-labeled dsRNA oligonucleotide, was used to demonstrate that the efficiency of WEB nucleofection with RNA molecules approached ∼90%. WEB nucleofection was utilized to transiently over-express CDX2 and MLL constructs within intact, developing hEBs, and the effects on generation of hEB-derived primitive and definitive hematopoiesis were assayed by colony-forming cell (CFC), and FACS analysis. CDX2 and MLL-nucleofected hEB each produced 5-10X greater amounts of multipotent, mixed CFU, in comparison to controls. Moreover, MLL-nucleofected hEB demonstrated a bias toward development of definitive erythroid progenitors. Hematopoietic regulation by over-expression or inhibition of miRNAs implicated in HOX regulation (e.g. mir-196, mir-10) is also currently being evaluated by WEB nucleofection. Our ability to specifically control multiple combinations of transgenic DNA, siRNA or miRNA molecules, temporally and spatially during hEB differentiation, provides novel opportunities to manipulate the CDX-HOX axis for generating and expanding multi-potent hematopoietic progenitors from hESC. The role of HOX-regulating factors and miRNAs involved in regulating the earliest steps of human hematopoietic commitment can now be directly evaluated.

Transient Over-Expression of HOX-Regulating Genes CDX2 and MLL during Human Embryoid Body Differentiation Greatly Augments the Generation of Multipotent Hematopoietic Progenitors.

Homeobox (HOX) genes play critical roles in normal anterior-posterior patterning of embryonic development, and in hematopoietic stem cell (HSC) development. Conversely, dysregulated expressions of HOX-regulating factors such as CDX2 (Caudal) and MLL (Mixed Lineage Leukemia) are directly linked to development of acute leukemia. Although CDX family (e.g. cdx4) and mll factors play important roles in murine HSC development, their role in normal human embryonic blood development is obscure. The role of CDX genes (e.g., CDX1, CDX2, CDX4), expressed exclusively during embryonic development, is difficult to evaluate in human hematopoietic development, since fetal tissue is difficult to obtain. Our group has developed a human embryonic stem cell (hESC) differentiation system that recapitulates the 2nd-6th gestational weeks of human yolk sac (YS) development, and initiates from an embryonic hemangioblastic progenitor of primitive and definitive hematopoiesis. The role of HOX-regulating genes (and also HOX-regulating microRNAs (miRNAs), e.g., mir196) that regulate the earliest stages of human hematopoietic development can therefore be studied directly in vitro using our hESC model. We tested the effects of pulsatile, transient over-expression of HOX-regulating factors and miRNAs on the generation of primitive and definitive hematopoeitic progenitors during human embryoid body (hEB) differentiation. Since expression of HOX-regulating genes and miRNAs follow temporal, transient expression patterns during normal embryonic, and also hEB development, we developed a methodology that allows similar transient expression of DNA and RNA molecules at multiple time points of advancing hEB differentiation. This method, termed whole embryoid body (WEB) nucleofection was optimized using GFP-expressing DNA constructs, GFP-silencing siRNA, and also miRNA molecules within intact, whole hEB. WEB nucleofection allowed expression in 15–90% of day 4–9 hEB cells without disrupting their three-dimensional structural integrity, and with minimal toxicity. GFP-nucleofected day 5–13 hEB demonstrated peak expression levels at 48 hrs post-nucleofection, and expression was sustained for approximately one week. A FITC-labeled dsRNA oligonucleotide, was used to demonstrate that the efficiency of WEB nucleofection with RNA molecules approached ∼90%. WEB nucleofection was utilized to transiently over-express CDX2 and MLL constructs within intact, developing hEBs, and the effects on generation of hEB-derived primitive and definitive hematopoiesis were assayed by colony-forming cell (CFC), and FACS analysis. CDX2 and MLL-nucleofected hEB each produced 5-10X greater amounts of multipotent, mixed CFU, in comparison to controls. Moreover, MLL-nucleofected hEB demonstrated a bias toward development of definitive erythroid progenitors. Hematopoietic regulation by over-expression or inhibition of miRNAs implicated in HOX regulation (e.g. mir-196, mir-10) is also currently being evaluated by WEB nucleofection. Our ability to specifically control multiple combinations of transgenic DNA, siRNA or miRNA molecules, temporally and spatially during hEB differentiation, provides novel opportunities to manipulate the CDX-HOX axis for generating and expanding multi-potent hematopoietic progenitors from hESC. The role of HOX-regulating factors and miRNAs involved in regulating the earliest steps of human hematopoietic commitment can now be directly evaluated.

Low nitric oxide: a key factor underlying copper-deficiency teratogenicity

Copper (Cu)-deficiency-induced teratogenicity is characterized by major cardiac, brain, and vascular anomalies; however, the underlying mechanisms are poorly understood. Cu deficiency decreases superoxide dismutase activity and increases superoxide anions, which can interact with nitric oxide (NO), reducing the NO pool size. Given the role of NO as a developmental signaling molecule, we tested the hypothesis that low NO levels, secondary to Cu deficiency, represent a developmental challenge. Gestation day 8.5 embryos from Cu-adequate (Cu+) or Cu-deficient (Cu−) dams were cultured for 48 h in Cu+ or Cu− medium, respectively. We report that NO levels were low in conditioned medium from Cu−/Cu− embryos and yolk sacs, compared to Cu+/Cu+ controls under basal conditions and with NO synthase (NOS) agonists. The low NO production was associated with low endothelial NOS phosphorylation at serine 1177 and cyclic guanosine-3′,5′-monophosphate (cGMP) concentrations in the Cu−/Cu− group. The altered NO levels in Cu-deficient embryos are functionally significant, as the administration of the NO donor DETA/NONOate increased cGMP and ameliorated embryo and yolk sac abnormalities. These data support the concept that Cu deficiency limits NO availability and alters NO-dependent signaling, which contributes to abnormal embryo and yolk sac development.

Characterization and Molecular Regulation of Embryonic Hemogenic Endothelium

Blood and blood vessels develop in parallel in mammalian embryos. At sites of definitive hematopoiesis, blood cells are thought to be derived from the endothelium. Previous work in our lab defined a population of Hoechst dye-effluxing (SP), Flk+, cKit+, CD45− hemogenic endothelial cells (HEC) residing in the developing yolk sac. The goal of this research is to determine whether such cells reside in the aorta-gonad-mesonephros (AGM) region, the first intra-embryonic site of definitive hematopoiesis, and function as HEC capable of giving rise to all blood lineages and have repopulation capabilities when transplanted into irradiated animal recipients. We are using both immunohistochemical (IHC) techniques and in vivo imaging to track cells throughout development and demonstrate their differentiation from cells residing in the endothelium to blood cells in circulation. Toward this goal, we have isolated SP cells from the AGM region at various stages of development that demonstrate acquired hematopoietic potential in vitro. Also, we have observed endothelial cells expressing stem markers and hematopoietic markers in close approximation to clusters of blood cells within developing vasculature. A greater understanding of the interdependent regulation of blood and blood vessel formation aids in the development of therapeutic strategies to treat human vascular and hematopoietic diseases. NIH #5 R01 EB005173-02

Myocardin‐related transcription factor B is required for normal mouse vascular development and smooth muscle gene expression

Smooth muscle gene expression is required for the proper development and function of multiple organ systems. Expression of smooth muscle genes is critical for contractile function and tissue architectural integrity. One critical transcription factor for smooth muscle gene expression is the Serum Response Factor (SRF). SRF is expressed ubiquitously, but tissue-specific transcriptional regulation is conferred by its binding to cofactors such as myocardin. Myocardin-related transcription factor B (MRTF-B) is a member of a family of genes (Myocardin(Myocd),Myocardin-related transcription factor A(MRTF-A),MRTF-B) that provides tissue-specificity and potentiate SRF-dependent transcription. Unlike myocardin, which is expressed specifically in smooth and cardiac muscle, MRTF-B is expressed in a wide variety of tissues. To examine the function of MRTF-B, we generated mice containing an insertional mutation of MRTF-B. MRTF-B homozygous mutants die in late gestation with vascular defects and liver hemorrhage. At E9.5, MRTF-B is expressed strongly in the septum transversum mesoderm critical for development of the vitelline system that produces the liver sinusoids and portal venous system. MRTF-B deficiency results in defective smooth muscle gene expression in the liver sinusoids, vitelline veins, and yolk sac, which contributes significantly to the lethal phenotype. These data support our hypothesis that MRTF-B has a unique role in regulating smooth muscle genes important for liver, yolk sac, and portal vascular development.

Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.

Fatty acid composition of Oncorhynchus mykiss during embryogenesis and other developmental stages

Fatty acid compositions of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) was determined during embryogenesis, yolk-sac fry, swim up fry and fry stages. Embryonic and yolk-sac development of fertilized eggs were followed until fry weighed 25 g and samples were taken from each stage to analyse fatty acid composition. Analyses of fatty acid composition were performed by gas chromatography mass spectrometry. Throughout all the developmental stages of O. mykiss important changes, such as an increase in the percentage of 18:2, 18:3 and 20:5 and as a decrease in the percentage of 14:0, 16:2, 20:0, 20:2, 20:3, 20:4 and 22:0 were observed in the amounts of fatty acid of developing eggs. During transition from the embryonic to the yolk-sac stage, an important increase in 20:0, 20:3, 20:5, 22:0 and a decrease in 18:3, 22:1 was determined between 45-day old embryo and yolk-sac fry. During transition from the 43 day yolk-sac stage to swim up stage, a decrease in the percentage of 18:0, 20:2 and an increase in the percentage of 14:0, 16:2, 18:2, 18:3 was seen. In all the observed stages, no qualitative but significant quantitative variations were determined in the fatty acid composition of O. mykiss. It may be concluded that 14:0, 16:0, 16:1, 18:0, 18:1 18, 20 and 22 polyunsaturated fatty acids are necessary for normal development of O. mykiss during their life stages.

Is the Fetal Heart a Hematopoietic Organ?

To the Editor: The article by Tomanek et al1 presents well the in vivo data on the requirement of VEGF family members for tubulogenesis and coronary artery formation. The authors observed that VEGF-Trap (a chimera of R1 and R2), apart from precluding formation of coronary arteries, causes a massive accumulation of erythrocytes in the subepicardium and in the interventricular septum. The important finding of this study was that erythrocytes that are components of blood islands derive from epicardial-derived precursors. The authors assume that proepicardial cells include a hemangioblast population. From this observation a conclusion can be drawn that either the proepicardium or the fetal heart exhibits a hematopoietic activity. Considering the topic of blood island derivation in the embryonic heart I would like to comment that although the derivation of the endothelial cell component in these structures has been proven, the origin of erythrocytes is …

Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development

AbstractWe elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESC338) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac–like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.

Disruption of the mouse inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene, associated lethality, and tissue distribution of 2-kinase expression

Many functions have been suggested for inositol 1,2,3,4,5,6-hexakisphosphate (InsP6), including mRNA export, nonhomologous end-joining, endocytosis, and ion channel regulation. However, it remains to be demonstrated that InsP6 is necessary for in vivo survival. We previously isolated a cDNA encoding the mammalian inositol 1,3,4,5,6-pentakisphosphate (InsP5) 2-kinase (2-kinase), the enzyme that converts InsP5 to InsP6. We used the sequence to search the BayGenomics databases and identify an ES cell line (XA232) that has a gene trap construct embedded in the 2-kinase gene. We obtained a mouse from this line, produced heterozygotes, and confirmed that the heterozygotes contain the trapping construct and have diminished 2-kinase activity. Breeding the XA232 heterozygotes produced no homozygous offspring; thus, loss of 2-kinase is lethal in mice. Dissections of embryonic day-8.5 uteri yielded no homozygous embryos; thus, the mice die before day 8.5 postcoitum. The gene trap construct contains a beta-galactosidase/neomycin reporter gene, allowing us to stain heterozygotes for beta-galactosidase to determine tissue-specific expression of 2-kinase protein. 2-kinase is expressed in the hippocampus, the cortex, the Purkinje layer of the cerebellum in the brain, in cardiomyocytes, and in the testes of adult mice. At day 9.5 postcoitum, 2-kinase was expressed in the notochord, the ventricular layer of the neural tube, and the myotome of the somites. Intense staining was also seen in the yolk sac, suggesting that InsP6 is necessary for yolk sac development or function. Furthermore, failure of yolk sac development or function is consistent with the early lethality of 2-kinase embryos.

Effect of ethanol on the development of visceral yolk sac

Prenatal ethanol exposure can cause development retardation and malformations in human offspring. Before the formation of chorioallantoic placenta, yolk sac plays an important role in transporting nutrients from the mother to the embryo. Functional suppression of yolk sac is found to be relevant to the malformations in mammalian embryos. Female 8.5-day C57BL/6J mouse embryos were cultured in vitro and exposed to different doses of ethanol. The development of visceral yolk sac (VYS) was examined with light and electron microscopes. The expression profiles of some vasculogenesis-related genes were detected with reverse transcription-PCR. A dose-dependent toxicity to the VYS was found, including reduced diameter, decreased protein and DNA contents, and suppressed development of vitelline vessels. The hypogenesis of VYS agreed with the retarded development and/or malformations found in the embryos. Histological and functional alterations were found in the ethanol-exposed VYS endodermal cells. The expressions of vasculogenesis-related genes, fetal liver kinase 1 (Flk1) and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie2), were repressed by ethanol. Impaired structural and functional development of VYS may contribute to the teratogenic action of ethanol in mice, which may also provide a clue to the study of fetal alcohol syndrome in humans.

Edd, the murine hyperplastic disc gene, is essential for yolk sac vascularization and chorioallantoic fusion.

EDD is the mammalian ortholog of the Drosophila melanogaster hyperplastic disc gene (hyd), which is critical for cell proliferation and differentiation in flies through regulation of hedgehog and decapentaplegic signaling. Amplification and overexpression of EDD occurs frequently in several cancers, including those of the breast and ovary, and truncating mutations of EDD are also observed in gastric and colon cancer with microsatellite instability. EDD has E3 ubiquitin ligase activity, is involved in regulation of the DNA damage response, and may control hedgehog signaling, but a definitive biological role has yet to be established. To investigate the role of Edd in vivo, gene targeting was used to generate Edd knockout (Edd(Delta/Delta)) mice. While heterozygous mice had normal development and fertility, no viable Edd-deficient embryos were observed beyond E10.5, with delayed growth and development evident from E8.5 onward. Failed yolk sac and allantoic vascular development, along with defective chorioallantoic fusion, were the primary effects of Edd deficiency. These extraembryonic defects presumably compromised fetal-maternal circulation and hence efficient exchange of nutrients and oxygen between the embryo and maternal environment, leading to a general failure of embryonic cell proliferation and widespread apoptosis. Hence, Edd has an essential role in extraembryonic development.

Two-step consumption of yolk granules during the development of quail embryos.

The mechanism of yolk consumption was studied morphologically and biochemically in Japanese quail Coturnix japonica. The amount of yolk granules in the yolk (or 'yolk cell') decreased in two steps during embryonic development. In the first step, during days 0-4 of incubation, the yolk-granule weight decreased at a rate of 13 mg/day. This decrease was due to segregation by endodermal cells that were newly formed in the developing yolk sac. In the second step after day 6, the decrease was drastic at a rate of 29.8 mg/day during days 6-12 and very slow thereafter. The decrease at the second step was due to the enzymatic digestion of yolk granules by cathepsin D that coexisted in yolk spheres. This digesting reaction was triggered by the solubilization of the granules with high concentrations of salts that were supplied after disruption of the limiting membrane of yolk spheres. The 'yolk cell' seemed to die around day 5 of incubation. Thus the digestion products might be taken up together with yolk lipids by endocytosis into the endodermal cells and transported to blood vessels.

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.

Flow regulates arterial-venous differentiation in the chick embryo yolk sac.

Formation of the yolk sac vascular system and its connection to the embryonic circulation is crucial for embryo survival in both mammals and birds. Most mice with mutations in genes involved in vascular development die because of a failure to establish this circulatory loop. Surprisingly, formation of yolk sac arteries and veins has not been well described in the recent literature. Using time-lapse video-microscopy, we have studied arterial-venous differentiation in the yolk sac of chick embryos. Immediately after the onset of perfusion, the yolk sac exhibits a posterior arterial and an anterior venous pole, which are connected to each other by cis-cis endothelial interactions. To form the paired and interlaced arterial-venous pattern characteristic of mature yolk sac vessels, small caliber vessels of the arterial domain are selectively disconnected from the growing arterial tree and subsequently reconnected to the venous system, implying that endothelial plasticity is needed to fashion normal growth of veins. Arterial-venous differentiation and patterning are controlled by hemodynamic forces, as shown by flow manipulation and in situ hybridization with arterial markers ephrinB2 and neuropilin 1, which show that expression of both mRNAs is not genetically determined but plastic and regulated by flow. In vivo application of ephrinB2 or EphB4 in the developing yolk sac failed to produce any morphological effects. By contrast, ephrinB2 and EphB4 application in the allantois of older embryos resulted in the rapid formation of arterial-venous shunts. In conclusion, we show that flow shapes the global patterning of the arterial tree and regulates the activation of the arterial markers ephrinB2 and neuropilin 1.