Defective vascular morphogenesis and mid-gestation embryonic death in mice lacking RA-GEF-1

A multitude of guanine nucleotide exchange factors (GEFs) regulate Rap1 small GTPases, however, their individual functions remain obscure. Here, we investigate the in vivo function of the Rap1 GEF RA-GEF-1. The expression of RA-GEF-1 in wild-type mice starts at embryonic day (E) 8.5, and continues thereafter. RA-GEF-1(-/-) mice appear normal until E7.5, but become grossly abnormal and dead by E9.5. This mid-gestation death appears to be closely associated with severe defects in yolk sac blood vessel formation. RA-GEF-1(-/-) yolk sacs form apparently normal blood islands by E8.5, but the blood islands fail to coalesce into a primary vascular plexus, indicating that vasculogenesis is impaired. Furthermore, RA-GEF-1(-/-) embryos proper show severe defects in the formation of major blood vessels. These results suggest that deficient Rap1 signaling may lead to defective vascular morphogenesis in the yolk sac and embryos proper.     

Role of vascular endothelial-cadherin in vascular morphogenesis

Vascular endothelial (VE)-cadherin is an adhesive transmembrane protein specifically expressed at interendothelial junctions. Its extracellular domain exhibits Ca2+-dependent homophilic reactivity, promoting cell-cell recognition. Mice deficient in VE-cadherin die at mid-gestation resulting from severe vascular defects. At the early phases of vascular development (E8.5) of VE-cadherin-deficient embryos, in situ differentiation of endothelial cells was delayed although their differentiation program appeared normal. Vascularization was defective in the anterior part of the embryo, while dorsal aortae and vitelline and umbilical arteries formed normally in the caudal part. At E9.25, organization of endothelial cells into large vessels was incomplete and angiogenesis was impaired in mutant embryos. Defects were more severe in extraembryonic vasculature. Blood islands of the yolk sac and clusters of angioblasts in allantois failed to establish a capillary plexus and remained isolated. This was not due to defective cell-cell recognition as endothelial cells formed intercellular junctions, as shown by electron microscopy. These data indicate that VE-cadherin is dispensable for endothelial homophilic adhesion but is required for vascular morphogenesis.     

Connexin31-deficiency in mice causes transient placental dysmorphogenesis but does not impair hearing and skin differentiation

Mutations in the human GJB3 gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis. To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZ reporter gene. Although homozygous Cx31-deficient adult mice (Gjb3(-/-)) were found among the offspring of heterozygous Cx31-deficient parents (Gjb3(+/-)), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of Gjb3(-/-) embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving Gjb3(-/-) embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult Gjb3(-/-) mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse.     

Emoxipin as an inhibitor of angiogenesis]

The effect of emoxypin on angiogenesis in rabbit cornea in aseptic inflammation induced by intracorneal implantation of a piece of quartz and on the development of the vessels of the chick embryo yolk sac was studied. 1% emoxypin pipetted thrice a day for 10-14 days inhibited corneal neovascularization and reduced the formation of new blood vessels. We observed an inhibitory effect on the development of vascular bed of the embryo yolk sac on incubation hour 64-72. The drug affected neither general growth of the embryos no the number of somites.     

Essential function of PTP-PEST during mouse embryonic vascularization, mesenchyme formation, neurogenesis and early liver development

PTP (protein-tyrosine phosphatase)-PEST is a ubiquitously expressed cellular regulator of integrin signalling. It has been shown to bind several molecules such as Shc, paxillin and Grb2, that are involved downstream of FAK (focal adhesion kinase) pathway. Through its specific association to p130cas and further dephosphorylation, PTP-PEST plays a critical role in cell-matrix interactions, which are essential during embryogenesis. We report here that ablation of the gene leads to early embryonic lethality, correlating well with the high expression of the protein during embryonic development. We observed an increased level of tyrosine phosphorylation of p130cas protein in E9.5 PTP-PEST(-/-) embryos, a first evidence of biochemical defect leading to abnormal growth and development. Analysis of null mutant embryos revealed that they reach gastrulation, initiate yolk sac formation, but fail to progress through normal subsequent developmental events. E9.5-10.5 PTP-PEST(-/-) embryos had morphological abnormalities such as defective embryo turning, improper somitogenesis and vasculogenesis, impaired liver development, accompanied by degeneration in both neuroepithelium and somatic epithelia. Moreover, in embryos surviving until E10.5, the caudal region was truncated, with severe mesenchyme deficiency and no successful liver formation. Defects in embryonic mesenchyme as well as subsequent failure of proper vascularization, liver development and somatogenesis, seemed likely to induce lethality at this stage of development, and these results confirm that PTP-PEST plays an essential function in early embryogenesis.     

Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells

Zonula occludens (ZO)-1/2/3 are the members of the TJ-MAGUK family of membrane-associated guanylate kinases associated with tight junctions. To investigate the role of ZO-1 (encoded by Tjp1) in vivo, ZO-1 knockout (Tjp1(-/-)) mice were generated by gene targeting. Although heterozygous mice showed normal development and fertility, delayed growth and development were evident from E8.5 onward in Tjp1(-/-) embryos, and no viable Tjp1(-/-) embryos were observed beyond E11.5. Tjp1(-/-) embryos exhibited massive apoptosis in the notochord, neural tube area, and allantois at embryonic day (E)9.5. In the yolk sac, the ZO-1 deficiency induced defects in vascular development, with impaired formation of vascular trees, along with defective chorioallantoic fusion. Immunostaining of wild-type embryos at E8.5 for ZO-1/2/3 revealed that ZO-1/2 were expressed in almost all embryonic cells, showing tight junction-localizing patterns, with or without ZO-3, which was confined to the epithelial cells. ZO-1 deficiency depleted ZO-1-expression without influence on ZO-2/3 expression. In Tjp1(+/+) yolk sac extraembryonic mesoderm, ZO-1 was dominant without ZO-2/3 expression. Thus, ZO-1 deficiency resulted in mesoderms with no ZO-1/2/3, associated with mislocalization of endothelial junctional adhesion molecules. As a result, angiogenesis was defected in Tjp1(-/-) yolk sac, although differentiation of endothelial cells seemed to be normal. In conclusion, ZO-1 may be functionally important for cell remodeling and tissue organization in both the embryonic and extraembryonic regions, thus playing an essential role in embryonic development.     

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.     

A mutant receptor tyrosine phosphatase,CD148, causes defects in vascular development

Vascularization defects in genetic recombinant mice have defined critical roles for a number of specific receptor tyrosine kinases. Here we evaluated whether an endothelium-expressed receptor tyrosine phosphatase, CD148 (DEP-1/PTPeta), participates in developmental vascularization. A mutant allele, CD148(DeltaCyGFP), was constructed to eliminate CD148 phosphatase activity by in-frame replacement of cytoplasmic sequences with enhanced green fluorescent protein sequences. Homozygous mutant mice died at midgestation, before embryonic day 11.5 (E11.5), with vascularization failure marked by growth retardation and disorganized vascular structures. Structural abnormalities were observed as early as E8.25 in the yolk sac, prior to the appearance of intraembryonic defects. Homozygous mutant mice displayed enlarged vessels comprised of endothelial cells expressing markers of early differentiation, including VEGFR2 (Flk1), Tal1/SCL, CD31, ephrin-B2, and Tie2, with notable lack of endoglin expression. Increased endothelial cell numbers and mitotic activity indices were demonstrated. At E9.5, homozygous mutant embryos showed homogeneously enlarged primitive vessels defective in vascular remodeling and branching, with impaired pericyte investment adjacent to endothelial structures, in similarity to endoglin-deficient embryos. Developing cardiac tissues showed expanded endocardial projections accompanied by defective endocardial cushion formation. These findings implicate a member of the receptor tyrosine phosphatase family, CD148, in developmental vascular organization and provide evidence that it regulates endothelial proliferation and endothelium-pericyte interactions.     

Apolipoprotein B-related gene expression and ultrastructural characteristics of lipoprotein secretion in mouse yolk sac during embryonic development.

In mice, the yolk sac appears to play a crucial role in nourishing the developing embryo, especially during embryonic days (E) 7;-10. Lipoprotein synthesis and secretion may be essential for this function: embryos lacking apolipoprotein (apo) B or microsomal triglyceride transfer protein (MTP), both of which participate in the assembly of triglyceride-rich lipoproteins, are apparently defective in their ability to export lipoproteins from yolk sac endoderm cells and die during mid-gestation. We therefore analyzed the embryonic expression of apoB, MTP, and alpha-tocopherol transfer protein (alpha-TTP), which have been associated with the assembly and secretion of apoB-containing lipoproteins in the adult liver, at different developmental time points. MTP expression or activity was found in the yolk sac and fetal liver, and low levels of activity were detected in E18.5 placentas. alpha-TTP mRNA and protein were detectable in the fetal liver, but not in the yolk sac or placenta. Ultrastructural analysis of yolk sac visceral endoderm cells demonstrated nascent VLDL within the luminal spaces of the rough endoplasmic reticulum and Golgi apparatus at E7.5 and E8.5. The particles were reduced in diameter at E13.5 and reduced in number at E18.5;-19.The data support the hypothesis that the yolk sac plays a vital role in providing lipids and lipid-soluble nutrients to embryos during the early phases (E7;-10) of mouse development. secretion in mouse yolk sac during embryonic development.     

Keratin 5-Cre-driven excision of nonmuscle myosin IIA in early embryo trophectoderm leads to placenta defects and embryonic lethality

In studies initially focused on roles of nonmuscle myosin IIA (NMIIA) in the developing mouse epidermis, we have discovered that a previously described cytokeratin 5 (K5)-Cre gene construct is expressed in early embryo development. Mice carrying floxed alleles of the nonmuscle myosin II heavy chain gene (NMHC IIA^flox/flox) were crossed with the K5-Cre line. The progeny of newborn pups did not show a Mendelian genotype distribution, suggesting embryonic lethality. Analysis of post-implantation conceptuses from embryonic day (E)9.5 to E13.5 revealed poorly developed embryos and defective placentas, with significantly reduced labyrinth surface area and blood vessel vascularization. These results suggested the novel possibility that the bovine K5 promoter-driven Cre-recombinase was active early in trophoblast-lineage cells that give rise to the placenta. To test this possibility, K5-Cre transgenic mice were crossed with the mT/mG reporter mouse in which activation of GFP expression indicates Cre transgene expression. We observed activation of K5-Cre-driven GFP expression in the ectoplacental cone, in the extraembryonic ectoderm, and in trophoblast giant cells in the E6.5 embryo. In addition, we observed GFP expression at E11.5 to E13.5 in both the labyrinth of the placenta and the yolk sac. NMIIA expression was detected in these same cell types in normal embryos, as well as in E13.5 yolk sac and labyrinth. These findings taken together suggest that NMHC IIA may play critical roles in the early trophoblast-derived ectoplacental cone and extraembryonic ectoderm, as well as in the yolk sac and labyrinth tissues that form later. Our findings are consistent with phenotypes of constitutive NMIIA knockout mice made earlier, that displayed labyrinth and yolk sac-specific defects, but our findings extend those observations by suggesting possible NMIIA roles in trophoblast lineages as well. These results furthermore demonstrate that K5-Cre gene constructs, previously reported to be activated starting at approximately E12.5 in the forming epidermis, may be widely useful as drivers for activation of cre/lox based gene excision in early embryo extraembronic trophoblast tissues as well.     

Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect

The gap junction protein connexin45-deficient (Cx45-KO) mice die shortly after the hearts begin to beat. In addition to the heart defect, they also show defective vascular development which may be closely related with the cardiac phenotype. Therefore, we created mice whose floxed-Cx45 locus could be removed conditionally. We utilized cardiac alpha-actin-Cre transgenic mice to investigate the specific cardiac muscular function of Cx45 in vivo. The resultant conditional mutants were lethal, showing conduction block similar to that of the Cx45-KO mice. Unlike Cx45-KO, development of the endocardial cushion was not disrupted in the conditional mutants. X-gal staining was detected in the embryonic cardiac myocytes as a hallmark of Cre-loxP mediated floxed-Cx45 deletion. These results reconfirm the requirement of Cx45 for developing cardiac myocytes. These also indicate that establishing the first contractions is a crucial task for the early hearts.     

Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development

Autotaxin (ATX), or nucleotide pyrophosphatase-phosphodiesterase 2, is a secreted lysophospholipase D that promotes cell migration, metastasis, and angiogenesis. ATX generates lysophosphatidic acid (LPA), a lipid mitogen and motility factor that acts on several G protein-coupled receptors. Here we report that ATX-deficient mice die at embryonic day 9.5 (E9.5) with profound vascular defects in yolk sac and embryo resembling the Galpha13 knockout phenotype. Furthermore, at E8.5, ATX-deficient embryos showed allantois malformation, neural tube defects, and asymmetric headfolds. The onset of these abnormalities coincided with increased expression of ATX and LPA receptors in normal embryos. ATX heterozygous mice appear healthy but show half-normal ATX activity and plasma LPA levels. Our results reveal a critical role for ATX in vascular development, indicate that ATX is the major LPA-producing enzyme in vivo, and suggest that the vascular defects in ATX-deficient embryos may be explained by loss of LPA signaling through Galpha13.     

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.     

Conditional deletion of Dicer in vascular smooth muscle cells leads to the developmental delay and embryonic mortality

Dicer is a RNAase III enzyme that cleaves double stranded RNA and generates small interfering RNA (siRNA) and microRNA (miRNA). The goal of this study is to examine the role of Dicer and miRNAs in vascular smooth muscle cells (VSMCs). We deleted Dicer in VSMCs of mice, which caused a developmental delay that manifested as early as embryonic day E12.5, leading to embryonic death between E14.5 and E15.5 due to extensive hemorrhage in the liver, brain, and skin. Dicer KO embryos showed dilated blood vessels and a disarray of vascular architecture between E14.5 and E15.5. VSMC proliferation was significantly inhibited in Dicer KOs. The expression of VSMC marker genes were significantly downregulated in Dicer cKO embryos. The vascular structure of the yolk sac and embryo in Dicer KOs was lost to an extent that no blood vessels could be identified after E15.5. Expression of most miRNAs examined was compromised in VSMCs of Dicer KO. Our results indicate that Dicer is required for vascular development and regulates vascular remodeling by modulating VSMC proliferation and differentiation.     

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.     

Expression patterns of gap junctional proteins connexin 32 and 43 suggest new communication compartments in the gastrulating rabbit embryo

A central problem in embryological research is the identification of mechanisms by which control over the development of a viable individual is maintained. An important role in this process is attributed to intercellular communication the preconditions of which were examined in the present study. Using a range of monoclonal antibodies, the expression patterns of the gap junctional proteins connexin 32 (Cx32) and connexin 43 (Cx43) were examined in whole-mount preparations and cryosections of gastrulating rabbit embryos between 6.0 and 7.5 days post conception. Distinct distribution patterns for Cx32 and Cx43, respectively, were found: Cx32 was exclusively expressed in the hypoblast and yolk sac epithelium (the lower layer of the embryo) whereas Cx43-expression was limited to the epiblast (in the upper layer) and its derivatives. Moreover, the dynamics of the Cx32 and Cx43 expression patterns indicate the existence of smaller tissue compartments within the three embryonic cell layers present at the beginning of gastrulation (epiblast, mesoderm and hypoblast). The most striking one of these smaller compartments is a belt-like area within the lower layer which straddles the epiblast-trophoblast border seen in the upper layer of the embryonic disc. The significance of these compartments for initiating and maintaining the gastrulation process is discussed.