Fetal and umbilical blood vessels form independently;however, the means by which they connect to each other to ensure fetal-maternal exchange during gestation is obscure. The goal of this new grant proposal is to elucidate the origin and mechanism of formation of a small hemogenic vessel, provisionally named the Vessel of Confluence (VOC), in the mouse embryo. The function of the VOC is to connect the umbilical artery to the embryonic dorsal aortae, thereby ensuring a continuous arterial system between the developing conceptus and its mother. How the VOC forms is a major question in reproductive biology and essential for successful gestational outcome. However, description of the VOC has no precedent in the literature. The VOC forms in close proximity to visceral endoderm/hypoblast, an extraembryonic tissue found in all amniotes. Closer inspection of the visceral endoderm revealed the unexpected presence of a stage- and site-specific gap within an otherwise continuous epithelium. This gap separated yolk sac blood islands from allantois-associated posterior visceral endoderm (aPVE), creating within the latter a distinct developmental domain that uniquely expressed Hedgehog (Hh). Unexpectedly, the aPVE contributed descendant cells to the VOC, whose site of formation was rich in Fibroblast Growth Factor (FGF) and heparan sulfate proteoglycan (HSPG) signaling. Pharmacological inhibition of FGF and HSPG resulted in failure of the VOC to form, while alterations in Hh signaling led to significant disruptions in vessel size and shape. On the basis of these data, we hypothesize that gap formation triggers an epithelial-to-mesenchymal transition (EMT) within the aPVE, leading to formation of the VOC and orchestrated by interaction between Hh, FGFs, and HSPGs. Using the mouse as our model system, we will combine genetics and molecular biology with classical techniques of embryology in living embryos to investigate establishment of the fetal-umbilical connection. Because FGF and Hh signaling pathways play well-established critical roles in all of the fundamental biological processes under study here, including apoptosis, epithelial-to-mesenchymal transition, and vasculo/hemogenesis, and because upstream and downstream pathways are well-characterized, they are highly promising candidates in which to gain entr?e into the fetal-umbilical interface, and dissect at a hitherto unprecedented pace the complex signaling pathways that guide VOC formation. In birthing rooms around the world, the umbilical cord is just cursorily examined, scrutinized only if the fetus is in obvious distress. Yet, given te cord's intimate anatomical association with the fetal vasculature, careful analysis of the neonate's umbilical cord must undoubtedly hold clues to disease, which if identified early, promise much-improved long-term prognosis. We anticipate that successful outcome of this study will alert scientists and clinicians to the VOC as a potential risk factor in neonatal defect that merits further screening and evaluation prior to routine disposal of the afterbirth.

Public Health Relevance

This new research proposal addresses establishment of the fetal-umbilical connection, which is critical for survival and development of the fetus during gestation. While at least half of all umbilical-associated birth defects are classified as Rare and Orphan Diseases by the National Institutes of Health (://rarediseases.info.nih.gov/), their underlying etiology is unknown, as the mechanism by which the umbilical vasculature joins to that of the fetus is completely obscure. Recently, we discovered a small arterial blood vessel, provisionally named the Vessel of Confluence (VOC), which, at a precise time and place, connects the umbilical vasculature to the arterial systems of the fetus and yolk sac. Unexpectedly, the origin of the VOC appears to lie within a segment of hitherto poorly-characterized hypoblast, an extraembryonic inducing tissue common to all Mammalia yet whose properties elsewhere in the conceptus are only just emerging. Referred to as allantois-associated posterior visceral endoderm (aPVE), we will test the hypothesis that this cryptic epithelium drives the formation of the VOC via two major signaling pathways, Fibroblast Growth Factor (FGF) and Hedgehog (Hh), both of which display unique localization patterns in this region. Intriguingly, Hh and FGFs have been implicated in two orphan diseases, omphalocoele (Hh) at the umbilical ring, and sirenomelia (FGF), in which a single umbilical artery is misconnected to the fetal midline aorta. We anticipate that our results will not only provide critical new insight into these severe birth defects, but serve as a fundamental first step in unifying the etiology of a large number of apparently unrelated Rare and Orphan Human Diseases.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Pregnancy and Neonatology Study Section (PN)
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Ilekis, John V
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University of Wisconsin Madison
Anatomy/Cell Biology
Schools of Medicine
United States
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