Characterization of the Role of Intrinsic and Extrinsic Factors During Murine Endoderm Development

Yin Yang1 (YY1) is a ubiquitously expressed factor that plays multiple roles in early mouse development. We have found that an essential role of YY1 in the visceral endoderm (VE) of the yolk sac is the maintenance of VEGF. Furthermore, we have demonstrated that paracrine signals downstream of VEGF support the VE, assessed by maintenance of HNF4α. Because the VE is essential for yolk sac development, and thus for embryonic survival, we used an inducible-knockout strategy to demonstrate that YY1 is essential in the definitive endoderm (DE) for invasion of hepatoblasts into the surrounding mesenchyme. By E14.5 YY1-/- livers are severely hypoplastic, and although YY1-deficient DE is capable of liver bud specification, the hepatoblasts fail to initiate invasion, upregulate VEGF or maintain HNF4α. The addition of exogenous VEGF to YY1-deficient liver buds rescues HNF4α expression, while addition of a VEGFR inhibitor to wild-type embryos mimics the specific temporal and spatial phenotypes of the YY1-/- hepatoblasts. Taken together, these studies demonstrate that a conserved role of YY1 in both the VE and liver bud is the production of VEGF. Careful analysis of early normal liver bud development reveals two temporally and spatially discreet waves of liver bud invasion. The first, initiated at E9.25, involves migration of the posterior liver bud while the second, initiated by E10.0, involves the anterior liver bud. Lineage analysis demonstrates that the anterior and posterior regions of the liver bud contribute to the caudal and rostral lobes, respectively, of the E10.25 liver. We have also previously observed differential effects upon inhibition of FGF signaling. When we impaired FGF in cultured embryos, we saw that the anterior portion of the liver bud was hypoplastic, lost molecular specification and underwent apoptosis while the posterior appeared normal. These studies, through examination of FGF and VEGF and the requirement for YY1, are the first to document that there are two spatially and temporally discreet waves of liver bud invasion that each contribute to distinct lobes