Deciphering the role of retinoic acid in hepatic patterning and induction in the mouse.

Retinoic acid (RA), a metabolite of vitamin A, is a small molecule and morphogen that is required for embryonic development. While normal RA signals are required for hepatic development in a variety of vertebrates, a role for RA during mammalian hepatic specification has yet to be defined. To examine the requirement for RA in murine liver induction, we performed whole embryo culture with the small molecule RA inhibitor, BMS493, to attenuate RA signaling immediately prior to hepatic induction and through liver bud formation. BMS493 treated embryos demonstrated a significant loss of hepatic specification that was confined to the prospective dorsal anterior liver bud. Examination of RA attenuated embryos demonstrates that while the liver bud displays normal expression of foregut endoderm markers and the hepato-pancreatobiliary domain marker, PROX1, the dorsal/anterior liver bud excludes the critical hepatic marker, HNF4α, indicating that RA signals are required for dorsal/anterior hepatic induction. These results were confirmed and extended by careful examination of Rdh10^trex/trex embryos, which carry a genetic perturbation in RA synthesis. At E9.5 Rdh10^trex/trex embryos display a similar yet more significant loss of the anterior/dorsal liver bud. Notably the anterior/dorsal liver bud loss correlates with the known dorsal-ventral gradient of the RA synthesis enzyme, Aldh1a2. In addition to altered hepatic specification, the mesoderm surrounding the liver bud is disorganized in RA abrogated embryos. Analysis of E10.5 Rdh10^trex/trex embryos reveals small livers that appear to lack the dorsal/caudal lobes. Finally, addition of exogenous RA prior to hepatic induction results in a liver bud that has failed to thicken and is largely unspecified. Taken together our ex vivo and in vivo evidence demonstrate that the generation of normal RA gradients is required for hepatic patterning, specification, and growth.

Patterning of the hepato-pancreatobiliary boundary by BMP reveals heterogeneity within the murine liver bud.

During development, the endoderm initiates organ-restricted gene expression patterns in a spatiotemporally controlled manner. This process, termed induction, requires signals from adjacent mesodermal derivatives. Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) emanating from the cardiac mesoderm and the septum transversum mesenchyme (STM), respectively, are believed to be simultaneously and uniformly required to directly induce hepatic gene expression from the murine endoderm. Using small molecule inhibitors of BMP signals during liver bud induction in the developing mouse embryo, we found that BMP signaling was not uniformly required to induce hepatic gene expression. Although BMP inhibition caused an overall reduction in the number of induced hepatoblasts, the STM-bounded posterior liver bud demonstrated the most severe loss of the essential hepatic transcription factor, hepatocyte nuclear factor 4-α (HNF4α), whereas the sinus venosus-lined anterior liver bud was less affected. We found that the posterior liver bud progenitors were anteriorly displaced and aberrantly activated pancreatobiliary markers, including sex-determining region Y-box 9 (SOX9). Additionally, we found that ectopically expressed SOX9 inhibited HNF4α and that BMP was indirectly required for hepatoblast induction. Finally, because previous studies have demonstrated that FGF signals are essential for anterior but not posterior liver bud induction, we examined synchronous BMP and FGF inhibition and found this led to a nearly complete loss of hepatoblasts. CONCLUSION: BMP signaling is required to maintain the hepato-pancreatobiliary boundary, at least in part, by indirectly repressing SOX9 in the hepatic endoderm. BMP and FGF signals are each required for the induction of spatially complementary subsets of hepatoblasts. These results underscore the importance of studying early inductive processes in the whole embryo. (Hepatology 2018;68:274-288).