Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA.
Department of Oral Immunology and Infectious Diseases, School of Dentistry, University of Louisville, Louisville, KY, USA.
Dev Biol. 2022 Nov;491:31-42. doi: 10.1016/j.ydbio.2022.08.003. Epub 2022 Aug 23.
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<sup>trex/trex</sup> embryos, which carry a genetic perturbation in RA synthesis. At E9.5 Rdh10<sup>trex/trex</sup> 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<sup>trex/trex</sup> 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.
视黄酸（RA）是维生素 A 的代谢产物，是一种小分子和形态发生素，对于胚胎发育是必需的。虽然各种脊椎动物的胚胎发育都需要正常的 RA 信号，但 RA 在哺乳动物肝脏特化过程中的作用尚未确定。为了研究 RA 在鼠类肝脏诱导中的需求，我们在胚胎整体培养中使用小分子 RA 抑制剂 BMS493，在肝诱导前及肝芽形成过程中减弱 RA 信号。BMS493 处理的胚胎显示出明显的肝特化丧失，仅限于前背侧肝芽。对 RA 减弱的胚胎进行检查表明，虽然肝芽显示出前肠内胚层标记物和肝胰胆管域标记物 PROX1 的正常表达，但背侧/前侧肝芽排除了关键的肝标记物 HNF4α，表明 RA 信号对于背侧/前侧肝诱导是必需的。这些结果通过仔细检查 Rdh10<sup>trex/trex</sup> 胚胎得到了证实和扩展，该胚胎携带 RA 合成的遗传扰动。在 E9.5 时，Rdh10<sup>trex/trex</sup> 胚胎显示出类似但更严重的前背侧肝芽丧失。值得注意的是，前背侧肝芽丧失与 RA 合成酶 Aldh1a2 的已知背腹梯度相关。除了肝特化改变外，肝芽周围的中胚层在 RA 消除的胚胎中也出现紊乱。对 E10.5 Rdh10<sup>trex/trex</sup> 胚胎的分析表明，肝脏较小，似乎缺乏背侧/尾侧叶。最后，在肝诱导前添加外源性 RA 会导致肝芽未能增厚，并且基本上未特化。总之，我们的体外和体内证据表明，正常 RA 梯度的产生对于肝脏的模式形成、特化和生长是必需的。
