Hhex-a Homeobox Gene Necessary for Liver Development

描述（由申请方提供）：具有同源异型盒基因Hhex无效突变的小鼠在妊娠中期死亡，具有多种发育缺陷，包括完全没有肝脏。最近，我们获得了两个品系的小鼠，在发育中的肝脏中缺失Hhex-在E9.0的肝芽和在E10.5 - 11的肝母细胞。我们发现，Hhex是必要的肝脏生长，肝细胞分化，肝外和肝内胆管发育。此外，我们发现Hhex的缺失与Hnf 4a，Hnf 1b（TCF 2）和Onecut 1（Hnf 6）的表达降低相关，表明Hhex直接或间接调节这些基因的表达。在肝脏中，Hnf 4a和Onecut 1是正常成肝细胞分化所必需的，并且已知它们调节彼此的表达。此外，有大量数据表明Onecut 1，Hnf 1b和Pkhd 1在高度保守的分子途径中发挥作用，这对肝脏，胰腺和肾脏中的正常上皮管形成和纤毛功能至关重要。此外，纤毛缺陷/缺失的小鼠具有异常的hedgehog信号传导。来自我实验室的新的初步数据显示，在肝脏特异性缺失Hhex的小鼠中，胆管纤毛短或缺失，并且囊肿相关基因Pkhd 1的表达显著降低。因此，我们假设Hhex调节发育中肝脏中由Hnf 4a，Onecut 1，Hnf 1b和Pkhd 1组成的分子途径。在不存在Hhex的情况下，这些基因的表达降低，这导致纤毛形成/信号传导缺陷、上皮管腔形成受干扰以及随后的囊肿形成和成肝细胞分化缺陷。我们建议通过实现以下具体目标来测试这一假设：目标1。建立Hhex，Hnf 4a和Onecut 1在调节肝母细胞命运中的遗传等级，并确定这种调节是否是细胞自主的和阶段特异性的;目的2。确定Foxa 3-Cre; Hhexd 2，3/-和Alfp-Cre; Hhexd 2，3/-小鼠中的囊肿形成和有缺陷的纤毛生物发生/功能是否是由于Onecut 1hHnf 1bhPkhd 1hcilia遗传途径的Hhex调节所致;目的3.确定Hhex如何在体外调节Hnf 4a，Onecut 1，Hnf 1b和Pkhd 1，以及这些基因和Hhex之间是否存在遗传相互作用;和目标4。确定在Hhex突变小鼠中观察到的有缺陷的纤毛生物发生是否扰乱了hedgehog信号传导。我们将使用在我的实验室中产生的独特的小鼠品系和肝源性细胞系来实现这些目标。我们已经概述了一个全面的体外和体内方法的实验方法，将确定新的机制控制肝细胞的细胞命运的决定，纤毛生物发生和信号，和囊肿的形成。我们产生的条件性基因敲除小鼠为肝脏研究界提供了重要的资源。它们为理解肝脏发育中早期细胞谱系决定的基础，理解上皮管腔形成和囊肿形成的分子基础以及测试多囊肝病的新疗法提供了机会。我们的研究还将确定在肝脏发育中起作用的新的遗传途径，这对肝脏再生和修复具有重要意义。公共卫生相关性：这些研究阐明的发育机制将为识别产生用于肝病细胞治疗的分化细胞所需的遗传因子提供关键知识。此外，这项研究将揭示胆道疾病的发病机制，包括胆道闭锁和多囊肝病。

DESCRIPTION (provided by applicant): Mice with a null mutation of the homeobox gene Hhex die mid-gestation with multiple developmental defects, including complete absence of the liver. Recently, we derived two strains of mice with a deletion of Hhex in the developing liver - at E9.0 in the liver bud and at E10.5 - 11 in hepatoblasts. We showed that Hhex is necessary for liver growth, hepatoblast differentiation, and both extra-hepatic and intra-hepatic bile duct development. Furthermore, we showed that deletion of Hhex is associated with decreased expression of Hnf4a, Hnf1b (TCF2) and Onecut1 (Hnf6), suggesting that Hhex regulates, either directly or indirectly, the expression of these genes. In the liver, Hnf4a and Onecut1 are necessary for normal hepatoblast differentiation and are known to regulate the expression of each other. Moreover, there are abundant data that Onecut1, Hnf1b and Pkhd1 function in a highly-conserved molecular pathway that is critical for normal epithelial tube formation and ciliary function in the liver, pancreas and kidney. Furthermore, mice with defective/absent cilia have aberrant hedgehog signaling. New preliminary data from my laboratory reveal that, in mice with a liver-specific deletion of Hhex, bile duct cilia are short or absent and the expression of the cyst-related gene Pkhd1 is markedly decreased. Therefore, we hypothesize that Hhex regulates molecular pathways consisting of Hnf4a, Onecut1, Hnf1b and Pkhd1 in the developing liver. In the absence of Hhex, the expression of these genes is decreased which leads to defective cilia formation/signaling, perturbed epithelial lumen formation with subsequent cyst formation, and defective hepatoblast differentiation. We propose to test this hypothesis by accomplishing the following specific aims: Aim 1. Establish the genetic hierarchy of Hhex, Hnf4a and Onecut1 in regulating hepatoblast cell fate and determine if this regulation is cell autonomous and stage-specific; Aim 2. Determine if cyst formation and defective ciliary biogenesis/function in Foxa3-Cre; Hhexd2, 3/- and Alfp-Cre; Hhexd2,3/- mice is due to Hhex regulation of the Onecut1hHnf1bhPkhd1hcilia genetic pathway; Aim 3. Determine how Hhex regulates Hnf4a, Onecut1, Hnf1b and Pkhd1 in vitro and if there is a genetic interaction between these genes and Hhex in vivo; and Aim 4. Establish if the defective ciliary biogenesis seen in Hhex-mutant mice perturbs hedgehog signaling. We will accomplish these aims using the unique mouse strains and liver-derived cell lines generated in my laboratory. We have outlined a comprehensive in vitro and in vivo approach experimental approach that will determine new mechanisms controlling hepatoblast cell-fate determination, cilia biogenesis and signaling, and cyst formation. The conditional knockout mice we have generated provide an important resource for the liver research community. They provide opportunities for understanding the basis of early cell-lineage decisions in liver development, for understanding the molecular basis of epithelial lumen formation and cystogenesis, and for testing new therapies for polycystic liver disease. Our studies will also identify new genetic pathways operative in liver development, which has important implications for liver regeneration and repair. PUBLIC HEALTH RELEVANCE: The developmental mechanisms elucidated by these studies will provide knowledge that is critical to the identification of genetic factors required to generate differentiated cells for cell therapy of liver diseases. Additionally, this research will shed important insight into the pathogenesis of diseases of the biliary tract, including biliary atresia and polycystic liver disease.