Hhex-a Homeobox Gene Necessary for Liver Development

描述(由申请人提供)：同源异型盒基因HHEX零突变的小鼠在妊娠中期死亡，并伴有多种发育缺陷，包括肝脏完全缺失。最近，我们在发育中的肝脏中获得了两个HHEX缺失的品系--在肝芽中的E9.0和在肝母细胞中的E10.5-11。我们发现HHEX对肝脏生长、肝母细胞分化以及肝外和肝内胆管发育都是必需的。此外，我们还发现HHEX的缺失与HNF4a、HNF1B(TCF2)和Onecut1(Hnf6)的表达降低有关，这表明HHEX直接或间接地调节这些基因的表达。在肝脏中，HNF4a和Onecut1是正常肝母细胞分化所必需的，并且已知相互调节彼此的表达。此外，有大量的数据表明，Onecut1、HNF1B和Pkhd1在一条高度保守的分子通路中发挥作用，该通路对肝、胰腺和肾脏的正常上皮管形成和纤毛功能至关重要。此外，纤毛有缺陷/缺失的小鼠有异常的刺猬信号。来自我的实验室的新的初步数据显示，在肝脏特异性HHEX缺失的小鼠中，胆管纤毛短小或缺失，囊性相关基因Pkhd1的表达显著减少。因此，我们推测HHEX在发育中的肝脏中调节由HNF4a、Onecut1、HNF1B和Pkhd1组成的分子通路。在没有HHEX的情况下，这些基因的表达减少，导致纤毛形成/信号缺陷，扰乱上皮腔形成和随后的囊形成，以及肝母细胞分化缺陷。我们建议通过实现以下特定目标来验证这一假说：目的1.建立HHEX、HNF4a和Onecut1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性和阶段特异性的；目的2.确定Foxa3-Cre；Hhed2，3/-和Alfp-Cre；Hhed2，3/-小鼠是否由于HHEX对ONecut1Hnf1bhPkhd1hcilia遗传途径的调控；目的3.确定HEX在体外如何调节HNF4a、ONecut1、HNF1B和Pkhd1，以及这些基因之间是否存在遗传相互作用；目的4.确定在HHEX突变小鼠身上发现的纤毛生物发生缺陷是否扰乱了刺猬信号。我们将使用我的实验室培育的独特的小鼠品系和肝脏来源的细胞系来实现这些目标。我们概述了一种全面的体外和体内实验方法，该方法将确定控制肝母细胞命运的新机制-纤毛生物发生和信号传递，以及包囊形成。我们培育的条件性基因敲除小鼠为肝脏研究界提供了重要的资源。它们为了解肝脏发育中早期细胞谱系决定的基础，了解上皮腔形成和囊变的分子基础，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过程中操作的新的遗传途径，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提供对识别遗传因素至关重要的知识，这些遗传因素需要产生分化细胞，用于肝病的细胞治疗。此外，这项研究将为胆道疾病的发病机制提供重要的见解，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.