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.
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.
| Arterbery, Adam S; Bogue, Clifford W (2016) Hhex Is Necessary for the Hepatic Differentiation of Mouse ES Cells and Acts via Vegf Signaling. PLoS One 11:e0146806 |
| Arterbery, Adam S; Bogue, Clifford W (2014) Endodermal and mesenchymal cross talk: a crossroad for the maturation of foregut organs. Pediatr Res 75:120-6 |
| Hunter, Michael P; Wilson, Christine M; Jiang, Xiaobing et al. (2007) The homeobox gene Hhex is essential for proper hepatoblast differentiation and bile duct morphogenesis. Dev Biol 308:355-67 |
| Cong, Rong; Jiang, Xiaobing; Wilson, Christine M et al. (2006) Hhex is a direct repressor of endothelial cell-specific molecule 1 (ESM-1). Biochem Biophys Res Commun 346:535-45 |
| Hallaq, Haifa; Pinter, Emese; Enciso, Josephine et al. (2004) A null mutation of Hhex results in abnormal cardiac development, defective vasculogenesis and elevated Vegfa levels. Development 131:5197-209 |
