Role of Wnt-Beta-catenin in liver development and metabolic zonation

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

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Abstract

ABSTRACT The Wnt-b-catenin pathway plays key roles in liver pathophysiology especially in regulating hepatocyte proliferation, survival, and differentiation. Our previous investigations have contributed to a better understanding of this signaling pathway in the process of liver development, metabolic zonation (or simply zonation) and liver repair. The current proposal is directed at improving our understanding of the role and regulation of the Wnt-b- catenin signaling in hepatic development and zonation. Previously, we have discovered an indispensable role of b-catenin in liver development when hepatoblast-specific b-catenin knockouts (KOs) manifested fetal lethality at embryonic day 17 (E17) due to decreased cell proliferation and defective hepatocyte maturation. Our preliminary data now suggests Wnts to be regulating b-catenin activation during liver development since conditional loss of redundant Wnt co-receptors LDL related protein 5 (Lrp5) and Lrp6 from hepatoblasts led to no viable KO. Interestingly, KO of Wntless (Wls) (required for cellular Wnt secretion) from liver endothelial cells or from macrophages were viable despite these cre lines being active during liver development. In aim 1 we will test the hypothesis that b-catenin control during prenatal hepatic development is Wnt-dependent. We will characterize hepatoblast-Lrp5-6-DKO comprehensively. We will also delete Wls simultaneously from both macrophages and endothelial cells. Our laboratory has also contributed to an understanding of the Wnt-b-catenin pathway in regulating the process of zonation, which encompasses heterogeneous gene expression by hepatocytes based on their location in liver lobule. We also identified Wnt2 and Wn9b expression in zone-3 endothelial cells to activate b-catenin signaling in zone-3 hepatocytes and control gene expression. Conditional loss from hepatocytes of b-catenin, Lrp5-6, or from endothelial cells of Wls or Wnt2-Wnt9b, exhibits loss of zone-3 genes and gain of non-zone-3 genes in zone-3 hepatocytes. Aim 2 will test two hypotheses. First, while Wnt-b-catenin target genes exist primarily in zone-3, there may be additional targets in zone-2 and zone-1 due to a gradually declining Wnt2 and Wnt9b gradient. Using newly obtained zone-specific Cre lines, we will delete b-catenin from each of the 3 zones individually and perform transcriptomic analysis on hepatocytes to identify b-catenin targets. Our 2nd hypothesis is that due to similarity of consensus sequence of Wnt response element (WRE) and DR-1 element, zonation is regulated by competition between b-catenin-TCF4 and HNF4a to bind and activate their target genes in different zones. We will test if presence of b-catenin drives TCF4 to bind its target genes at the WRE and induce their transcriptional activation while binding DR-1 element on HNF4a target genes and blocking their expression. In the absence of b-catenin, TCF4 alone serves as a transcription repressor of Wnt target genes & allows TCF4 to be displaced by pan-zonally present nuclear HNF4a which can activate its target genes. We will use RNA-seq, ATAC-seq and ChIP-seq on zone-specific hepatocytes with normal, absent or activated b- catenin to show this competition. We will also address any impact of b-catenin modulation in one zone on the adjacent zones for changes in gene expression, chromatin accessibility and promoter occupancy by HNFa, TCF4 and b-catenin. Lastly, aim 3 will address the mechanism of constitutive baseline expression of Wnt2 and Wnt9b in endothelial cells in zone-3 only. We posit that hypoxia signaling in endothelial cells controls the expression of Wnt2 and Wnt9b in these cells. We will generate and characterize endothelial cell KOs of Hif1a- Hif2a and of Aryl Hydrocarbon Receptor Nuclear Translocator for Wnt-b-catenin signaling and zonation via gene expression, single cell spatial transcriptomics and functional studies. Overall, our studies will uncover novel regulation of liver development and zonation which would have implications in hepatic health and disease.