Role of the Wnt/ β-catenin Pathway in Liver Development and Zonation

Abstract

Understanding normal liver development is critical for the production of cell therapies for treating liver disease and also for the construction of in vitro liver models. During the progress of the research described in this thesis I specifically addressed the role of the Wnt/β-catenin pathway in liver development and also the effect of ectopic expression of liver-enriched transcription factors on the conversion of pancreatic cells to hepatocyte-like cells.The Wnt pathway is known to play a critical role in the maintenance of the zonation of ammonia metabolizing enzymes in the adult liver. Herein we determined the spatiotemporal role of the Wnt/β-catenin pathway in the development of zonation in embryonic mouse liver. We initially examined the expression of Wnt ligands (Wnt4, Wnt5a, Wnt5b and Wnt9), receptors (Frizzled1, Frizzled2 and LRP5) and two hepatic zonation markers, the periportal marker and urea cycle enzyme carbamoylphosphate synthetase (CPS) and the perivenous enzyme Glutamine synthetase (GS) at different stages of liver development by in situ hybridization, immunohistochemistry and western blotting. We observed temporal changes in the Wnt signalling molecules. In addition, we found that CPS became expressed between E14.5 and E15.5 and gradually the number of cells expressing the enzyme increased during development. Nuclear β-catenin and GS were expressed between E17.5 and E18.5 of liver development in a complementary fashion to the CPS-expressing cells.In order to examine the effect of activating the Wnt/β-catenin pathway during liver development, conditional deletion of Apc was induced at different time points during liver development and the expression of periportal and perivenous hepatocyte and ductal markers determined by PCR and immunohistochemical techniques. Conditional loss of Apc resulted in the expression of nuclear16β-catenin throughout the developing liver. This was accompanied by a reduction in the expression of CPS and up-regulation of GS expression. When Apc loss was induced at around E16.5 and liver samples were collected at E18.5, we also observed an upregulation of the ductal phenotype.To extend these studies we examined the direct role of the Wnt/β-catenin pathway on development of liver zonation regulation by infecting in vitro cultures of embryonic mouse livers with adenoviral vectors expressing Wnt1 and Wnt5a. The results demonstrated that ectopic expression of Wnt1 and Wnt5a enhances the perivenous phenotype.Transdifferentiation is the conversion of one differentiated cell type to another and a number of in vitro models have been developed for converting pancreatic cells to hepatocytes, including culturing the rat pancreatic amphicrine call line AR42J-B13 (B13) with synthetic glucocorticoid dexamethasone (Dex). CCAAT-enhancer binding proteins (C/EBP)-β has been identified as part of the molecular basis of the switch however it did not cause the same degree of morphological flattening as Dex. To test the role of hepatic nuclear factor (HNF4)-in Dex induced transdifferentiation, we co-expressed C/EBP and HNF4 in B13 cells by using adenoviral infection. The results showed that ectopic expressed HNF4 and C/EBPcould induce the expression of hepatic genes and morphological changes of B13 cells reminiscent of hepatocytes.

Date of Award	1 Sept 2012
Original language	English
Awarding Institution	University of Bath
Supervisor	David Tosh (Supervisor)