PgmNr Z6096: The zebrafish prox1a controls liver development by regulating Wnt signaling pathway.

Authors:
B. Zhang; Y. Hu; Z. Luo; M. Wang


Institutes
Peking University, Beijing, CN.


Abstract:

Prox1, encoding a homeobox transcription factor, has been shown to be essential for liver development in mice, though its molecular mechanism needs further investigation and its function in zebrafish hepatogenesis has not been reported. In this study, we generated prox1a knock-out zebrafish by using CRISPR/Cas9 system. prox1a homozygous mutants exhibited severe edema around the gut upon 4-5 dpf, with smaller liver and abnormal intrahepatic structure. The number of mature hepatocytes was reduced, whereas early endoderm development and hepatic bud formation was not affected, as shown by the in situ hybridization results of the corrresponding marker genes. prox1a ablation resulted in defective hepatocyte proliferation but not apoptosis in the liver outgrowth stage. Moreover, cholangiocyte differentiation was totally blocked in the mutant liver, as shown by using both Notch-reporter transgenic fish and cytokeratin staining. Endothelial cells also failed to invade into the mutant liver to form vascular networks. As a result, the prox1a mutants lack intrahepatic biliary ducts and blood vessels. Liver-specific RNA seq was performed to explore downstream effectors of prox1a. About 1000 genes were identified as significantly differentially expressed in the mutant liver. Functional clustering analysis on these genes revealed that, majority of the downregulated group was involved in metabolic process, while Wnt signaling was highlighted in the upregultated pathways. Dissecting different components of this pathway uncovered bifurcating outcomes. Upon prox1a ablation, excessive expression of Wnt ligands, receptors, as well as inhibitors integrate to activate non-canonical Wnt signaling but suppress canonical Wnt signaling. Reduced expression of canonical Wnt target genes cmyc and ccnd1 led to defective hepatocyte proliferation, rendering smaller liver size. However, enhanced non-canonical Wnt signaling activity interfered with cell polarity, displayed by aberrant subcellular localization of aPKC, an apical membrane protein. Lack of correct cell polarity resulted in loose organization and irregular orientation of hepatoblast/hepatocyte, and may influence interations between different cell types, on which both cholangiocyte differentiation and endothelial cell invasion are dependent. In conclusion, our data illustrated that prox1a controls zebrafish liver development at multiple levels, including cell proliferation, polarity and differentiation, through regulating and integrating Wnt signaling pathway.



ZFIN Genetics Index
1. prox1a