Zebrafish hhex-null mutant develops an intrahepatic intestinal tube due to de-repression of cdx1b and pdx1

The hepatopancreatic duct (HPD) system links the liver and pancreas to the intestinal tube and is composed of the extrahepatic biliary duct, gallbladder, and pancreatic duct. Haematopoietically expressed-homeobox (Hhex) protein plays an essential role in the establishment of HPD; however, the molecular mechanism remains elusive. Here, we show that zebrafish hhex-null mutants fail to develop the HPD system characterized by lacking the biliary marker Annexin A4 and the HPD marker sox9b. The hepatobiliary duct part of the mutant HPD system is replaced by an intrahepatic intestinal tube characterized by expressing the intestinal marker fatty acid-binding protein 2a (fabp2a). Cell lineage analysis showed that this intrahepatic intestinal tube is not originated from hepatocytes or cholangiocytes. Further analysis revealed that cdx1b and pdx1 are expressed ectopically in the intrahepatic intestinal tube and knockdown of cdx1b and pdx1 could restore the expression of sox9b in the mutant. Chromatin-immunoprecipitation analysis showed that Hhex binds to the promoters of pdx1 and cdx1b genes to repress their expression. We therefore propose that Hhex, Cdx1b, Pdx1, and Sox9b form a genetic network governing the patterning and morphogenesis of the HPD and digestive tract systems in zebrafish.