Study On Cell Fate Regulation And Cell Lineage Tracing During Liver-biliary System Development And Liver Regeneration

The formation of an organism relies on the generation of diverse cell types.Within the field of developmental biology one goal is to understand how a cell's fate is determined.Studies on cell lineage and cell fate regulators will contribute to address this issue.Hhex encodes a homeodomain-containing protein that functions as both a transcriptional repressor and activator,and is necessary for early development of the liver.In zebrafish,the hhex gene is highly expressed in the liver bud,pancreatic bud and hepatopancreatic duct(HPD)cells.However the role of Hhex in the establishment of the hepatopancreatic ductal system is not known.Here,we derived zebrafish hhex mutants with CRISPR/Cas9 system for investigation of the role of Hhex in the development of hepatopancreatic ductal system.The hhex mutant was characterized by whole-mount in situ hybridization(WISH)and immunostaining using a series of molecular markers.Luciferase assay was performed to identify Hhex downstream genes essential for the development of the hepatopancreatic ductal system.Immunostaining revealed that the hhex mutant fails to develop the HPD system.WISH and histologic analysis with transgenic fishes revealed that the hhex mutant develops an intrahepatic intestinal tube.Cell lineage analysis showed that this intrahepatic intestinal tube is not originated from hepatocytes or intrahepatic duct cells.WISH using the cdx1b and pdxl probes showed that this intrahepatic intestinal tube is derived from the extension of the main intestine.Finally we show that Hhex functions as a repressor to the promoter of cdx1b and pdx1 genes to safeguard the development of the hepatopancreatic ductal system.This study reveals that transcription regulators Hhex,Sox9b,Cdxlb and Pdxl form a genetic network governing the patterning and morphogenesis of the HPD system in zebrafish.The liver is thought to utilize stem cells,also known as "oval cells",for regeneration following various types of injury.However,this notion was based largely on in vitro studies and transplantation models.Considering the importance of partial hepatectomy(PH)in clinical medicine,there are few studies on the contribution to liver regeneration from various cell types after PH.Here,we used biomolecule and genetic tools to mark and track the origin and contribution of various cell populations to liver regeneration in vivo following partial hepatectomy in zebrafish.Zebrafish Bhmt and Fabp 10a are both specifically expressed in hepatocytes.Immunostaining was performed using antibodies against them,showing that a certain number of newborn hepatocytes which were Fabp 10a-Bhmt+gathering at wound sites but not in the parenchyma area.Basing on CreErt2/LoxP system,Genetic lineage tracing showed that these newborn hepatocytes generated from BECs and maybe other kind of cells,but not preexisting hepatocytes.Our research shows that virtually all new hepatocytes come from preexisting hepatocytes in parenchyma area,whereas a certain number of hepatocytes come from BECs at the wound sites.