Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish

Background & aims: The liver has high regenerative capacity, but it is not clear whether most biliary cells (particularly larger cholangiocytes) transdifferentiate into hepatocytes in regenerating liver. We investigated how this process might contribute to liver regeneration in zebrafish.

Methods: Zebrafish transgenic lines were generated using the standard I-SceI meganuclease transgenesis technique. Hepatocytes of the Tg(lfabp:mCherry-NTR)(cq2) animals were ablated by the administration of metronidazole. We investigated transdifferentiation of biliary cells to hepatocytes and expression of markers using whole mount antibody staining, fluorescent in situ hybridization, and Cre/loxP-based genetic lineage tracing analyses. The role of biliary cells in hepatocyte regeneration was explored using zebrafish larvae with defects in biliary cell development.

Results: After extreme loss of hepatocytes, nearly all the biliary cells steadily lost their tubular morphology, proliferated, and expressed hepatocyte-specific markers. Cre/loxP-based inducible lineage tracing showed that new hepatocytes mainly arose from transdifferentiation of biliary cells; this process required Notch signaling and, in turn, activation of Sox9b in cholangiocytes. Activation of early endoderm and hepatoblast markers in most of the cholangiocytes indicated that biliary transdifferentiation includes a step of dedifferentiation into a bipotential intermediate. Defects in development of biliary cells impaired hepatocyte regeneration.

Conclusions: Using our zebrafish liver regeneration model, we found that biliary cells can transdifferentiate into hepatocytes and are the major contributors to hepatocyte regeneration after extreme hepatocyte loss.