Cholangiocytes contribute to hepatocyte regeneration after partial liver injury in larval zebrafish

Liver possesses robust regenerative ability, characterized by flexibility in the cellular source of regeneration based on the extent of the injury. After partial hepatectomy or minor injuries, hepatocytes, the primary liver cells, undergo self-duplication to replenish the liver mass. In contrast, when the damage is extensive, or hepatocyte proliferation is impaired, cholangiocytes contribute to hepatocyte recovery. This current paradigm of regenerative flexibility in the liver has been established for animals with little or no growth. However, the regenerative mechanisms during periods of growth in young animals remain unexplored. Here, we establish two new partial liver injury protocols in the zebrafish model of rapid growth during late larval stage and observe emergence of de novo hepatocytes in the presence of spared hepatocytes. Using single-cell RNA sequencing and lineage tracing, we identify cholangiocytes as the source of de novo hepatocytes. Our study offers a new perspective on the current paradigm of liver regenerating by proposing cholangiocyte-to-hepatocyte transdifferentiation as the default mechanism of hepatocyte recovery in late larval stage zebrafish. Overall design: We conducted single-cell RNA Sequencing using the 10x platform of larval zebrafish liver. This was performed in the basal state (Control1, Control2), after partial ablation (MTZ_0dppa, MTZ_1dppa and MTZ_9dppa) and after partial hepatectomy (PHx_4dpi and PHx_11dpi). The two control samples were not treated with 4-OHT, and were also not injured. The control samples are from 13 dpf zebrafish raised under normal rearing condition. For all experiments Tg(fabp10a:CellCousin) line was used.

肝脏具备极强的再生能力，其再生的细胞来源具有灵活性，该特性由损伤程度决定。在部分肝切除术或轻度损伤后，作为肝脏主要功能细胞的肝细胞（hepatocytes）会通过自我复制以补充肝组织质量。与之相对，当损伤范围较大或肝细胞增殖功能受损时，胆管上皮细胞（cholangiocytes）会参与肝细胞的修复过程。目前，这一肝脏再生灵活性的经典范式仅在几乎无生长或生长缓慢的动物中得到确立，但幼龄动物处于生长阶段时的肝脏再生机制仍未被探明。

本研究在处于晚期幼虫阶段、处于快速生长状态的斑马鱼（zebrafish）模型中建立了两种全新的部分肝损伤造模方案，并观察到在残留肝细胞存在的情况下，新生肝细胞（de novo hepatocytes）的产生。研究人员借助单细胞RNA测序（single-cell RNA sequencing）与谱系示踪（lineage tracing）技术，确认胆管上皮细胞是新生肝细胞的来源。本研究提出，在晚期幼虫阶段的斑马鱼中，胆管上皮细胞向肝细胞转分化是肝细胞修复的默认机制，为当前的肝脏再生研究范式提供了全新视角。

整体实验设计：我们采用10x平台（10x platform）对斑马鱼幼虫的肝脏进行单细胞RNA测序。测序样本涵盖正常状态下的对照组（Control1、Control2）、部分消融组（MTZ_0dppa、MTZ_1dppa及MTZ_9dppa）以及部分肝切除组（PHx_4dpi及PHx_11dpi）。两组对照组均未接受4-OHT处理，也未受到损伤，样本取自正常饲养条件下的13 dpf斑马鱼。所有实验均使用Tg(fabp10a:CellCousin)转基因品系。