The basic helix‐loop‐helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: Analysis of stellate cell entry into the developing liver

Hepatic stellate cells (HSCs) represent a versatile mesenchymal cell type that plays vital roles in liver function and injury response. In healthy livers, HSCs serve as the main vitamin A-storing cells. Upon liver injury, these quiescent cells transform into activated, proliferative myofibroblast-like cells to generate scar tissue (reviewed by 1). Sustained activation of HSCs is a central event in liver fibrosis and has been linked to the progression of hepatitis and steatohepatitis (reviewed by 2). Despite the importance of HSCs in liver physiology and disease, our knowledge of HSC biology is far from complete. Characterization of HSC development could provide clues for understanding their activation during liver injury. However, tracking HSCs during development has not yet been feasible in mammalian models. The embryonic origin of HSCs is elusive because they express marker genes of all three germ layers (3, 4). Genetic lineage-tracing analysis in mice using a CreERT2 transgene knocked into the Wilms’ tumor suppressor (Wt1) locus indicated that HSCs derive from the septum transversum-derived mesothelium (5). Interestingly, in chick, the mesothelium contributes not only to HSCs, but also to sinusoidal endothelial cells (SECs) (6). HSCs and SECs exhibit close physical association and common expression of angiogenic factors (7). These observations have led to the hypothesis that HSCs and SECs originate from a common embryonic precursor. However, no studies have specifically addressed this issue. Finding promoters that selectively drive transgene expression in HSCs could facilitate both their in vivo observation and their genetic manipulation. Previous studies used the promoters of the mesoderm-associated α-smooth muscle actin gene and the neural crest-related glial fibrillary acidic protein/GFAP gene to direct gene expression in HSCs in transgenic mice (8-10). However, identifying HSC-specific promoters remains challenging. The bHLH transcription factor gene heart and neural crest derivatives expressed transcript 2/hand2 is expressed in the lateral plate mesoderm and the neural crest (11), tissues that may give rise to HSCs. We previously reported the generation of the Tg(hand2:EGFP) zebrafish line that expresses EGFP under the control of the hand2 regulatory sequences (12). During liver budding morphogenesis, Tg(hand2:EGFP) is expressed in the lateral plate mesoderm surrounding the liver primordium. However, Tg(hand2:EGFP) expression was not characterized during later stages of liver development. The teleost zebrafish (Danio rerio) has emerged as a valuable vertebrate model system for studying liver development and disease. The zebrafish liver contains the same main cell types as the mammalian liver, including hepatocytes, biliary cells, and endothelial cells (13). Although the basic architecture of the fish liver differs from that of the mammalian liver, mechanisms of liver development and diseases are conserved (reviewed by 14). In addition, the zebrafish model presents important advantages that complement those of other animal models. The rapid external development and translucence of the embryos and larvae make them well suited for in vivo imaging analyses. Using transgenic approaches, investigators have generated zebrafish that express fluorescent proteins in different hepatic cell types, allowing easy visualization of hepatic cell behaviors in the animal, and greatly facilitating genetic and chemical screens to identify regulators of liver development and disease pathogenesis (reviewed by 14). Although intensive studies have been conducted on parenchymal cells in the zebrafish liver, no report has yet focused on HSCs. In this study, we report that the Tg(hand2:EGFP) line marks HSCs during both embryonic and adult stages. Zebrafish HSCs share significant similarities with mammalian HSCs, including their morphology, localization, lipid storage, and gene expression. They respond to acute alcohol exposure by changing morphology, upregulating extracellular matrix protein production and increasing proliferation. By tracking HSCs throughout development, we show that zebrafish HSCs enter the liver after SECs do. Study of cloche/clo mutants which lack SECs indicates that although SECs are not required for HSC differentiation or their entry into the liver, they influence the localization of HSCs inside the liver. We also reveal that inhibition of vascular endothelial growth factor (VEGF) signaling impairs entry of HSCs into the developing liver. Taken together, our work presents a new in vivo model to study HSC biology and provides novel insights into the molecular and cellular mechanisms underlying HSC development.