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

Abstract

 

Hepatic stellate cells (HSCs) are liver-specific mesenchymal cells that play vital roles in liver development and injury. Our knowledge of HSC biology is limited by the paucity ofin vivodata. HSCs and sinusoidal endothelial cells (SECs) reside in close proximity, and interactions between these two cell types are potentially critical for their development and function. Here, we introduce a transgenic zebrafish line,Tg(hand2:EGFP), that labels HSCs. We find that zebrafish HSCs share many similarities with their mammalian counterparts, including morphology, location, lipid storage, gene-expression profile, and increased proliferation and matrix production, in response to an acute hepatic insult. Using theTg(hand2:EGFP) line, we conducted time-course analyses during development to reveal that HSCs invade the liver after SECs do. However, HSCs still enter the liver in mutants that lack most endothelial cells, including SECs, indicating that SECs are not required for HSC differentiation or their entry into the liver. In the absence of SECs, HSCs become abnormally associated with hepatic biliary cells, suggesting that SECs influence HSC localization during liver development. We analyzed factors that regulate HSC development and show that inhibition of vascular endothelial growth factor signaling significantly reduces the number of HSCs that enter the liver. We also performed a pilot chemical screen and identified two compounds that affect HSC numbers during development.

Conclusion: 

Our work provides the first comprehensive description of HSC development in zebrafish and reveals the requirement of SECs in HSC localization. TheTg(hand2:EGFP) line represents a unique tool forin vivoanalysis and molecular dissection of HSC behavior.

Abbreviations: clo, cloche; DMSO, dimethyl sulfoxide; dpf, days postfertilization; ECs, endothelial cells; ECM, extracellular matrix; EdU, 5-ethynyl-2′deoxyuridine; EGFP, enhanced green fluorescent protein; EMT, epithelial-to-mesenchymal transition; EtOH, ethanol; FACS, fluorescence-activated cell sorting; GFAP, glial fibrillary acidic protein; hand2, heart and neural crest derivatives expressed transcript 2; hpf, hours postfertilization; HSC, hepatic stellate cell; KDR, kinase insert domain receptor; MA, methoprene acid; MO, morpholino; Prox1, Prospero-related homeobox gene 1; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; SEC, sinusoidal endothelial cell; UCSF, University of California San Francisco; VEGF, vascular endothelial cell growth factor; WT, wild type; Wt1, Wilms' tumor suppressor gene.