Specification of fetal liver endothelial progenitors to functional zonated adult sinusoids requires c-Maf induction [development stage]

Endothelial cell heterogeneity shapes the unique attributes of organ-specific blood vessels; however, how intra-organ vascular cell diversity is established remains unsolved. Liver vascular network is patterned by sinusoidal and hepatocyte co-zonation, however how intra-liver vessels acquire their hierarchical specialized functions is unknown. Here, we resolve the heterogeneity of hepatic vascular cells during development through functional and single-cell RNA sequencing. We show that acquisition of sinusoidal endothelial cell identity is initiated during early development and is completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. Notably, we identify the peri-natal induction of the transcription factor c-Maf as a critical switch for sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Notably, c-Maf overexpression in generic differentiated endothelial cells switches on a liver sinusoidal transcriptional zonation program that maintains hepatocyte function. Therefore, c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of sinusoidal cell identity and lays the foundation for designing strategies to initiate vasculature-driven liver repair. Single cell RNA sequencing of liver endothelial cells along developmental time points E12, E14, E16, E18, P2, P8, P15 and P30.内皮细胞异质性（Endothelial cell heterogeneity）塑造了器官特异性血管的独特属性，然而器官内血管细胞多样性的建立机制仍未阐明。肝脏血管网络的构建模式由血窦（sinusoidal）与肝细胞的共分区调控，但肝内血管如何获得其层级化的特化功能仍属未知。本研究通过功能实验与单细胞RNA测序（single-cell RNA sequencing），解析了发育过程中肝脏血管细胞的异质性。研究发现，血窦内皮细胞身份的获取始于发育早期，并于出生后完成，其起源于胚胎第12天（E12）的未分化血管祖细胞群。值得注意的是，本研究发现转录因子（transcription factor）c-Maf的围产期诱导是决定血窦内皮细胞身份的关键开关。内皮特异性敲除c-Maf会破坏肝脏血窦发育，异常扩增出生后肝脏的造血活性，促进过度的出生后血窦增殖，并加重肝脏对化学损伤的促纤维化易感性。值得注意的是，在普通分化的内皮细胞中过表达c-Maf，可激活维持肝细胞功能的肝脏血窦转录分区程序。因此，c-Maf作为一种可诱导的、器官内特异性且响应微环境的血窦细胞身份分子决定因子，为设计启动血管介导的肝脏修复策略奠定了基础。本研究对发育时间节点E12、E14、E16、E18、P2、P8、P15及P30的肝脏内皮细胞进行了单细胞RNA测序。