Genome-wide strategies reveal downstream target genes of Npas4l associated with vascular development in zebrafish [ChIP-Seq]

The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification in vertebrates remains incomplete. cloche/npas4l mutants have broadly been used as an avascular model in zebrafish, but little is known about the molecular mechanism of action of Npas4l. Here, to identify the direct and indirect target genes of this transcription factor, we combined complementary genome-wide approaches, including transcriptome analyses and chromatin immunoprecipitation (ChIP). The cross-analysis of these datasets indicate that Npas4l functions as a master regulator by directly inducing a wave of transcription factor genes crucial for hematoendothelial specification in vertebrates, including etv2, tal1, and lmo2. We identified additional target genes acting downstream of npas4l and investigated the function of a subset of them using CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel role for tspan18b in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to investigate novel genes with a potential role in vascular development and EC fate determination in vertebrates. ChIP-seq examination of Npas4l-HA of zebrafish embryos compared to input.

血管网络的发育对于滋养组织、维持终生器官功能至关重要。内皮细胞（Endothelial cells，ECs）是血管的基本构成单元，但目前我们对脊椎动物内皮细胞特化的认知仍存在不足。cloche/npas4l突变体已被广泛用作斑马鱼的无血管模型，但对于Npas4l的分子作用机制仍知之甚少。为鉴定该转录因子的直接与间接靶基因，本研究结合了包括转录组分析与染色质免疫共沉淀（Chromatin Immunoprecipitation，ChIP）在内的互补性全基因组研究策略。对上述数据集的交叉分析表明，Npas4l作为主调控因子，可直接诱导一系列对脊椎动物造血内皮细胞特化至关重要的转录因子基因的表达，其中包括etv2、tal1与lmo2。本研究还鉴定了npas4l下游的其他靶基因，并利用CRISPR/Cas9技术对其中部分基因的功能进行了探究。对tspan18b突变体的表型表征揭示了tspan18b在发育性血管生成中的全新功能，证实了本研究生成的数据集的可靠性。综上，这些数据可为未来旨在探究脊椎动物血管发育与内皮细胞命运决定中具有潜在功能的新基因的研究提供宝贵的资源。本研究对斑马鱼胚胎中Npas4l-HA的ChIP-seq检测结果与对照输入样本进行了比对分析。