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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 remains incomplete. Zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we have combined complementary genome-wide approaches, including transcriptome analyses and chromatin immunoprecipitation. The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes that are crucial for hematoendothelial specification, such as etv2 , tal1 and lmo2 We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

During development, hematopoietic stem cells (HSCs) derive from specialized endothelial cells (ECs) called hemogenic endothelium (HE) via a process called endothelial-to-hematopoietic transition (EHT). Hypoxia-inducible factor-1α (HIF-1α) has been reported to positively modulate EHT in vivo, but current data indicate the existence of other regulators of this process. Here we show that in zebrafish, Hif-2α also positively modulates HSC formation. Specifically, HSC marker gene expression is strongly decreased in hif -1aa;hif-1ab ( hif-1α ) and in hif-2aa;hif-2ab ( hif-2α ) zebrafish mutants and morphants. Moreover, live imaging studies reveal a positive role for hif-1α and hif-2α in regulating HE specification. Knockdown of hif-2α in hif-1α mutants leads to a greater decrease in HSC formation, indicating that hif-1α and hif-2α have partially overlapping roles in EHT. Furthermore, hypoxic conditions, which strongly stimulate HSC formation in wild-type animals, have little effect in the combined absence of Hif-1α and Hif-2α function. In addition, we present evidence for Hif and Notch working in the same pathway upstream of EHT. Both notch1a and notch1b mutants display impaired EHT, which cannot be rescued by hypoxia. However, overexpression of the Notch intracellular domain in ECs is sufficient to rescue the hif-1α and hif-2α morphant EHT phenotype, suggesting that Notch signaling functions downstream of the Hif pathway during HSC formation. Altogether, our data provide genetic evidence that both Hif-1α and Hif-2α regulate EHT upstream of Notch signaling., (© 2018 by The American Society of Hematology.)