Hif-1α and Hif-2α regulate hemogenic endothelium and hematopoietic stem cell formation in zebrafish.

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 () and in () zebrafish mutants and morphants. Moreover, live imaging studies reveal a positive role for and in regulating HE specification. Knockdown of in mutants leads to a greater decrease in HSC formation, indicating that and 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 and mutants display impaired EHT, which cannot be rescued by hypoxia. However, overexpression of the Notch intracellular domain in ECs is sufficient to rescue the and 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.