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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.)

The mechanisms that allow cells to bypass anti-vascular endothelial growth factor A (VEGFA) therapy remain poorly understood. Here we use zebrafish to investigate this question and first show that vegfaa mutants display a severe vascular phenotype that can surprisingly be rescued to viability by vegfaa messenger RNA injections at the 1-cell stage. Using vegfaa mutants as an in vivo test tube, we found that zebrafish Vegfbb, Vegfd, and Pgfb can also rescue these animals to viability. Taking advantage of a new vegfr1 tyrosine kinase-deficient mutant, we determined that Pgfb rescues vegfaa mutants via Vegfr1. Altogether, these data reveal potential resistance routes against current anti-VEGFA therapies. In order to circumvent this resistance, we engineered and validated new dominant negative Vegfa molecules that by trapping Vegf family members can block vascular development. Thus, our results show that Vegfbb, Vegfd, and Pgfb can sustain vascular development in the absence of VegfA, and our newly engineered Vegf molecules expand the toolbox for basic research and antiangiogenic therapy., (© 2016 by The American Society of Hematology.)