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During embryogenesis, the initial vascular network forms by the process of vasculogenesis, or the specification of vascular progenitors de novo. In contrast, the majority of later-forming vessels arise by angiogenesis from the already established vasculature. Here, we show that new vascular progenitors in zebrafish embryos emerge from a distinct site along the yolk extension, or secondary vascular field (SVF), incorporate into the posterior cardinal vein, and contribute to subintestinal vasculature even after blood circulation has been initiated. We further demonstrate that SVF cells participate in vascular recovery after chemical ablation of vascular endothelial cells. Inducible inhibition of the function of vascular progenitor marker etv2/etsrp prevented SVF cell differentiation and resulted in the defective formation of subintestinal vasculature. Similar late-forming etv2+ progenitors were also observed in mouse embryos, suggesting that SVF cells are evolutionarily conserved. Our results characterize a distinct mechanism by which new vascular progenitors incorporate into established vasculature.

During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development.

Vasculogenesis involves the differentiation of vascular endothelial progenitors de novo from undifferentiated mesoderm, their migration and coalescence to form the major embryonic vessels and the acquisition of arterial or venous identity. Vascular Endothelial Growth Factor (Vegf) signaling plays multiple roles during vascular development. However, its function during embryonic vasculogenesis has been controversial. Previous studies have implicated Vegf signaling in either regulating arteriovenous specification or overall vascular endothelial differentiation. To clarify the role of Vegf in embryonic vasculogenesis and identify its downstream targets, we used chemical inhibitors of Vegf receptor (Vegfr) signaling in zebrafish embryos as well as zebrafish genetic mutants. A high level of chemical inhibition of Vegfr signaling resulted in the reduction of overall vascular endothelial marker gene expression, including downregulation of both arterial and venous markers, ultimately leading to the apoptosis of vascular endothelial cells. In contrast, a low level of Vegfr inhibition specifically blocked arterial specification while the expression of venous markers appeared largely unaffected or increased. Inhibition of Vegfr signaling prior to the initiation of vasculogenesis reduced overall vascular endothelial differentiation, while inhibition of Vegfr signaling starting at mid-somitogenesis stages largely inhibited arterial specification. Conversely, Vegf overexpression resulted in the expansion of both arterial and pan-endothelial markers, while the expression of several venous-specific markers was downregulated. We further show that Vegf signaling affects overall endothelial differentiation by modulating the expression of the ETS transcription factor etv2/ etsrp. etv2 expression was downregulated in Vegfr- inhibited embryos, and expanded in Vegfaa-overexpressing embryos. Furthermore, vascular-specific overexpression of etv2 in Vegfr-inhibited embryos rescued defects in vascular endothelial differentiation. Similarly, vegfaa genetic mutants displayed a combination of the two phenotypes observed with chemical Vegfr inhibition: the expression of arterial and pan-endothelial markers including etv2 was downregulated while the expression of most venous markers was either expanded or unchanged. Based on these results we propose a revised model which explains the different phenotypes observed upon inhibition of Vegf signaling: low levels of Vegf signaling promote overall vascular endothelial differentiation and cell survival by upregulating etv2 expression, while high levels of Vegf signaling promote arterial and inhibit venous specification.

ETS transcription factors play an important role in the specification and differentiation of endothelial cells during vascular development. Despite previous studies, the role of the founding member of the ETS family, Ets1, in vascular development in vivo is only partially understood. Here, we generated a zebrafish ets1 mutant by TALEN genome editing and tested functional redundancy between Ets1 and a related ETS factor Etv2 / Etsrp / ER71. While zebrafish ets1(−/−) mutants have a normal functional vascular system, etv2(−/−);ets1(−/) embryos had more severe angiogenic defects and lower expression levels of kdr and kdrl, the two zebrafish homologs of the mammalian Vascular Endothelial Growth Factor Receptor 2 VEGFR2/Flk1, than etv2(−/−)embryos. Expression of constitutively active Mitogen-Activated Protein Kinase1 (MAP2K1) within endothelial cells partially rescued this angiogenic defect. Interestingly, ets1(−/−) embryos displayed extensive apoptosis within the trunk vasculature despite exhibiting normal vascular patterning. Loss of Ets1 combined with a partial knockdown of Etv2 function resulted in a decrease in endothelial cell numbers in the axial vasculature, which argues for a role of Ets1 in promoting vasculogenesis. We also demonstrate that although both Ets1 and Etv2 can induce ectopic vascular marker expression in zebrafish embryos, Ets1 activity is dependent on MAPK-mediated phosphorylation of its Thr30 and Ser33 residues, while Etv2 activity is not. Together, our results identify a novel function of Ets1 in regulating endothelial cell survival during vasculogenesis in vivo. Based on these findings, we propose a revised model of how Ets1 and Etv2 play unique and partially redundant roles to promote vascular development.

Cell fate decisions involved in vascular and hematopoietic embryonic development are still poorly understood. An ETS transcription factor Etv2 functions as an evolutionarily conserved master regulator of vasculogenesis. Here we report a single-cell transcriptomic analysis of hematovascular development in wild-type and etv2 mutant zebrafish embryos. Distinct transcriptional signatures of different types of hematopoietic and vascular progenitors are identified using an etv2ci32Gt gene trap line, in which the Gal4 transcriptional activator is integrated into the etv2 gene locus. We observe a cell population with a skeletal muscle signature in etv2-deficient embryos. We demonstrate that multiple etv2ci32Gt; UAS:GFP cells differentiate as skeletal muscle cells instead of contributing to vasculature in etv2-deficient embryos. Wnt and FGF signaling promote the differentiation of these putative multipotent etv2 progenitor cells into skeletal muscle cells. We conclude that etv2 actively represses muscle differentiation in vascular progenitors, thus restricting these cells to a vascular endothelial fate., The signals restricting specification of vascular progenitors are unclear. Here, the authors use scRNAseq to identify transitional steps during blood and vascular development in zebrafish and identify Etv2 as repressing skeletal muscle differentiation in vascular progenitors.