Etv2 and Fli1b Function Together as Key Regulators of Vasculogenesis and Angiogenesis

Abstract

Objective The E26 transformation-specific domain transcription factor Etv2/Etsrp/ER71 is a master regulator of vascular endothelial differentiation during vasculogenesis, although its later role in sprouting angiogenesis remains unknown. Here, we investigated in the zebrafish model a role for Etv2 and related E26 transformation-specific factors, Fli1a and Fli1b in developmental angiogenesis. Approach and Results Zebrafish fli1a and fli1b mutants were obtained using transposon-mediated gene trap approach. Individual fli1a and fli1b homozygous mutant embryos display normal vascular patterning, yet the angiogenic recovery observed in older etv2 mutant embryos does not occur in embryos lacking both etv2 and fli1b. Etv2 and fli1b double-deficient embryos fail to form any angiogenic sprouts and show greatly increased apoptosis throughout the axial vasculature. In contrast, fli1a mutation did not affect the recovery of etv2 mutant phenotype. Overexpression analyses indicate that both etv2 and fli1b, but not fli1a, induce the expression of multiple vascular markers and of each other. Temporal inhibition of Etv2 function using photoactivatable morpholinos indicates that the function of Etv2 and Fli1b during angiogenesis is independent from the early requirement of Etv2 during vasculogenesis. RNA-Seq analysis and chromatin immunoprecipitation suggest that Etv2 and Fli1b share the same transcriptional targets and bind to the same E26 transformation-specific sites. Conclusions Our data argue that there are 2 phases of early vascular development with distinct requirements of E26 transformation-specific transcription factors. Etv2 alone is required for early vasculogenesis, whereas Etv2 and Fli1b function redundantly during late vasculogenesis and early embryonic angiogenesis.

Hyperactive FOXO1 results in lack of tip stalk identity and deficient microvascular regeneration during kidney injury

“…There are multiple ETS family members, but of these ETS-1 and ERG are highly expressed in the vasculature and have been shown to be regulated by VEGF signaling [27, 28]. The FOXO family of transcription factors has been implicated in metabolism, proliferation and longevity in endothelial cells [29, 30].…”

ETS transcription factors in embryonic vascular development

“…Despite dramatic defects in early vasculogenesis, vascular development partially recovers even in etv2 null mutant zebrafish embryos at later stages, and only aberrant angiogenesis is observed during later stages. This later recovery is mediated by related ETS transcription factors such as Fli1b which functions redundantly with Etv2 during later stages of vasculogenesis and during angiogenesis [10]. Overexpression of Etv2 results in dramatic expansion of hemangioblast and vascular endothelial marker expression in either zebrafish or Xenopus embryos, demonstrating that a single transcription factor is sufficient to induce vascular endothelial fate in different cell types [18, 21].…”

“…In contrast, a separate study demonstrated that Flk1:Cre; Etv2 conditional mouse knockout embryos displayed disorganized vasculature and reduced hematopoietic progenitors cells and died at 10.5 [11]. Conditional Etv2 knockdown in zebrafish embryos using photoactivatable caging strand hybridized to a morpholino also demonstrated lack of vascular phenotype when Etv2 function is inhibited at 18-somite or later stages after the initial vasculature has formed [10, 22, 36]. However, recent studies show that Etv2 functions redundantly with other ETS transcription factors during later angiogenesis stages [10], which may explain the absence of the phenotype in mouse Flk1:Cre conditional Etv2-knockout embryos.…”

“…Conditional Etv2 knockdown in zebrafish embryos using photoactivatable caging strand hybridized to a morpholino also demonstrated lack of vascular phenotype when Etv2 function is inhibited at 18-somite or later stages after the initial vasculature has formed [10, 22, 36]. However, recent studies show that Etv2 functions redundantly with other ETS transcription factors during later angiogenesis stages [10], which may explain the absence of the phenotype in mouse Flk1:Cre conditional Etv2-knockout embryos. Inducible Etv2 inactivation at the 18-somite stage resulted in angiogenic defects in fli1b −/− zebrafish mutant embryos, which otherwise were phenotypically normal (Fig.…”

“…However, runt-related transcription factor 1 ( runx1 ) expression was not rescued by Fli1a unless it was co-injected with scl -α and scl -β mRNAs, suggesting that Fli1a is insufficient to initiate hematopoietic stem cell (HSC) specification, but that it is capable of mediating the endothelial program [43]. However, in two separate studies, Fli1a overexpression failed to induce vascular endothelial markers in zebrafish embryos [10, 42]. Rather, Fli1a likely plays an important later role in the maintenance of the endothelial lineage.…”