ETS transcription factor Etsrp / Etv2 is required for lymphangiogenesis and directly regulates vegfr3 / flt4 expression

Abstract

The molecular mechanisms initiating the formation of the lymphatic system, lymphangiogenesis, are still poorly understood. Here we have identified a novel role in lymphangiogenesis for an ETS transcription factor, Etv2/Etsrp, a known regulator of embryonic vascular development. Through the use of fully validated photoactivatable morpholinos we show that inducible Etv2 inhibition in zebrafish embryos at 1 day post-fertilization (dpf) results in significant inhibition of lymphangiogenesis, while development of blood vessels is unaffected. In Etv2-inhibited embryos and larvae, the number of lymphatic progenitors is greatly reduced, the major lymphatic vessel, the thoracic duct, is absent or severely fragmented, and lymphangiogenesis-associated marker expression, including lyve1b, prox1a, and vegfr3/flt4, is strongly downregulated. We also demonstrate that lymphatic progenitors in Etv2 deficient embryos fail to respond to Vegfc signaling. Chromatin immunoprecipitation and sequencing (ChIP-Seq) studies using differentiated mouse embryonic stem (ES) cells as well as luciferase reporter studies in the ES cells and in zebrafish embryos argue that Etv2 directly binds the promoter/enhancer regions of Vegfc receptor Vegfr3/Flt4 and lymphatic marker Lyve1, and promotes their expression. Together these data support a model where Etv2 initiates lymphangiogenesis by directly promoting the expression of flt4 within the posterior cardinal vein.

Zebrafish etv2 knock‐in line labels vascular endothelial and blood progenitor cells

“…9 Whole mount in situ hybridization at later stages is typically challenging in zebrafish embryos due to poor probe penetration, therefore it is possible that etv2 continues to be expressed at low levels in the vasculature throughout adulthood, and this low level expression is amplified in etv2 ci32Gt ; UAS:GFP line. In addition, it enables visualization and study of etv2-deficient cells that would ordinarily express etv2.…”

“…We have previously reported similar although weaker etv2 mRNA expression in the vasculature at 2-2.5 dpf. 9 Although endogenous etv2 expression in most blood vessels was largely not detectable at 3 dpf or later stages (data not shown), it is challenging detecting expression using whole-mount in situ hybridization in older embryos due to the limited probe penetration (see Discussion). In contrast, vascular endothelial specific expression in Tg(−2.3 etv2:GFP) line was downregulated between 48 and 72 hpf, while nonspecific GFP expression in epithelial cells and a subset of neurons was apparent ( Figure 6).…”

“…Both lines also showed GFP expression in the lymphatic vasculature ( Figure 6). 9 Although endogenous etv2 expression in most blood vessels was largely not detectable at 3 dpf or later stages (data not shown), it is challenging detecting expression using whole-mount in situ hybridization in older embryos due to the limited probe penetration (see Discussion). 9 Although endogenous etv2 expression in most blood vessels was largely not detectable at 3 dpf or later stages (data not shown), it is challenging detecting expression using whole-mount in situ hybridization in older embryos due to the limited probe penetration (see Discussion).…”

“…These etv2 functions are conserved between vertebrate embryos, and mouse Etv2 mutant embryos show failure of yolk sac and intraembryonic vasculogenesis and hematopoiesis. 7,9 Furthermore, not all distal regulatory elements involved in the dynamic regulation of etv2 expression may be present in these reporters. A few such zebrafish reporter lines have been previously generated and include the TgBAC(etv2:GFP), which was generated by BAC recombineering, 7 and the Tg(−2.3 etv2:GFP) reporter line, which contains 2.3 kb region of the etv2 promoter and the first intron.…”

“…However, the TgBAC(etv2:GFP) line shows nonspecific expression in the neural tube while the Tg(−2.3 etv2:GFP) line shows nonspecific expression in epithelial and neuronal cells. 7,9 Furthermore, not all distal regulatory elements involved in the dynamic regulation of etv2 expression may be present in these reporters.…”