Fli1 ⁺ cells transcriptional analysis reveals an Lmo2–Prdm16 axis in angiogenesis

Abstract

A network of molecular factors drives the development, differentiation, and maintenance of endothelial cells. Friend leukemia integration 1 transcription factor (FLI1) is a bona fide marker of endothelial cells during early development. In zebrafish <i>Tg(</i><i>f</i><i>li1:EGFP)</i>^<i>y1</i> , we identified two endothelial cell populations, high-<i>fli1</i>⁺ and low-<i>fli1</i>⁺, by the intensity of green fluorescent protein signal. By comparing RNA-sequencing analysis of non-<i>fli1</i> expressing cells (<i>fli1</i>^-) with these two (<i>fli1</i>⁺) cell populations, we identified several up-regulated genes, not previously recognized as important, during endothelial development. Compared with <i>fli1</i>^- and low-<i>fli1</i>⁺ cells, high-<i>fli1</i>⁺ cells showed up-regulated expression of the zinc finger transcription factor PRDI-BF1 and RIZ homology domain containing 16 (<i>prdm16</i>). <i>Prdm16</i> knockdown (KD) by morpholino in the zebrafish larva was associated with impaired angiogenesis and increased number of low-<i>fli1</i>⁺ cells at the expense of high-<i>fli1</i>⁺ cells. In addition, <i>PRDM16</i> KD in endothelial cells derived from human-induced pluripotent stem cells impaired their differentiation and migration in vitro. Moreover, zebrafish mutants (mut) with loss of function for the oncogene LIM domain only 2 (<i>lmo2</i>) also showed reduced <i>prdm16</i> gene expression combined with impaired angiogenesis. <i>Prdm16</i> expression was reduced further in endothelial (CD31⁺) cells compared with CD31^- cells isolated from <i>l</i><i>mo2</i>-mutants (<i>l</i><i>mo2-</i>mut) embryos. Chromatin immunoprecipitation-PCR demonstrated that Lmo2 binds to the promoter and directly regulates the transcription of <i>prdm16</i> This work unveils a mechanism by which <i>prdm16</i> expression is activated in endothelial cells by Lmo2 and highlights a possible therapeutic pathway by which to modulate endothelial cell growth and repair.