Prdm16 and Notch functionally and physically interact during artery development

Proper vascular function requires correct arteriovenous (AV) endothelial cell (EC) differentiation. While Notch and its effector Hey2 favor arterial specification, transcription factor (TF) Coup-TFII inhibits Notch activity to induce venous identity. TFs competing with Coup-TFII to orchestrate arterial specification upstream of Notch remain largely unknown. Transcriptional profiling of human and mouse ECs and whole mount in situ hybridization in zebrafish embryos revealed that the TF Prdm16 is exclusively expressed by arterial ECs throughout development, independent of species and hemodynamic factors. Accordingly, prdm16 deficiency in zebrafish perturbed AV specification and caused aberrant shunts between the arterial and venous circulation in an EC- autonomous manner. Prdm16 regulated arterial Notch activity both in vitro and in vivo and simultaneous knockdown of notch aggravated the vascular defects observed in prdm16 morphant zebrafish. In vitro studies revealed that Prdm16 did not alter the levels of the transcriptionally active, intracellular domain of the Notch1 receptor (N1ICD). Rather, it boosted Notch signaling by physically and functionally interacting with both N1ICD and the Notch downstream effector Hey2. Together, these data demonstrate the hitherto unexplored role for Prdm16 during arterial development, by its dual activity on arterial canonical Notch during embryogenesis.