Analysis of Dll4 regulation reveals a combinatorial rolefor Sox and Notch in arterial development

Abstract

The mechanisms by which arterial fate is established and main-tained are not clearly understood. Although a number of signalingpathways and transcriptional regulators have been implicated inarterio-venous differentiation, none are essential for arterialformation, and the manner in which widely expressed factorsmay achieve arterial-specific gene regulation is unclear. Using bothmouse and zebrafish models, we demonstrate here that arterialspecification is regulated combinatorially by Notch signaling andSoxF transcription factors, via direct transcriptional gene activa-tion. Through the identification and characterization of two arte-rial endothelial cell-specific gene enhancers for the Notch ligandDelta-like ligand 4 (Dll4), we show that arterial Dll4 expressionrequires the direct binding of both the RBPJ/Notch intracellulardomain and SOXF transcription factors. Specific combinatorial,but not individual, loss of SOXF and RBPJ DNA binding ablatesall Dll4 enhancer-transgene expression despite the presence ofmultiple functional ETS binding sites, as does knockdown of sox7;sox18 in combination with loss of Notch signaling. Furthermore,triple knockdown of sox7, sox18 and rbpj also results in ablationof endogenous dll4 expression. Fascinatingly, this combinatorialablation leads to a loss of arterial markers and the absence of a de-tectable dorsal aorta, demonstrating the essential roles of SoxF andNotch, together, in the acquisition of arterial identity