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

Abstract

The mechanisms by which arterial fate is established and maintained are not clearly understood. Although a number of signaling pathways and transcriptional regulators have been implicated in arterio-venous differentiation, none are essential for arterial formation, and the manner in which widely expressed factors may achieve arterial-specific gene regulation is unclear. Using both mouse and zebrafish models, we demonstrate here that arterial specification is regulated combinatorially by Notch signaling and SoxF transcription factors, via direct transcriptional gene activation. Through the identification and characterization of two arterial endothelial cell-specific gene enhancers for the Notch ligand Delta-like ligand 4 (Dll4), we show that arterial Dll4 expression requires the direct binding of both the RBPJ/Notch intracellular domain and SOXF transcription factors. Specific combinatorial, but not individual, loss of SOXF and RBPJ DNA binding ablates all Dll4 enhancer-transgene expression despite the presence of multiple 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 ablation of endogenous dll4 expression. Fascinatingly, this combinatorial ablation leads to a loss of arterial markers and the absence of a detectable dorsal aorta, demonstrating the essential roles of SoxF and Notch, together, in the acquisition of arterial identity.arterial-specific enhancer | CSL

Dynamic regulation of VEGF-inducible genes by an ERK-ERG-p300 transcriptional network

“…We found that the DLL4 locus contains multiple conserved ERG-bound enhancers, including regions ∼12 kb upstream of the transcriptional start site (TSS) and within intron 3 (Fig. 8A), both of which were previously shown to have arterialspecific activity in vivo (Sacilotto et al, 2013;Wythe et al, 2013). We also identified an ERG-bound enhancer, conserved in cows and humans, ∼3.0 kb upstream of the gene H2.0-like homeobox (HLX) ( Fig.…”

Fast revascularization of the injured area is essential to support zebrafish heart regeneration

“…These observations support the notion that, as shown in the caudal fin regeneration model (24), once the vascular plexus is formed, some vessels gain arterial identity. To further test whether arterial specification is a late event during revascularization, we developed an arterial-specific reporter line by using a previously described enhancer of the mouse Dll4 gene (26), Tg(Dll4in3:TagRFP). This line expresses TagRFP specifically in arterial endothelium in both embryos ( Fig.…”

Sox7 controls arterial specification in conjunction withhey2andefnb2function

“…Recently, in vitro studies reported that SoxF transcription factors can bind the arterial-specific enhancer of the notch ligand dll4 (Sacilotto et al, 2013). As Hey2 and Efnb2a/b have been suggested to act downstream of Notch signaling (Lawson et al, 2001) and as we show here that sox7 genetically interacts with both factors, we wondered whether the arterial defects in sox7 mutants are a consequence of altered Notch signaling levels.…”

“…One subgroup of the Sox family that is of particular interest for vascular development is the SoxF group, consisting of SOX7, SOX17 and SOX18. In vitro studies revealed that SoxF transcription factors can bind the arterial-specific enhancer of the Notch ligand dll4 (Sacilotto et al, 2013). Furthermore, Sox17 has recently been shown to play a key role in endoderm formation, hematopoietic stem cell regulation and the acquisition of arterial identity by functioning upstream of Notch signaling (Hudson et al, 1997;Kanai-Azuma et al, 2002;He et al, 2011;Corada et al, 2013).…”