Prdm16 and Notch functionally and physically interact during artery development

ABSTRACTRationaleProper functionality of the circulatory system requires correct arteriovenous (AV) endothelial cell (EC) differentiation. While Notch signaling and its downstream effector Hes- Related Family bHLH Transcription Factor with YRPW Motif (Hey)2 favor arterial specification, transcription factor (TF) chicken ovalbumin upstream transcription factor 2 (Coup-TFII) inhibits canonical Notch activity to induce venous identity. However, transcriptional programs that compete with Coup-TFII to orchestrate arterial specification upstream of Notch remain largely unknown. We identified positive regulatory domain-containing protein (Prdm)16 as an arterial EC- specific TF, but its role during arterial EC specification and development remains unexplored.ObjectiveTo unravel the role of Prdm16 during arterial endothelial lineage specification and artery formation.Methods and ResultsTranscriptomic data of freshly isolated arterial and venous ECs from humans and mice revealed that Prdm16 is exclusively expressed by arterial ECs throughout development. This expression pattern was independent of hemodynamic factors and conserved in zebrafish. Accordingly, loss of prdm16 in zebrafish perturbed AV endothelial specification and caused AV malformations in an EC-autonomous manner. This coincided with reduced canonical Notch activity in arterial ECs and was amplified when prdm16 and notch pathway members were concomitantly knocked down. In vitro studies further indicated that Prdm16 not only amplified Notch signaling, but also physically and functionally interacted with Hey2 to drive proper arterial specification.ConclusionWe showed that Prdm16 plays a pivotal role during arterial development through its physical and functional interaction with canonical Notch. As both Hey2 and Prdm16 have been associated with diverse vascular disorders including migraine and atherosclerosis, Prdm16 represents an attractive new target to treat these vascular disorders.

Notch controls arterialization by regulating the cell cycle and not differentiation

Arteries are thought to be formed by the induction of a highly conserved arterial genetic program in a subset of vessels experiencing an increase in pulsatile and oxygenated blood flow. Both VEGF and Notch signalling have been shown to be essential for the initial steps of arterial specification. Here, we combined inducible genetic mosaics and transcriptomics to modulate and understand the function of these signalling pathways on cell proliferation, arterial-venous differentiation and mobilization. We observed that endothelial cells with high VEGF or Notch signalling are not genetically pre-determined and can form both arteries and veins. Importantly, cells completely lacking the Notch-Rbpj transcriptional activator complex can form arteries when the Myc-dependent metabolic and cell-cycle activity is suppressed. Thus, arterial development does not require the induction of a Notch-dependent arterial differentiation program, but rather the timely suppression of the endothelial metabolism and cell-cycle, a process preceding arterial mobilization and complete differentiation.

Akt is required for artery formation during embryonic vascular development

SUMMARYOne of the first events in the development of the cardiovascular system is morphogenesis of the main embryonic artery, the dorsal aorta (DA). The DA forms via a conserved genetic process mediated by the migration, specification, and organization of endothelial progenitor cells into a distinct arterial lineage and vessel type. Several angiogenic factors activate different signaling pathways to control DA formation, however the physiological relevance of distinct kinases in this complex process remains unclear. Here, we identify the role of Akt during early vascular development by generating mutant zebrafish lines that lack expression of akt isoforms. Live cell imaging coupled with single cell RNA sequencing of akt mutants reveal that Akt is required for proper development of the DA by sustaining arterial cell progenitor specification and segregation. Mechanistically, inhibition of active FOXO in akt mutants rescues impaired arterial development but not the expression of arterial markers, whereas Notch activation rescues arterial marker expression. Our work suggests that Akt activity is critical for early artery development, in part via FOXO and Notch-mediated regulation.

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.

Coronary arterial development is regulated by a Dll4-Jag1-EphrinB2 signaling cascade

Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase MFng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.