Heart field origin of great vessel precursors relies on nkx2.5-mediated vasculogenesis

Abstract

The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that make indispensable contributions to the carotid arteries and great vessels of the heart, including the aorta and pulmonary artery1, 2. During embryogenesis, the PAAs appear in a craniocaudal sequence to connect pre-existing segments of the primitive circulation after de novo vasculogenic assembly from angioblast precursors3, 4. Despite the unique spatiotemporal characteristics of PAA development, the embryonic origins of PAA angioblasts and the genetic factors regulating their emergence remain unknown. Here, we identify the embryonic source of PAA endothelium as nkx2.5+ progenitors in lateral plate mesoderm long considered to adopt cell fates within the heart exclusively5, 6. Further, we report that PAA endothelial differentiation relies on Nkx2.5, a canonical cardiac transcription factor not previously implicated in blood vessel formation. Together, these studies reveal the heart field origin of PAA endothelium and attribute a novel vasculogenic function to the cardiac transcription factor nkx2.5 during great vessel precursor development.

Origin, Specification, and Plasticity of the Great Vessels of the Heart

“…To test this idea, we analyzed the formation of the ventral head endothelium in tcf21 mutant, nkx2.5 mutant, and tcf21 ; nkx2.5 double mutant embryos transgenic for flk:mCherry to label endothelial cells and nkx2.5:ZsYellow to label pPAA progenitors. In contrast to nkx2.5 morpholino knockdown experiments [8], which have recently been reported to be error prone [37], we find that the pPAAs are present at 60 hpf in tcf21 mutant, nkx2.5 mutant, and tcf21 ; nkx2.5 double mutant embryos, although pPAAs occasionally failed to lumenize in nkx2.5 mutant, tcf21 mutant, and tcf21 ; nkx2.5 double mutant embryos (Figure 5R). This result indicates, unexpectedly, that tcf21 and nkx2.5 are not required for pPAA formation.…”

“…Consistent with observations in chicken and mice [20–22], we find that the transcription factor tcf21 (Epicardin/Capsulin/Pod1) is expressed bilaterally at the 12-somite stage (15 hpf) in two domains within the head mesoderm—an anterior domain and a posterior domain (Figure 1A). Using tcf21:mCherry-NTR (referred to as tcf21:mCherry if not indicated otherwise), a transcriptional reporter for tcf21 + cells [23], we find that at the 14-somite stage (16 hpf), the anterior tcf21 expression domain is bounded anteriorly by the prechordal plate mesoderm marked by pitx2 [24] and overlaps posteriorly with the cardiac mesoderm marked by a transcriptional reporter for nkx2.5 ( nkx2.5: ZsYellow ) [8, 25] (Figures 1D, 1J, and 1K). The posterior domain of tcf21 expression overlaps anteriorly with the nkx2.5 -expressing domain (Figures 1D and 1E; Movie S3).…”

“…One candidate for such a transcription factor is Nkx2.5, as a subset of the tcf21 + cells co-express nkx2.5 before mesoderm migration into the pharyngeal arches (Figures 1D and 1E). Moreover, nkx2.5 -expressing ( nkx2.5 +) cells also contribute to the pPAAs [8], and perduring fluorescent proteins expressed from the tcf21 and nkx2.5 promoters co-label the ventral head endothelium (Figures 1L, 1M, 5A–5D, and 5D′). To test this idea, we analyzed the formation of the ventral head endothelium in tcf21 mutant, nkx2.5 mutant, and tcf21 ; nkx2.5 double mutant embryos transgenic for flk:mCherry to label endothelial cells and nkx2.5:ZsYellow to label pPAA progenitors.…”

“…In mice, clonal analysis suggests that distinct progenitor pools give rise to distinct portions of head and heart muscles [5–7]. In mice and fish, nkx2.5 -expressing mesoderm gives rise to heart muscle as well as pPAA endothelium [8]. Together, these observations suggest that the head muscles, the heart, and the pharyngeal arch arteries share a common origin.…”

Molecular Basis for Dysregulated Activation of NKX2‐5 in the Vascular Remodeling of Systemic Sclerosis

“…Chromatin was immunoprecipitated using specific antibodies. Five primer pairs 1, 2, 3, 4, 5 were designed to cover the 1.6‐kb genomic region of the downstream enhancer. Results are shown as the fold enrichment of each protein in every genomic segment.…”