Hox/Meis-dependent gene-regulatory transition underlies cardiopharyngeal neural crest diversification

Abstract

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox-positive pharyngeal NCCs to Hox-negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and gene-regulatory network architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox-codes. A Meis2–Sox9–Scx gene-regulatory network defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox-dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification. How neural crest cells remodel their regional identity to generate distinct cardiovascular tissues has remained unresolved. By combining single-cell multiomics, spatial transcriptomics, enhancer analyses, and lineage tracing, this study identifies a Hox/Meis-dependent regulatory transition that governs cardiopharyngeal neural crest diversification. A comprehensive spatiotemporal atlas links early pharyngeal neural crest cells to late cardiac neural crest derivatives. Loss of Hox transcriptional programs is coupled to a Meis-interacting transcriptional co-factor switch that rewires gene regulatory networks during cardiac cushion differentiation. A Meis2–Sox9–Scx regulatory cascade defines an intermediate progenitor giving rise to coronary artery smooth muscle and semilunar valve tissues. A comprehensive spatiotemporal atlas links early pharyngeal neural crest cells to late cardiac neural crest derivatives. Loss of Hox transcriptional programs is coupled to a Meis-interacting transcriptional co-factor switch that rewires gene regulatory networks during cardiac cushion differentiation. A Meis2–Sox9–Scx regulatory cascade defines an intermediate progenitor giving rise to coronary artery smooth muscle and semilunar valve tissues. Loss of Hox-dependent regional identity enables pharyngeal neural crest cells to acquire new fates during heart development.