Nkx2-5 defines distinct scaffold and recruitment phases during formation of the cardiac Purkinje fiber network

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PF share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and PF network morphogenesis are unknown. Using genetic fate mapping and temporal clonal analysis we identify cardiomyocytes committed to the PF lineage as early as E7.5. A polyclonal PF network emerges by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors. At late fetal stages, the segregation of conductive cells increases during a phase of rapid recruitment to build the definitive PF network. In Nkx2-5 haploinsufficient embryos, PF differentiation is impaired leading to failure to extend the scaffold. In particular, late fetal recruitment is abolished resulting in PF hypoplasia and persistence of bipotent progenitors at birth. Our results reveal how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.