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

Caroline Choquet,Robert G Kelly,Lucile Miquerol

doi:10.1038/s41467-020-19150-9

Abstract

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. Here, we apply genetic fate mapping and temporal clonal analysis to identify murine cardiomyocytes committed to the PF lineage as early as E7.5. We find that 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 through a non-cell autonomous mechanism. We also show that PF differentiation is impaired in Nkx2-5 haploinsufficient embryos leading to failure to extend the scaffold. In particular, late fetal recruitment fails, resulting in PF hypoplasia and persistence of bipotent progenitors. Our results identify how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.

Highlights

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network
The terminal conduction system consists of Purkinje fiber cells (PFs) characterized by fast conduction properties necessary to activate the ventricular myocardium from the apex to the base and ensure the efficient expulsion of the blood
Cx40-CreERT223 mice were crossed with Rosa26-YFP conditional lineage reporter mice and Cre recombination was activated by tamoxifen injections at different embryonic days, prior to analysis at postnatal day 21 (P21; Supplementary Fig. 1)

Summary

Introduction

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. To identify the timing of lineage segregation of contractile and conductive cardiomyocytes within the trabecular progenitor cell population, an in vivo temporal clonal analysis is required. By performing temporal clonal analysis of cardiac progenitors, we identify distinct phases of PF network morphogenesis: an initial bipotent-progenitor-dependent scaffolding phase and a later conductive progenitor recruitment phase. Extending our analysis to Nkx[2,3,4,5] heterozygous mutant mice we define the temporal requirement for Nkx[2,3,4,5] during the segregation of trabecular cells towards the conductive lineage and gain mechanistic insight into the etiology of PF hypoplasia

Methods

Results

Conclusion