Nr2f1a and Isl1 repress acquisition of epicardial identity in venous atrial cardiomyocytes

Regulatory networks that maintain cardiomyocyte identity are not completely understood. Here, we examined the relationship of the transcription factors (TFs) Nr2f1a and Isl1, which respectively repress and promote pacemaker cardiomyocyte (PC) differentiation within the venous pole of zebrafish atria. Using zebrafish nr2f1a;isl1 mutants, we found that loss of Isl1 exacerbates the inability to maintain atrial cardiomyocyte (AC) identity found in nr2f1a mutant hearts. Subsequently, while nr2f1a;isl1 mutants have a failure of PC differentiation and inability of ACs to transdifferentiate into PC identity, ACs in nr2f1a;isl1 mutant hearts lose myocardial marker expression, gain epicardial cell (EC) gene expression, and exit the myocardial layer. Single cell RNA-seq analysis of hearts supports that the ACs and ECs of nr2f1a;isl1 mutant embryos share common myocardial and EC transcriptomic signatures. Depletion of tcf21 and tbx18 in nr2f1a;isl1 mutants was sufficient to prevent the acquisition of EC identity within putatively transdifferentiating ACs. Thus, our results reveal reiterative requirements for Nr2f and Isl1 TFs in binary fate decisions within the genetic hierarchy that maintains distinct AC, PC, and EC identities at the venous pole of vertebrate hearts.