Segregation of morphogenetic regulatory function of Shox2 from its cell fate guardian role in sinoatrial node development.

Shox2 plays a vital role in the morphogenesis and physiological function of the sinoatrial node (SAN), the primary cardiac pacemaker, manifested by the formation of a hypoplastic SAN and failed differentiation of pacemaker cells in Shox2 mutants. Shox2 and Nkx2-5 are co-expressed in the developing SAN and regulate the fate of the pacemaker cells through a Shox2-Nkx2-5 antagonistic mechanism. Here we show that simultaneous inactivation of Nkx2-5 in the SAN of Shox2 mutants (dKO) rescued the pacemaking cell fate but not the hypoplastic defects, indicating uncoupling of SAN cell fate determination and morphogenesis. Single-cell RNA-seq revealed that the presumptive SAN cells of Shox2-/- mutants failed to activate pacemaking program but remained in a progenitor state preceding working myocardium, while both wildtype and dKO SAN cells displayed normal pacemaking cell fate with similar cellular state. Shox2 thus acts as a safeguard but not a determinant to ensure the pacemaking cell fate through the Shox2-Nkx2-5 antagonistic mechanism, which is segregated from its morphogenetic regulatory function in SAN development.