Tbx1-dependent and independent pathways promote six gene expression downstream of retinoic acid signaling to determine cardiomyocyte number in zebrafish

Tight regulation of retinoic acid (RA) levels is critical for normal heart development in vertebrates, with early RA signaling restricting the size of the cardiac progenitor field within the anterior lateral plate mesoderm (ALPM). However, the regulatory networks by which RA signaling limits the size of the cardiac progenitor field and consequently cardiomyocyte (CM) number are not fully understood. Here, we identified that the expression of the transcription factors six1b and six2a, whose orthologs regulate outflow tract (OFT) development in mice, are expanded within the ALPM of RA-deficient zebrafish embryos. At 48 h post-fertilization (hpf), RA-deficient six1b;six2a double mutants, but not single six1b or six2a mutants, had a reduction in the number of surplus CMs relative to RA-deficient wild-type embryos. The expansion of six1b, as well as fgf8a, within the ALPM were dependent on tbx1, a factor that is also expanded within the ALPM of RA-deficient zebrafish embryos. However, the restriction of six2a expression by RA was independent of Tbx1. Consistent with a bifurcation of pathways downstream of RA signaling, loss of function experiments demonstrates that tbx1 expansion alone does not contribute to the surplus CMs in RA-deficient embryos. Together, our data indicate that both Tbx1-dependent and independent pathways restrict Six dosage downstream of RA within the ALPM to pattern the CM progenitor field and establish proper heart size.