GSE126647
Pbx4 limits vertebrate heart size and promotes pharyngeal arch artery development through partitioning progenitor fates within the second heart field and restricting proliferation
Development of the vertebrate heart requires the appropriate integration of temporally-distinct differentiating progenitor populations. While factors that promote accrual of later-differentiating second heart field (SHF)-derived cells to the outflow tract (OFT) have been intensively investigated, few signals are understood that specifically restrict the size of the vertebrate OFT. Here, we show that improper specification and proliferation of SHF progenitors in zebrafish lazarus (lzr) mutants, which lack the transcription factor Pbx4, produces enlarged hearts with a specific increase in ventricular cardiomyocytes and smooth muscle cells within the OFT. Specifically, optogenetic lineage-tracing demonstrates Pbx4 initially promotes the proper partitioning of the SHF into anterior progenitors, which contribute to the OFT, and adjacent endothelial cell progenitors, which contribute to posterior pharyngeal arch arteries. Subsequently, Pbx4 also limits the size of the SHF through repressing SHF progenitor proliferation. Single-cell RNA sequencing of nkx2.5+ cells revealed that the enlarged SHF progenitor population within Pbx4-deficient embryos assimilates characteristics of normally distinct proliferative and more anterior, differentiated cardiomyocyte populations. Therefore, the generation of proper OFT size and great arteries in vertebrates requires Pbx-dependent stratification of unique progenitor differentiation states to facilitate homeotic-like transformations and limit progenitor production within the SHF. Overall design: To determine the effects of Pbx4 loss on cardiac progenitors and differentiated cardiomyocytes, we utilized  single-cell RNA-Seq (scRNA-Seq) data sets collected from  control and Pbx4-depleted embryos nkx2.5:ZsYellow+ cells at 28 hours post-ferlilization. The 10x Genomics platform was used for these studies (version 2 library chemistry).