The cardiovascular system comprises diverse cellular lineages that emerge from progenitor cells in the anterior lateral plate mesoderm (ALPM). In zebrafish, nkx2.5 expression initiates in the bilateral field of ALPM progenitors during early somitogenesis before their spatial segregation into two main subpopulations. The cardiac fraction, including first (FHF) and second heart field (SHF) progenitors, migrates to the midline and gives rise to ventricular myocardium. The pharyngeal fraction remains lateral and sequesters in the cores of the pharyngeal arches (PAs). Progenitors in PA2 give rise to craniofacial muscles and three lineages in the outflow tract, while those in PA3-6 differentiate into pharyngeal arch artery (PAA) endothelium. The mechanisms underlying nkx2.5 + progenitor cell segregation into the various downstream fates remain unclear. Here, we demonstrate that nkx2.5 + progenitors in the ALPM are heterogenous with predetermined lineage outcomes. Specifically, single-cell RNA sequencing of nkx2.5:ZsYellow + progenitors revealed six transcriptionally unique populations, including a cardiomyocyte cluster (FHF-derived) and a large lateral plate mesoderm (LPM) population that further subclustered into three distinct groups. LPM-1 differentially expressed aplnra , hey1 , and the retinoic acid (RA)-eliminating enzyme cyp26c1 , LPM-2 expressed bmp4, bmp6 , and cdh11 , and LPM-3 expressed the transcriptional repressor prdm1a and the RA-producing enzymes rhd10a and aldh1a2 . In situ hybridization uncovered regionalization in the nkx2.5 + ALPM with LPM-1 cells occupying the anterior-medial and central region of the field and LPM-3 the posterior domain. Targeted photoconversion of nkx2.5:Kaede revealed that LPM-1 are PA2 progenitors while LPM-3 are progenitors for PA3-6. We are currently performing spatial transcriptomics to generate a comprehensive expression map of the ALPM, localizing and tracing LPM-2 progenitors, and performing functional studies that suggest a tbx1 -RA-prdm1a signaling pathway is required for LPM-3 specification. In conclusion, our data highlight progenitor heterogeneity in the ALPM as a source of the lineage diversification required to build the cardiovascular system.
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