Single-Cell Transcriptomic Analysis Reveals Early Transcriptional Heterogeneity of Cardiac-Associated Cell Populations During Zebrafish Embryogenesis

Understanding the development and differentiation of cardiac progenitor cells during the initial stages of embryogenesis is central to a complete understanding of vertebrate heart development. In zebrafish, cardiac specification begins during gastrulation; however, the single-cell transcriptional dynamics of initial cardiac lineage commitment remain not fully defined. In this case, we integrated single-cell RNA sequencing datasets of zebrafish embryos at 4 and 6 h post-fertilisation (hpf) to investigate early cardiac lineage specification. The unsupervised clustering of the integrated dataset identified 12 distinct cell clusters, which made it possible to identify a transcriptionally distinct population of cells characterised by the coordinated expression of transcription factors associated with cardiac development. A further subclustering of the cells expressing cardiac-associated transcription factors showed a significant level of early diversification of the cardiac progenitor group. A projection onto low-dimensional embedding revealed a structured transcriptional organisation of the cardiac subclusters, marked by the differential expression of key cardiac transcription factors, including Gata5, Gata6, Hand2, Nkx2.5, and Tbx5a. A pseudotemporal trajectory analysis uncovered a continuous developmental progression within the cardiac lineage and indicated the gene-specific dynamic regulation and temporal hierarchy of cardiac transcriptional programs. Collectively, these results indicate that zebrafish cardiac progenitors are transcriptionally diverse and acquire cardiac fate through a sustained, continuous regulatory process rather than an abrupt fate transition. This work provides an informative, high-resolution model of early cardiac lineage specification and highlights the power of single-cell transcriptomics for analysing dynamic events in vertebrate embryogenesis.