PgmNr Z555: Identifying novel regulators of early cardiac development in zebrafish using single-cell mRNA-seq and ATAC-seq.

Authors:
X. Yuan 1,2 ; M. Song 3 ; A. Aleksandrova 1 ; W. Devine 4 ; B. Bruneau 4,5 ; M. Wilson* 1,2 ; I. Scott* 1,2 ; *co-corresponding authors


Institutes
1) The Hospital for Sick Children, Toronto, ON, Canada; 2) University of Toronto, Toronto, ON,Canada; 3) Peking University, Beijing, China; 4) Gladstone Institute, San Francisco, CA, USA; 5) UCSF, San Francisco, CA, USA.


Abstract:

Heart disease remains a leading cause of death worldwide. A comprehensive understanding of the mechanisms underlying heart formation is crucial for uncovering causes of congenital heart disease and developing regenerative therapies. However there is currently a lack of markers that will enhance our understanding of the very earliest steps of vertebrate heart development, prior to the onset of nkx2.5 expression in the cardiac crescent. Recently a mouse enhancer (Smarcd3-F6) was shown to label cardiac progenitor cells (CPCs) in vivo. Here we asked whether this mouse Smarcd3-F6 enhancer could serve as an early CPC marker in zebrafish. We created a stable zebrafish Smarcd3-F6:EGFP transgenic line and characterized the labeled population using immunostaining, RNA-seq and ATAC-seq (genome-wide profiling of open chromatins). The Smarcd3-F6 enhancer was active at early gastrula stage and enriched for CPCs. Despite sharing no sequence conservation with zebrafish, Smarcd3-F6:EGFP activity required the cardiac master regulator Gata5. Both RNA-seq and ATAC-seq results showed cardiac development-related pathways were enriched in Smarcd3-F6:EGFP labeled cells. Several ATAC-seq peaks near known cardiac genes drove early cardiac expression when tested in embryos, suggesting they could be novel markers facilitating early cardiac development studies. In order to further dissect the Smarcd3-F6+ cell population, we conducted single-cell mRNA-seq and identified a cluster of cells co-expressing known cardiac markers at the end of gastrulation, which represented the potential CPC lineage. Using in situ hybridization we have tested the expression of over 20 novel genes identified from the single-cell cardiac cluster and confirmed the vast majority of them showed early expression in the cardiac domain of zebrafish embryos. Currently we are performing single-cell mRNA-seq at multiple time points during gastrulation to assess the dynamics of CPC development. On-going experiments will also characterize the roles the novel genes play in cardiac development via CRISPR/Cas9 mutagenesis. In addition to improving our understanding of early cardiac gene regulation in zebrafish, our work underscores the conserved regulatory logic of vertebrate heart development.