scRNA-seq reveals the diversity of the developing cardiac cell lineage and molecular players in heart rhythm regulation
We utilized scRNA-seq to delineate the diversity of cell types in the zebrafish heart. Transcriptome profiling of over 50,000 cells at 48 and 72 hpf defined at least 18 discrete cell lineages of the developing heart. Utilizing well-established gene signatures, we identified a population of cells likely to be the primary pacemaker and characterized the transcriptome profile defining this critical cell type. Two previously uncharacterized genes, atp1b3b and colec10, were found to be enriched in the sinoatrial cardiomyocytes. CRISPR/Cas9-mediated knockout of these two genes significantly reduced heart rate, implicating their role in cardiac development and conduction. Additionally, we describe other cardiac cell lineages, including the endothelial and neural cells, providing their expression profiles as a resource. Our results established a detailed atlas of the developing heart, providing valuable insights into cellular and molecular mechanisms, and pinpointed potential new players in heart rhythm regulation. Overall design: Whole hearts were extracted from double transgenic individuals [sqet31Et x Tg(myl7:mRFP) and sqet33mi59BEt x Tg(myl7:mRFP)]. The term “pseudo-replicates” was used to denote samples of two different genotypic backgrounds (sqet31Et and sqet33mi59BEt) at the same developmental stage (in total 4 samples, 2 per each developmental stage). Embryos were anesthetized with Tricaine (0.16 mg/ml in egg water) and large-scale extraction was performed according to a previously published protocol, with minor adjustments. Hearts were manually separated from the remaining tissue under a fluorescence stereomicroscope and collected into 0,5 ml of EDM (L-15/10% FBS). Single-cell heart suspension was obtained according to Bresciani et al. Ultimately, cells were resuspended by gentle pipetting, loaded on the cell counting chamber, visually inspected under the microscope (Zeiss Apotome), and quantified by Countess automated cell counter (Invitrogen). After viability and quantity check, dissociated cells derived from embryonic zebrafish hearts at 48 hpf and 72 hpf were loaded on 10x Chromium Controller Chip G (10x Genomics) and processed according to the Chromium Next GEM Single Cell 3' Reagent Kits v.3.1. Final libraries were sequenced (read 1 - 28 cycles, i7 index - 8 cycles, i5 index - 0 cycles,  read 2 - 91 cycles) on Nextseq 500 (Illumina).