Epicardial heterogeneity during zebrafish heart development

The epicardium, a cell layer enveloping the heart muscle, drives embryonic heart development and heart repair in the adult zebrafish. Previous studies found the epicardium to consist of multiple cell populations with distinct phenotypes and functions. Here, I investigated epicardial heterogeneity in the developing zebrafish heart, focusing on the developmental gene program that is also reactivated during adult heart regeneration. Transcription factor 21 (Tcf21), T-box 18 (Tbx18) and Wilms’ tumor suppressor 1b (Wt1b) are often used interchangeably to identify the zebrafish epicardium. Analyzing newly generated reporter lines and endogenous gene expression, I showed that the epicardial expression of tcf21, tbx18 and wt1b during development is heterogeneous. I then collected epicardial cells from newly generated reporter lines at 5 days-post-fertilization and performed single-cell RNA sequencing. I identified three distinct epicardial subpopulations with specific gene expression profiles. The first subpopulation expressed tcf21, tbx18 and wt1b and appeared to represent the main epicardial layer. The second subpopulation expressed tbx18, but not tcf21 or wt1b. Instead, it expressed smooth muscle markers and seemed restricted to the bulbus arteriosus. The third epicardial subpopulation only expressed tcf21 and resided within the epicardial layer. I compared the single-cell subpopulations with transcriptomic bulk data and visualized the expression of marker genes to investigate their spatial distribution. Using ATAC sequencing, I additionally identified open regulatory regions located in proximity to subpopulation-specific marker genes and showed subpopulation-specific activity in vivo. My results detail distinct cell populations in the developing zebrafish epicardium, likely to fulfil distinct and specific cellular functions. Future experiments will involve targeting signature genes enriched within each epicardial subpopulation, such as those encoding Adrenomedullin a (first subpopulation), Alpha Smooth Muscle Actin (second subpopulation) and Claudin 11a (third subpopulation), employing cell type-specific genome editing to test whether and how the identified heterogeneity underlies distinct epicardial cell fates and functions. Taken together, my work adds significantly to the understanding of the cellular and molecular basis of epicardial development and can offer novel insights in the context of heart regeneration.