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.

Indiana University-Purdue University Indianapolis (IUPUI) / Insulin-like Growth Factor I (IGF-I) is a growth factor that stimulates both mitogenic and anabolic responses in articular chondrocytes. While it has been shown that exogenous IGF-I can regulate chondrocyte integrins, little is known regarding regulatory effects of IGF-I produced from a transiently expressed plasmid based adeno-associated virus (pAAV) vector. Because chondrocytes are using cellular machinery to overexpress IGF-I, it is of interest to see whether or not pAAV IGF-I will significantly upregulate or downregulate chondrocyte integrins. Additionally, it is of interest to know whether chondrocyte adhesion through integrins will have any regulatory effects on the production of IGF-I from the transgene. Therefore, this study will ascertain if pAAV IGF-I will have similar effects that exogenous IGF-I has on integrin regulation and if integrin silencing mechanisms will affect the production of IGF-I from the transgene. To test these hypotheses, adult articular chondrocytes were doubly transfected with the pAAV vector for IGF-I and short interference ribonucleic acid (siRNA) for integrins beta 1 and alpha V. Gene products were monitored at the transcriptional levels using quantitative real time polymerase chain reactions (qPCR) and IGF-I protein production was monitored at the translational level using enzyme linked immunoabsorbant assays (ELISAs). Adult articular chondrocytes doubly transfected were encapsulated in a three dimensional hydrogel system to simulate an in vivo environment. Samples were collected for analysis at days 2, 4, and 6 post encapsulation. Results show that IGF-I treatment with the pAAV vector does not cause significant changes in the transcriptional regulation of the beta 1 integrin in a three dimensional hydrogel system. The pAAV IGF-I vector did not cause significant regulatory changes on integrin alpha V at any time point during the experiment. Additionally, by knocking down the expression levels of integrins by using siRNA, it was shown that integrin knockdown does not have a significant regulatory effect on transcriptional or translational expression levels of IGF-I from the pAAV vector.