Applying state of the art technologies in the zebrafish to elucidate the mechanisms underpinning cardiovascular regeneration
Item statusRestricted Access
Embargo end date12/01/2024
Ross Stewart, Katherine Margaret
While the adult mammalian heart lacks the ability to undergo regeneration following injury, both larval and adult zebrafish can fully recover from a range of cardiac insults. Over the past two decades, our understanding of the complexities of injury response mechanisms by multiple cardiac tissues during zebrafish heart regeneration has grown exponentially. In particular, it is becoming increasingly clear that myocardial tissue repair depends on successful neovascularisation by cardiac endothelial cells (cECs) coupled with a carefully timed immune response. However, zebrafish cardiac regeneration studies, and particularly those at single cell resolution, have predominantly focussed exclusively on the role of cardiomyocytes, and not on the regenerating cECs. Despite the importance of neovascularisation by the cECs, the precise mechanisms underpinning the process remain unclear. I hypothesised that understanding and interrogating the gene regulatory networks of regenerating zebrafish cardiac vasculature would provide insight into mechanisms that are blocked or insufficient in mammalian species. This project aimed to make use of publicly available single cell RNA sequencing (scRNAseq) datasets to map differential gene expression signatures at key timepoints following adult zebrafish cardiac injury and to identify and validate selected targets with a potential novel role in endogenous cardiovascular repair. Of the cEC genes that were upregulated after injury compared to the uninjured control, four were selected for further functional validation: ifitm1, serpine1, nppc, and igfbp7. These genes have previously been associated with a range of human cardiac and ischaemic conditions and were specifically investigated in this project as potential drivers of regenerative neovascularisation. VEGFC and PLVAP (which has two zebrafish paralogues, plvapa and plvapb) were also included in this study. These were concurrently identified by the Brittan laboratory as potential players in cEC responses in the injured mammalian heart following a scRNAseq meta-analysis of developing and adult mouse and human hearts in healthy and diseased/injured states. In collaboration with the Minchin laboratory and using an endothelial reporter transgenic zebrafish line (Tg(fli1a:eGFP)y1), a CRISPR/Cas9 mutagenesis screen was performed as part of a functional validation of ifitm1, serpine1, nppc, igfbp7, vegfc, plvapa, and plvapb. I aimed to assess both developmental changes in knockdown fish and regenerative responses following vascular injury. Developmentally, mutant larvae showed a diversity of phenotypes, with many CRISPants exhibiting pericardiac oedema and aberrations in vascular patterning. To assess mutant regenerative abilities, I developed and characterised a novel vascular regeneration assay in the trunk of the larval zebrafish. In 2 days post fertilisation (dpf) Tg(fli1a:eGFP)y1 zebrafish, laser-induced injury to an intersegmental vessel (ISV) elicited a strong vascular response, showing vascular sprouting by 4 hours post injury (hpi) and achieving full regeneration by 48 hpi. The immune response to the ISV laser injury was also characterised in a macrophage / neutrophil double transgenic zebrafish line (Tg(mpeg1:mCherry)gl23 / Tg(BACmpx:GFP)i114), which showed a similar immune response profile to the well-established larval tail fin resection model and a fully-characterised laser-mediated injury to the larval heart, recently developed in the Denvir laboratory. The ISV laser injury system was then applied to the ifitm1, serpine1, nppc, igfbp7, plvapa, and plvapb CRISPants. As with the mutagenesis screen, there was a range of regenerative phenotypes. Up to 12 hpi, most CRISPants showed wild type vascular sprouting, but by 24 – 48 hpi, many of the knockdown fish in all gene groups showed disorganised or incomplete vascular repair, which points to the possible involvement of these genes in the vascular regeneration programme of the zebrafish. While the ISV laser model in the larval zebrafish has the benefit of being relatively high throughput, it is considered to be more accurate to assess the regenerative contribution of a gene in an adult vascular regeneration model. To date, the only existing models to achieve this in the adult zebrafish heart include in vivo injuries, which disallow live imaging due to the opacity of the adult fish. Studies have shown that the adult zebrafish heart maintains its contractile properties in an ex vivo environment, so this project aimed to discover whether the heart’s vascular regenerative properties were also preserved ex vivo. I optimised an explant culturing protocol and adapted the laser injury model to ablate a fixed area of vasculature on the surface of excised adult Tg(fli1a:eGFP)y1 hearts. Indeed, vascular regeneration was observed over 48 hours. The advantage of the ex vivo heart culture system was that it provided continuous live imaging opportunities of the same heart as it regenerated. However, since the heart continues to beat in culture, gaining high-quality images proved difficult. Multiple imaging platforms were investigated to optimise live imaging strategies in collaboration with the Lee laboratory at the Institute of Genetics and Cancer, and the Taylor laboratory at Glasgow University. We aimed to produce clear images without generating prohibitively large datasets or causing undue phototoxicity to the tissue, with the view to apply this novel adult injury and imaging system with cEC isolation and RNA extraction to adult CRISPants in the future. This thesis describes and provides a workflow for using open access single cell transcriptomic data to identify novel neovasculogenic gene targets, and a means to functionally validate them. I present two vascular regeneration assays in the zebrafish, and I provide preliminary evidence of a direct functional role of ifitm1, serpine1, nppc, and igfbp7, in the endogenous endothelial response to vascular injury. I have also shown progress towards optimising live imaging strategies of the beating zebrafish heart ex vivo, which in the future could be applied to the unique adult laser injury model in gene of interest CRISPants. Through further understanding and validation of the genes driving neovasculogenesis, we will gain a fuller picture of the endogenous regeneration programme of the zebrafish heart, which may be applied therapeutically to humans suffering cardiac damage.