Single-cell transcriptomics of zebrafish heart regeneration with subcellular spatial resolution
[eng] Adult zebrafish (Danio rerio), in contrast to mammals, can efficiently regenerate large portions of the heart after experimental damage or amputation. This process has been extensively studied we are beginning to understand the key events necessary for this regenerative response to happen. How...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2024 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/216917 |
| Acceso en línea: | https://hdl.handle.net/2445/216917 http://hdl.handle.net/10803/692661 |
| Access Level: | acceso abierto |
| Palabra clave: | Transcripció genètica Seqüència de nucleòtids Peix zebra Regeneració (Biologia) Cor Genetic transcription Nucleotide sequence Zebra danio Regeneration (Biology) Heart |
| Sumario: | [eng] Adult zebrafish (Danio rerio), in contrast to mammals, can efficiently regenerate large portions of the heart after experimental damage or amputation. This process has been extensively studied we are beginning to understand the key events necessary for this regenerative response to happen. However, the cellular interplay and molecular mechanisms that govern the proliferation and migration of cardiomyocytes, as well as the termination of the regenerative process, have been only fragmentary explored. Understanding these mechanisms and exploiting them in the human context could open new therapeutic approaches to promote myocardial regeneration and prevent the development of heart failure. With this aim, we sought to determine the transcriptomic profile of healthy and regenerating zebrafish hearts at single-cell resolution, using novel transcriptomic approaches, and to analyze the importance of candidate genes in the regeneration process. For this purpose, in the first part of the current thesis, we developed a tissue dissociation protocol using a cold active protease, from B. licheniformis, to minimize collagenase-associated artifacts introduced in single-cell RNA sequencing (scRNA-seq) analyses. We also made use of a droplet-based microfluidics encapsulation device that is particularly suitable for capturing both large (cardiomyocytes) and small (fibroblasts, endothelial, myeloid) size cells. Using these protocols, we have investigated changes in cell types and cardiomyocyte cell states in uninjured (Sham) zebrafish hearts, and after 7- and 30- days post amputation (DPA) . Due to the high sensitivity of scRNA-seq, we could find changes in gene expression that were not detected in previous bulk RNA-seq analyses. Our results identified novel markers to describe cardiomyocyte cell-states that are present in both healthy and regenerating myocardium. We then studied the transcriptomic changes of these cell-states, and we identified candidate genes with possible implication in heart regeneration. In the second part of the current thesis, we sought to examine the importance of candidate genes in zebrafish heart regeneration. Our previous results showed that anxa2a, mustn1b, fhl1a and casq2 are dynamically expressed in cardiomyocytes and upregulated at 7 DPA. We followed a CRISPR/Cas9-based approach for loss-of-function experiments of these genes. Knock-out zebrafish did not exhibit any developmental defects or abnormal adult phenotypes but failed to regenerate their myocardium after apex resection. For further characterization of the role(s) that these genes play during zebrafish heart regeneration, we developed two novel experimental platforms: 1) a cardiomyocyte-specific Ca2+ indicator reporter transgenic zebrafish line for monitoring calcium handling during zebrafish heart regeneration, and 2) an in vitro platform for measuring cell-autonomous behaviors in response to matrices of controlled stiffness. In the third part, we have examined the transcriptomic changes in periostin b (postnb) knock-out animals that exhibit increased cardiomyocyte proliferation but fail to regenerate the ventricle upon injury. We found similar cellular composition between postnb-/- and wild type ventricles. However, postnb-/- mutant hearts showed changes in the proportions of cardiomyocytes, macrophages, fibroblasts, and epicardial cells at 7 DPA. To include spatial information about the expression of genes and infer gene regulatory networks, we performed spatial transcriptomics experiments. Our results demonstrate the suitability and efficiency of the technique for finding gene specific domains in the regenerating zebrafish heart. |
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