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Adult zebrafish are frequently described to be able to "completely" regenerate the heart. Yet, the extent to which cardiomyocytes lost to injury are replaced is unknown, since only indirect evidence for cardiomyocyte proliferation exists. We established stereological methods to quantify the number of cardiomyocytes at several time-points post cryoinjury. Intriguingly, after cryoinjuries that killed about 1/3 of the ventricular cardiomyocytes, pre-injury cardiomyocyte numbers were restored already within 30 days. Yet, many hearts retained small residual scars, and a subset of cardiomyocytes bordering these fibrotic areas remained smaller, lacked differentiated sarcomeric structures, and displayed defective calcium signaling. Thus, a subset of regenerated cardiomyocytes failed to fully mature. While lineage-tracing experiments have shown that regenerating cardiomyocytes are derived from differentiated cardiomyocytes, technical limitations have previously made it impossible to test whether cardiomyocyte trans-differentiation contributes to regeneration of non-myocyte cell lineages. Using Cre responder lines that are expressed in all major cell types of the heart, we found no evidence for cardiomyocyte transdifferentiation into endothelial, epicardial, fibroblast or immune cell lineages. Overall, our results imply a refined answer to the question whether zebrafish can completely regenerate the heart: in response to cryoinjury, preinjury cardiomyocyte numbers are indeed completely regenerated, while restoration of cardiomyocyte differentiation and function, as well as resorption of scar tissue, is less robustly achieved.

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death.

Wu, Chi Chung., "September 2010.", Thesis (M.Phil.)--Chinese University of Hong Kong, 2010., Includes bibliographical references (leaves 86-96)., s in English and Chinese., p.i, (Chinese version) --- p.iii, Acknowledgments --- p.iv, List of Abbreviations --- p.v, List of Tables --- p.vii, List of Figures --- p.viii, Chapter 1. --- INTRODUCTION, Chapter 1.1. --- Neurodegenerative disorders 一 a brief overview --- p.1, Chapter 1.2. --- Polyglutamine diseases --- p.1, Chapter 1.3. --- Polyglutamine protein conformers and toxicity --- p.5, Chapter 1.4. --- in vivo modeling of polyglutamine diseases in Drosophila, Chapter 1.4.1. --- GAL4/UAS transgene expression system in Drosophila --- p.13, Chapter 1.4.2. --- Temporal control of transgene expression systemin Drosophila --- p.15, Chapter 1.4.3. --- Drosophila as a model to study polyglutamine diseases --- p.16, Chapter 1.5. --- in vitro polyglutamine diseases models --- p.19, Chapter 1.6. --- Aim of study --- p.23, Chapter 2. --- MATERIALS AND METHODS, Chapter 2.1. --- Drosophila culture and manipulation, Chapter 2.1.1. --- Drosophila culture --- p.25, Chapter 2.1.2. --- Pseudopupil assay of adult retinal degeneration --- p.25, Chapter 2.2. --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Chapter 2.2.1. --- Protein extraction from adult Drosophila heads --- p.26, Chapter 2.2.2. --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.27, Chapter 2.2.3. --- Western blotting --- p.28, Chapter 2.2.4. --- Immunodetection --- p.29, Chapter 2.3. --- Solubilization of SDS-insoluble protein --- p.31, Chapter 2.4. --- Filter retardation assay --- p.31, Chapter 2.5. --- Immunoprecipitation --- p.32, Chapter 2.6. --- Nucleocytoplasmic fractionation --- p.33, Chapter 2.7. --- PCR cloning, Chapter 2.7.1 . --- Drosophila DNA preparation --- p.34, Chapter 2.7.2. --- Construction of pGEX4T3-MJDflQ27/81 expression plasmid --- p.34, Chapter 2.8. --- in vitro aggregation assay, Chapter 2.8.1. --- Expression and purification of GST-MJDAQ27/81 protein --- p.36, Chapter 2.8.2. --- in vitro aggregation --- p.37, Chapter 2.8.3. --- Native slot-blot --- p.38, Chapter 2.9. --- Reagents and buffers, Chapter 2.9.1. --- Reagents for Drosophila culture --- p.39, Chapter 2.9.2. --- Reagents for SDS-PAGE --- p.39, Chapter 2.9.3. --- Reagents for filter retardation assay --- p.42, Chapter 2.9.4. --- Reagents for immunoprecipitation --- p.43, Chapter 2.9.5. --- Reagents for nucleocytoplasmic fractionation --- p.43, Chapter 2.9.6. --- Reagents for PCR cloning --- p.44, Chapter 2.9.7. --- Reagents for in vitro aggregation assay --- p.46, Chapter 3. --- Establishment of a GAL80ts-mediated transgenic Drosophila model of Machado-Joseph Disease (MJD), Chapter 3.1. --- Introduction --- p.48, Chapter 3.2. --- Results, Chapter 3.2.1. --- GAL80ts-mediated expression of expanded full-length MJD protein caused progressive neuronal degenerationin Drosophila --- p.49, Chapter 3.2.2. --- Detection of SDS-insoluble expanded full-length MJD protein and its correlation with neuronal degeneration, Chapter 3.2.2.1. --- Progressive neuronal degeneration is not mediated by progressive accumulation of expanded full-length MJD protein --- p.51, Chapter 3.2.2.2. --- SDS-soluble expanded full-length MJD protein does not correlate with progressive neuronal degeneration --- p.53, Chapter 3.2.2.3. --- Progressive accumulation of SDS-insoluble expanded full-length MJD protein correlate with progressive neuronal degeneration --- p.55, Chapter 3.3. --- Discussion --- p.57, Chapter 4. --- Detection of conformational changes of expanded full-length MJD protein and its association with neuronal degeneration, Chapter 4.1. --- Introduction --- p.60, Chapter 4.2. --- Results, Chapter 4.2.1. --- Expanded full-length MJD protein underwent conformational changes from monomer to fibrils and such conformational changes correlated with neuronal degeneration --- p.61, Chapter 4.2.2. --- Mechanistic studies of how conformational changes of expanded full-length MJD protein triggers neuronal degeneration, Chapter 4.2.2.1. --- Expanded full-length MJD protein gradually accumulated in the nucleus during the course of neurodegeneration --- p.62, Chapter 4.2.2.2. --- Fibrillar expanded full-length MJD protein caused transcriptional dysregulation of endogenous Hsp70 gene --- p.66, Chapter 4.2.3. --- Consolidation of the role of fibrillar expanded full-length MJD protein in neuronal degeneration --- p.67, Chapter 4.3. --- Discussion --- p.72, Chapter 5. --- Attempts to generate new conformation-specific antibody against recombinant expanded full-length MJD proteins, Chapter 5.1. --- Introduction --- p.75, Chapter 5.2. --- Results, Chapter 5.2.1. --- Recombinant expanded full-length MJD protein underwent conformational changes during in vitro aggregation --- p.75, Chapter 5.3. --- Discussion --- p.77, Chapter 6. --- GENERAL DISCUSSION --- p.81, Chapter 7. --- CONCLUSION --- p.84, Chapter 8. --- REFERENCES --- p.86, http://library.cuhk.edu.hk/record=b5894444, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)