Analysis of a conditional gene trap reveals that tbx5a is required for heart regeneration in zebrafish.
Ontology highlight
ABSTRACT:
SUBMITTER: Grajevskaja V
PROVIDER: S-EPMC6014646 | biostudies-literature | 2018
REPOSITORIES: biostudies-literature
Publications
Analysis of a conditional gene trap reveals that tbx5a is required for heart regeneration in zebrafish.
Grajevskaja Viktorija V Camerota Diana D Bellipanni Gianfranco G Balciuniene Jorune J Balciunas Darius D
PloS one 20180622 6
The ability to conditionally inactivate genes is instrumental for fine genetic analysis of all biological processes, but is especially important for studies of biological events, such as regeneration, which occur late in ontogenesis or in adult life. We have constructed and tested a fully conditional gene trap vector, and used it to inactivate tbx5a in the cardiomyocytes of larval and adult zebrafish. We observe that loss of tbx5a function significantly impairs the ability of zebrafish hearts to ...[more]
Similar Datasets
Project description:During development, mesodermal progenitors from the first heart field (FHF) form a primitive cardiac tube, to which progenitors from the second heart field (SHF) are added. The contribution of FHF and SHF progenitors to the adult zebrafish heart has not been studied to date. Here we find, using genetic tbx5a lineage tracing tools, that the ventricular myocardium in the adult zebrafish is mainly derived from tbx5a+ cells, with a small contribution from tbx5a- SHF progenitors. Notably, ablation of ventricular tbx5a+-derived cardiomyocytes in the embryo is compensated by expansion of SHF-derived cells. In the adult, tbx5a expression is restricted to the trabeculae and excluded from the outer cortical layer. tbx5a-lineage tracing revealed that trabecular cardiomyocytes can switch their fate and differentiate into cortical myocardium during adult heart regeneration. We conclude that a high degree of cardiomyocyte cell fate plasticity contributes to efficient regeneration.
| S-EPMC5789846 | biostudies-literature
Project description:Mammalian hearts cannot regenerate. In contrast, zebrafish hearts regenerate even when up to 20% of the ventricle is amputated. The mechanism of zebrafish heart regeneration is not understood. To systematically characterize this process at the molecular level, we generated transcriptional profiles of zebrafish cardiac regeneration by microarray analyses. Distinct gene clusters were identified based on temporal expression patterns. Genes coding for wound response/inflammatory factors, secreted molecules, and matrix metalloproteinases are expressed in regenerating heart in sequential patterns. Comparisons of gene expression profiles between heart and fin regeneration revealed a set of regeneration core molecules as well as tissue-specific factors. The expression patterns of several secreted molecules around the wound suggest that they play important roles in heart regeneration. We found that both platelet-derived growth factor-a and -b (pdgf-a and pdgf-b) are upregulated in regenerating zebrafish hearts. PDGF-B homodimers induce DNA synthesis in adult zebrafish cardiomyocytes. In addition, we demonstrate that a chemical inhibitor of PDGF receptor decreases DNA synthesis of cardiomyocytes both in vitro and in vivo during regeneration. Our data indicate that zebrafish heart regeneration is associated with sequentially upregulated wound healing genes and growth factors and suggest that PDGF signaling is required.
| S-EPMC1523227 | biostudies-literature
Project description:Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.
| S-EPMC3458342 | biostudies-literature
Project description:tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt-Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations.
| S-EPMC3511548 | biostudies-literature
Project description:Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3K27M, that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3K27M-expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity.
| S-EPMC6803378 | biostudies-literature
Project description:Unlike its mammalian counterpart, the adult zebrafish heart is able to fully regenerate after severe injury. One of the most important events during the regeneration process is cardiomyocyte proliferation, which results in the replacement of lost myocardium. Growth factors that induce cardiomyocyte proliferation during zebrafish heart regeneration remain to be identified. Signaling pathways important for heart development might be reutilized during heart regeneration. IGF2 was recently shown to be important for cardiomyocyte proliferation and heart growth during mid-gestation heart development in mice, although its role in heart regeneration is unknown. We found that expression of igf2b was upregulated during zebrafish heart regeneration. Following resection of the ventricle apex, igf2b expression was detected in the wound, endocardium and epicardium at a time that coincides with cardiomyocyte proliferation. Transgenic zebrafish embryos expressing a dominant negative form of Igf1 receptor (dn-Igf1r) had fewer cardiomyocytes and impaired heart development, as did embryos treated with an Igf1r inhibitor. Moreover, inhibition of Igf1r signaling blocked cardiomyocyte proliferation during heart development and regeneration. We found that Igf signaling is required for a subpopulation of cardiomyocytes marked by gata4:EGFP to contribute to the regenerating area. Our findings suggest that Igf signaling is important for heart development and myocardial regeneration in zebrafish.
| S-EPMC3694143 | biostudies-literature
Project description:BACKGROUND: We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology. DESCRIPTION: To increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish. CONCLUSION: zTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.
| S-EPMC2970601 | biostudies-literature
Project description:Ischemic cardiopathy is the leading cause of death in the world, for which efficient regenerative therapy is not currently available. In mammals, after a myocardial infarction episode, the damaged myocardium is replaced by scar tissue featuring collagen deposition and tissue remodelling with negligible cardiomyocyte proliferation. Zebrafish, in contrast, display an extensive regenerative capacity as they are able to restore completely lost cardiac tissue after partial ventricular amputation. Due to the lack of genetic lineage tracing evidence, it is not yet clear if new cardiomyocytes arise from existing contractile cells or from an uncharacterised set of progenitors cells. Nonetheless, several genes and molecules have been shown to participate in this process, some of them being cardiomyocyte mitogens in vitro. Though questions as what are the early signals that drive the regenerative response and what is the relative role of each cardiac cell in this process still need to be answered, the zebrafish is emerging as a very valuable tool to understand heart regeneration and devise strategies that may be of potential value to treat human cardiac disease. Here, we performed a genome-wide transcriptome profile analysis focusing on the early time points of zebrafish heart regeneration and compared our results with those of previously published data. Our analyses confirmed the differential expression of several transcripts, and identified additional genes the expression of which is differentially regulated during zebrafish heart regeneration. We validated the microarray data by conventional and/or quantitative RT-PCR. For a subset of these genes, their expression pattern was analyzed by in situ hybridization and shown to be upregulated in the regenerating area of the heart. The specific role of these new transcripts during zebrafish heart regeneration was further investigated ex vivo using primary cultures of zebrafish cardiomyocytes and/or epicardial cells. Our results offer new insights into the biology of heart regeneration in the zebrafish and, together with future experiments in mammals, may be of potential interest for clinical applications. In order to study zebrafish heart regeneration, a time course experiment was realized where amputated heart regenerating were compared to control heart. Samples in triplicate were extracted at 1, 3, 5 and 7 days post-amputation.
2010-03-09 | E-GEOD-17993 | biostudies-arrayexpress
Project description:Adult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. Here, we set out to characterize the ECM protein composition in adult zebrafish hearts, and whether it changed during the regenerative response. For this purpose, we first established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days postamputation) time point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.
| S-EPMC6731076 | biostudies-literature
Project description:Aims: Nitrite is reduced to nitric oxide (NO) under physiological and pathological hypoxic conditions to modulate angiogenesis and improve ischemia-reperfusion injury. Although adult mammals lack the ability to regenerate the heart after injury, this is preserved in neonates and efforts to reactivate this process are of great interest. Unlike mammals, the adult zebrafish maintain the innate ability to regenerate their hearts after injury, providing an important model to study cardiac regeneration. We thus explored the effects of physiological levels of nitrite on cardiac and fin regeneration and downstream cellular and molecular signaling pathways in response to amputation and cryoinjury. Results: Nitrite treatment of zebrafish after ventricular amputation or cryoinjury to the heart in hypoxic water (?3 parts per million of oxygen) increases cardiomyocyte proliferation, improves angiogenesis, and enhances early recruitment of thrombocytes, macrophages, and neutrophils to the injury. When tested in a fin regeneration model, neutrophil recruitment to the injury site was found to be dependent on NO. Innovation: This is the first study to evaluate effects of physiological levels of nitrite on cardiac regeneration in response to cardiac injury, with the observation that nitrite in water accelerates zebrafish heart regeneration. Conclusion: Physiological and therapeutic levels of nitrite increase thrombocyte, neutrophil, and macrophage recruitment to the heart after amputation and cryoinjury in zebrafish, resulting in accelerated cardiomyocyte proliferation and angiogenesis. Translation of this finding to mammalian models of injury during early development may provide an opportunity to improve outcomes during intrauterine fetal or neonatal cardiac surgery.
| S-EPMC6985782 | biostudies-literature