Autophagy is essential for cardiac morphogenesis during vertebrate development
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ABSTRACT: This study examined the effects of genetic knockdown of autophagy genes on vertebrate cardiac development We performed microarray studies comparing the hearts of control zebrafish embryos to the hearts of embryos with decreased expression of the autophagy genes atg5, becn1 or atg7. The results provide insight into the role of autophagy in developmental morphogenesis. Hearts were purified from 3 day-old zebrafish embryos injected with control or autophagy gene-specific morpholino oligonucleotides. RNA was prepared from all samples and hybridized to zebrafish-specific Affymetricx arrays.
Project description:This study examined the effects of genetic knockdown of autophagy genes on vertebrate cardiac development We performed microarray studies comparing the hearts of control zebrafish embryos to the hearts of embryos with decreased expression of the autophagy genes atg5, becn1 or atg7. The results provide insight into the role of autophagy in developmental morphogenesis.
Project description:The aim of this study was to identify genes that are regulated by ccm2 signaling with the zebrafish heart at around 72 hours post fertilization. In total, 4 samples based on cDNA from extracted cardiac tissue were analyzed: 2 samples of ccm2 m201 mutant embryos and 2 WT control samples.
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 post-amputation) 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.
Project description:The aim of this study was to identify genes that are regulated by Bmp signaling with the zebrafish heart at the heart cone stage around 21-23 hours post fertilization. In total, 4 samples based on cDNA from extracted cardiac tissue were analyzed: 2 samples of Tg(hsp70l:bmp2b)fr13 transgenic embryos heatshocked at 18 hpf and 2 WT control samples.
Project description:Heart formation requires input from two populations of progenitor cells - the first and second heart fields - that differentiate at distinct times and create different cardiac components. The cardiac outflow tract (OFT) is built through recruitment of late-differentiating, second heart field (SHF) -derived cardiomyocytes to the arterial pole of the heart. Mechanisms responsible for selection of an appropriate number of OFT cells from the SHF remain unclear, although several lines of evidence emphasize the importance of FGF signaling in promoting this process. Here, we examine the impact of inhibition of FGF signaling on cardiac transcription profiles in an effort to identify genes operating downstream of FGF during OFT development. We compared hearts from embryos treated with the FGFR inhibitor SU5402 to the hearts from sibling embryos treated with DMSO. Two replicates were performed.
Project description:We report global RNA expression profiles from whole zebrafish hearts 24 hours after ventricle amputation. Zebrafish were exposed to atropine or water following surgery. 15 zebrafish hearts were pooled per microarray chip. Amputated hearts of zebrafish exposed to atropine was compared to hearts of zebrafish exposed to water.
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 heartregeneration has been comparatively rarely explored. Here, we set out to characterize theECM protein composition inadult 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 dayspost-amputation) time-point analyzed. Regeneration associated withsharp increases inspecific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopythat the changes in ECM composition translatedto decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.
Project description:The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration. In order to monitor the whole regeneration process, we recovered samples at different time points post-injury: 4 h, 1 day, 3 days, 7 days, 14 days and 90 days (respectively 4 hpi, 1 dpi, 3 dpi, 7 dpi, 14 dpi and 90 dpi). Cryoinjured hearts were compared to healthy hearts from control fish in 3 independent experiments.
Project description:Retinoic acid (RA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin activate distinct ligand-dependent transcription factors, and both cause cardiac malformation and heart failure in zebrafish embryos. We hypothesized that they cause this response by hyperactivating a common set of genes critical for heart development. To test this, we used microarrays to measure transcripts changes in hearts isolated from zebrafish embryos 1,2,4 and 12 h after exposure to 1μM RA. We used hierarchical clustering to compare the transcriptional responses produced in the embryonic heart by RA and TCDD. We could identify no early responses in common between the two agents. However, at 12 h both treatments produced a dramatic downregulation of a common cluster of cell cycle progression genes, which we term the Cell Cycle Gene Cluster (CCGC). This was associated with a halt in heart growth. These results suggest that RA and TCDD ultimately trigger a common transcriptional response associated with heart failure, but not through the direct activation of a common set of genes. Among the genes rapidly induced by RA was Nr2F5, a member of the COUP-TF family of transcription repressors. We found that induction of Nr2F5 was both necessary and sufficient for the cardiotoxic response to RA. Experiment Overall Design: For RA study, total of 24 samples were collected and analyzed by microarray. Samples of zebrafish embryonic hearts were collected at four timepoints: 1, 2, 4, 12 hours post RA dosing. For each timepoint, there are three replicates of microarray studies. Each replicate includes microarray studies of one sample of RA treated heats and one sample of vehicle control (DMSO) treated hearts.
Project description:This experiment sought to understand the transcriptomic changes that occur in the larval zebrafish heart following injury. 600 hearts were laser injured at 3 days post fertilisation, extracted 48 hours later and pooled into three groups of 200. RNA was extracted from the whole heart(s) and sent for sequencing along with 3 groups of 200 uninjured hearts, extracted and processed identically. RNA sequencing, quality control and alignment was performed by the commercial company GENEWIZ.