Project description:Amputation of heart tissue followed by regeneration of the heart. Samples were taken at 0 hpa (hours post-amputation), 6 hpa, 12 hpa, 24 hpa, 3 dpa and 5 dpa.
Project description:In the present study, we have used the zebrafish to investigate the anti-inflammatory effects of glucocorticoids. An inflammatory response was induced by amputation of the tail fin of 3dpf zebrafish larvae, and the effect of treatment with the glucocorticoid beclomethasone was investigated. A microarray study was performed to study transcriptional changes 4 hours after amputation and the beclomethasone-induced effects on these changes. Our results show that beclomethasone attenuates almost the entire transcriptional response to amputation. Interestingly, the amputation-induced expression of genes involved in leukotriene biosynthesis appears to be resistant to beclomethasone treatment.
Project description:Zebrafish caudal fin regeneration is an established model to study tissue regeneration. In order to identify novel molecular signaling pathways critical for regeneration, we developed a rapid throughput in vivo regeneration assay. We screened a 2000 member structurally diverse small molecule library, followed by assessment of regenerative progression at three days post amputation. A cluster of glucocorticoids was identified among the âpositive hitsâ. To identify the molecular targets of the activated glucocorticoid receptor, microarray analysis was performed using RNA isolated from the regenerates of control and glucocorticoid exposed zebrafish. We identified 673 transcripts that were differentially regulated. The level of expression and spatial expression pattern of select genes were completed by qPCR and by in situ hybridization, respectively. Altogether, these studies demonstrate the power of chemical genetics to identify chemical probes and their targets which will provide a path towards defining conserved regenerative pathways. Experiment Overall Design: The caudal fin of zebrafish larvae at 2days post fertilization were amputated and exposed to vehicle control alone or Beclomethasone . Regenerating fins were isolated at 1days post amputation. Three replicates were collected at each time point. 150 fins were pooled to comprise one replicate.
Project description:We investigated the role and programming of early immune responses during zebrafish heart regeneration. We found zebrafish treated with anti-inflammatory steroid, beclomethasone, would suffer significantly reduction of its heart regenerative ability, leading to excessive collagen deposition in wound. Microarray approach was used to evaluate the differential expression of heart transcriptome between normal and impaired healing zebrafish, which provides an overall assessment of significant genes that were important for triggering regeneration in the hemostasis-inflammation phase. Three sets of samples each containing 10 zebrafish hearts were used: sham operation, DMSO-1dpa, beclomethasone-1dpa. (dpa= days post amputation) Objects were pre-exposed to water containing 0.25M-NM-<M beclomethasone or DMSO for 1 day. After ventricular resection surgery, objects were sacrificed with their ventricles harvested at the next day (i.e. 1dpa)
Project description:Adult zebrafish can completely regenerate their caudal fin following amputation. This complex process is initiated by the formation of an epithelial would cap over the amputation site by 12 hours post amputation (hpa). Once the cap is formed, mesenchymal cells proliferate and migrate from sites distal to the wound plane and accumulate under the epithelial cap forming the blastemal structure within 48 hpa. Blastemal cells proliferate and differentiate, replacing the amputated tissues, which are populated with angiogenic vessels and innervating nerves during the regenerative outgrowth phase which is completed around 14 days post amputation (dpa). Regenerative outgrowth does not occur in TCDD-exposed zebrafish. To identify the molecular pathways that are perturbed by TCDD exposure, male zebrafish were i.p. injected with 50 ng/g TCDD or vehicle and caudal fins were amputated. Regenerating fin tissue was collected at 1, 3 and 5 dpa for mRNA abundance analysis. Microarray analysis and quantitative real time PCR revealed that wound healing and regeneration alone altered the expression of nearly 900 genes by at least two fold between 1 and 5 dpa. TCDD altered the abundance of 370 genes at least two fold. Among these, several known aryl hydrocarbon responsive genes were identified in addition to several genes involved in extracellular matrix composition and metabolism. The profile of misexpressed genes is suggestive of impaired cellular differentiation and extracellular matrix composition potentially regulated by Sox9b. Experiment Overall Design: Regenerating fins were isolated at 1, 3 and 5 days post amputation. Three replicates were collected at each time point. 10 fins were pooled to comprise one replicate. Fish were dosed at 0 days post amputation with vehicle control alone or 50 ng/g TCDD. Experiment Overall Design: 1 Day Post Amputation Vehicle Exposed: GSM85187, GSM85188, and GSM85189 Experiment Overall Design: 1 Day Post Amputation TCDD Exposed: GSM85190, GSM85191, and GSM85192 Experiment Overall Design: 3 Days Post Amputation Vehicle Exposed: GSM85193, GSM85194, and GSM85195 Experiment Overall Design: 3 Day Post Amputation TCDD Exposed: GSM85196, GSM85197, and GSM85198 Experiment Overall Design: 5 Days Post Amputation Vehicle Exposed: GSM85199, GSM85200, and GSM85201 Experiment Overall Design: 5 Days Post Amputation TCDD Exposed: GSM85202, GSM85203, and GSM85204
Project description:The purpose of this experiment is to understand which transcripts are differentially expressed following exposure to TCDD. We used microarrays to understand global changes in gene signal intensity Adult zebrafish (danio rerio) were injected (i.p) to vehicle control or TCDD (70 ng/g) one day prior to heart ventricle amputation. A subset of fish were exposed but the hearts were not amputated. Amputated hearts were collected at 6 hours post amputation and 7 days post amputation and processed for microarray analysis