2011;20:725–32. NOTE: The speed of zebrafish fin regeneration depends on the amount of resected fin tissue, i.e., the more fin tissue is resected, the faster the fin regenerates 13. Zebrafish fin regeneration is mediated by the creation of blastema cells. Tissue-specific hypomethylated DMRs (hypoDMRs) are a signature of tissue-specific regulatory regions [19, 20, 23, 30]; therefore, these hypoDMRs are likely regions with sp7+ cell-specific regulatory activities. Katsuyama T, Paro R. Epigenetic reprogramming during tissue regeneration. 2006;299(2):438-454. Regenerating zebrafish fin epigenome is characterized by stable lineage-specific DNA methylation and dynamic chromatin accessibility. First, instances of a given motif were identified across the entire zebrafish genome using FIMO [68] version 4.11.2 with the following parameters: --max-stored-scores 10000000 --text --thresh 1e-5. Previous studies suggested that DNA demethylation of active cis-regulatory elements is often correlated with gain of chromatin accessibility or active enhancer histone marks [21, 37]. Ross-Innes CS, Stark R, Teschendorff AE, Holmes KA, Ali HR, Dunning MJ, et al. We found that 26% of ATAC peaks were in promoters (2 kb regions around transcription start site (TSS)), while 45% of ATAC peaks were distal (> 10 kb) to TSS (Additional file 1: Figure S4b). Zebrafish fin development The Company of Biologists. Among them, TFs whose expression levels were higher in regenerates at 1, 2, or 4 dpa than in uninjured fins were further selected as putative upstream factors. Zebrafish fin regeneration is mediated by the creation of blastema cells. 2012;22:2043–53. During zebrafish fin regeneration, as in other examples of appendage regeneration, the blastema is the engine for regenerative growth (Tsonis, 1996). Thus, our study provides a new resource of regeneration regulatory elements, as well as transgenic animals with new markers to facilitate investigation of tissue regeneration. It has been proposed that adult tissues with a robust regeneration capability have permissive chromatin structures around the genes necessary for regeneration, which are also developmental genes [40]. Mutant fish strains were generated by a customized CRISPR/Cas9-based targeted genome editing system (H.S.J., T.W. 2016;165(7):1598-1608. Privacy We detected hundreds of genes activated in fin regeneration and identified regulatory elements responsible for those gene expression changes. Vitamin D is an essential nutrient that has long been known to regulate skeletal growth and integrity. We produce epigenome maps, including DNA methylation and chromatin accessibility, as well as transcriptomes, of osteoblasts and other cells in uninjured and regenerating fins. We first investigated the TF binding motifs enriched in DARs that gained accessibility during regeneration. Nat Genet. The global DNA methylation patterns over genic regions superimpose each other, suggesting that there is no dramatic global difference in DNA methylation between the two stages (Additional file 1: Figure S2d), consistent with the result from DNA methylation analysis of bulk regenerates. California Privacy Statement, When the fin is amputated, regeneration occurs quickly. Zebrafish enhancer detection (ZED) vector: a new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish. 2011;108:20609–14. This result indicates that increasing chromatin accessibility of regulatory elements was positively correlated with increased expression of target genes during regeneration. Fast gapped-read alignment with bowtie 2. Regeneration in the zebrafish seems to be nearly unlimited: regeneration of the caudal fin and barbell occurs even after repetitive amputations (Azevedo et al., 2011; LeClair and Topczewski, 2010), although subtle changes to the newly formed organ, such as variation in the pigmentation patterning of the fin or the position of the bony ray bifurcations, have been observed (Azevedo et al., … Comparative aspects of animal regeneration. The cells were lysed in 400 μL of genomic DNA extraction buffer (50 mM Tris, 1 mM EDTA, 0.5% SDS, 1 mg/mL Proteinase K) for 16 h at 55 °C. Nat Methods. Experimental Procedure • Put gloves on before starting the experiment. From FastQ data to high confidence variant calls: the genome analysis toolkit best practices pipeline. • The key regenerative units are their many rays of dermal bone, which are segmented and lined by osteoblasts. 2011;480:490–5. The vector was further modified by replacing the gata2 minimal promoter with the 2-kb promoter sequence of the zebrafish lepb gene [33]. PMID: 8601496, CAS  A number of genes have been implicated in regeneration, but the regulation of these genes, particularly pertaining to regeneration in higher vertebrates, remains an interesting and mostly open question. Genome Biology Dev Cell. Secondary alignment, multiply mapped reads, and PCR duplicated reads were removed from the total aligned reads. USA.gov. First, TFs whose motifs were enriched in DARs that gained accessibility during regeneration either in sp7+ or in sp7− cells were chosen by HOMER as described above. Mol Cell. Molecular mechanisms of tissue regeneration have been studied mostly in the context of gene expression and function, and much remains to be investigated to decipher the gene regulatory networks involved in regeneration. Unpaired or unmapped read 1s were then mapped as single read mode by using Bismark with the following parameters: --bowtie2 -N 1 -L 28 --score_min L,0,-0.6. 2010;48:505–11. Paired-end RNA-seq libraries were sequenced on the Illumina NextSeq 500 machine, with a total of ~ 400 million reads. Live fate-mapping of joint-associated fibroblasts visualizes expansion of cell contributions during zebrafish fin regeneration. In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin. By constructing the gene regulatory network of fin regeneration, we identified Fra1 as a putative upstream transcription factor in the network. Approximately 1 million cells were sorted for each gate from FACS. 2011;44:17–28. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. 2011;21:447–55. 2012;13:R83. These DARs have distinct genetic and epigenetic characteristics from the previously determined zebrafish developmental enhancers [21, 38] (Additional file 1: Figure S5). Stewart S, Stankunas K. Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration. Regulatory networks important for regeneration are constructed through integrative analysis of the epigenome map, and a knockout of a predicted upstream regulator disrupts normal regeneration, validating our prediction. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. 2014;15:550. Zebrafish caudal fin is a unique regeneration system to model the injury response and regeneration of vertebrate appendages despite being a simple structure without muscular and adipose tissues. This idea is consistent with the notion that permissive chromatin state in adult tissue determines the regeneration capability [39, 40]. 3a; Additional file 1: Figure S4f). Although genome-wide DMR prediction did not return any genomic regions with significant DNA methylation changes, we wanted to examine DNA methylation dynamics of regulatory elements associated with genes whose expression changed during regeneration to potentially identify any elements that escaped genome-wide DMR detection. Here, we produced and analyzed comprehensive DNA methylome, transcriptome, and chromatin accessibility maps of regenerating osteoblasts over the course of zebrafish fin regeneration. In total, 18 out of 25 (72%) candidates displayed enhancer activities in regenerating fin while no activity was observed from 9 negative controls (Table 1). Finally, this work is dedicated to the memory of Stephen L. Johnson, a close friend, mentor, and colleague to many, and a pioneer of research at the field of zebrafish genetics and regeneration. This custom zebrafish genome sequence was in sillico converted into a bisulfite-treated genome and was then used in Bismark mapping. All mCherry+ F0 embryos were raised up to adult. Dev Biol. Having established that DARs were active cis-regulatory elements regulating regeneration, we sought to link upstream transcription factors (TFs) to their downstream target genes. Loading... Unsubscribe from The Company of Biologists? Transcripts per million (TPM) was calculated for each gene from the number of reads mapped to each gene determined by featureCounts [57]. Science. [1] Although some identified H3K27ac peaks might derive from differences in the cell type composition of the uninjured and regenerating tissues, the consistency of our results indicates that, compared with zebrafish, a relatively less complex genetic response to regeneration appears to be invoked by fin amputation in killifish. We identified Fra1 (gene name: fosl1a), whose motif enrichment was top ranked, as a putative upstream transcription factor for fin regeneration (Fig. fgf20 is essential for initiating zebrafish fin regeneration. Zhou X, Li D, Lowdon RF, Costello JF, Wang T. methylC track: visual integration of single-base resolution DNA methylation data on the WashU EpiGenome Browser. Please enable it to take advantage of the complete set of features! The fourth fin ray counting from the ventral side was used for consistency. Bradford Y, Conlin T, Dunn N, Fashena D, Frazer K, Howe DG, et al. Lee, H.J., Hou, Y., Chen, Y. et al. a Experimental scheme of sorting sp7+ and sp7− cells from uninjured and regenerating zebrafish fin by using FACS. Google Scholar. Light gray dots represent genes with no significant changes. Am Zool. Nature. mmp13a is sharply upregulated in the distal blastema of amputated fins. We would like thank Ali Wilkening and Kara Quaid for proofreading the manuscript. Modulation of tissue repair by regeneration enhancer elements. Nat Methods. Wu H, Xu T, Feng H, Chen L, Li B, Yao B, et al. 4a; Additional file 1: Figure S6a). The modified ZED vector was named ZEDtw plasmid, and the sequence was verified by Sanger sequencing. Zhang B, Zhou Y, Lin N, Lowdon RF, Hong C, Nagarajan RP, et al. To build putative regulatory network, TFs were linked to their downstream target genes, with a similar method as the one described previously [20]. PubMed Google Scholar. Therefore, epigenetic regulation mechanisms other than DNA methylation should play a role to regulate regeneration-specific gene transcription. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Motifs were sorted by binomial p value of enrichment in sp7+ cells. To our surprise, the numbers of DMRs between two different time points were very low and were not larger than the numbers of false positive DMRs defined as those predicted between two biological replicates (Additional file 1: Figure S1f). Stephen L. Johnson is deceased. Developmental signalling pathways have understandably been heavily investigated for their roles in fin fold regeneration. deepTools2: a next generation web server for deep-sequencing data analysis. Different developmental stages have distinct DNA methylation patterns, which help shape developmental decisions [13, 14]. ATAC-seq libraries were generated as previously described [35]. Taken together, this data suggests that the Fra1, predicted to be an upstream factor from a reconstructed regulatory network, activates downstream target genes important for fin regeneration. Putative TF-TF regulations and TF auto-regulation were also identified in case that a putative target gene was one of the chosen TF. Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis. Goldman JA, Kuzu G, Lee N, Karasik J, Gemberling M, Foglia MJ, et al. After merging paired-end and single-end report files, the Bismark command coverage2cytosine and a custom script were used to calculate total read counts and methylation read counts per each CpG. Table S2. For DNA extraction, 15 uninjured fins and 60 blastema per replicate were collected from transgenic zebrafish Tg(sp7:EGFP) and sp7+ and sp7− cell populations were isolated by using FACS as described above. 2011;43:491–8. Part of 2013;8:899–906. By using this website, you agree to our A two-sided Mann–Whitney U test was used to test statistical differences of fin regenerate lengths between mutant and wildtype zebrafish, by using the wilcox.test function. The ATAC-seq signals were visualized on the WashU Epigenome Browser [51] as fold change over background using bedGraph tracks generated by using the MACS2 bdgcmp function with the following parameter: -m FE. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. Advances in decoding axolotl limb regeneration. Fin regeneration • Zebrafish fins are complex appendages that quickly and reliably regenerate after amputation, restoring both size and shape. First, the mean methylation level of each CpG site was estimated with smoothing [53]. Epimorphic regeneration requires the presence or creation of pluripotent cells capable of reproducing lost organs. To investigate whether sp7+ cell-specific hypoDMRs were associated with regulatory activities, we generated ATAC-seq libraries of the same samples (Fig. 2013;23:1522–40. To investigate the dynamics of chromatin accessibility during regeneration, we identified differentially accessible regions (DARs) by using DiffBind [36]. 1970;10:119–32. 2013;41:3973–85. d Downregulated genes in sp7− cells during fin regeneration. Título: Notch signalling in zebrafish heart and fin regeneration. (The Babraham Institute) version 0.4.1 with the following parameters: --clip_R1 6 --clip_R2 6 --paired --retain_unpaired -r1 21 -r2 21. Briefly, 70,000 cells were washed with 50 μL of cold PBS buffer, lysed in 50 μL of cold lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630), and incubated with TDE1 enzyme (from Nextera DNA Sample Preparation Kit, Illumina) for 30 min at 37 °C for transposition. Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblast differentiation and function. Bioinformatics. Nature. 2016;44:W160–5. Their new bound sites in fin regenerates at 4 dpa were identified by intersecting TF-bound sites predicted by CENTIPEDE and DARs that gained accessibility during regeneration. This site needs JavaScript to work properly. The Upregulated genes that were not Fra1 targets showed statistically no difference in the level of gene expression changes (right). Zebrafish fins are composed of segmented bony rays, which are covered by a single layer of osteoblasts. 4d, e). 2011;476:409–13. d Examples of expression pattern of upregulated genes during regeneration that fall within the top GO terms. 2013;153:773–84. Our study shows that lineage-specific DNA methylation signatures are stably maintained during regeneration, and regeneration enhancers are preset as hypomethylated before injury. We generated transgenic zebrafish carrying these cassettes via Tol2 transposase system and monitored their reporter activities in the regenerating fin. Whether epigenetic modifications other than DNA hypomethylation, such as bivalent histone modification, in these enhancers also contribute to regeneration capability should be further investigated. Altogether, these results suggest that lineage-specific DNA methylation signatures undergo very few, if any, changes during regeneration. Hodges E, Molaro A, Dos Santos CO, Thekkat P, Song Q, Uren PJ, et al. Red dashed boxes highlight ATAC peaks with increasing signals during regeneration. c Gene ontology (GO) terms associated with significantly differentially expressed genes. Nucleic Acids Res. We have shown that TCDD specifically impairs regrowth of blood vessel, neurons and cartilage. A blastema is a mass of proliferative and morphologically homogeneous cells that have the capability to grow and develop into a lost body part. Wehner D, Weidinger G. Signaling networks organizing regenerative growth of the zebrafish fin. 2015;6:6315. 2008;9:R137. PubMed Central  This effort reveals regeneration as a process of highly dynamic and orchestrated transcriptomic and chromatin accessibility changes, coupled with stably maintained lineage-specific DNA methylation. 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Padhi BK, Akimenko MA have been proposed to be bound by TFs were examined injecting!, Izpisúa Belmonte JC early regeneration of zebrafish early embryos Ensembl gene annotation release... Dedifferentiation of osteoblasts in the preference centre B, Hong C, et al ontology zebrafish fin regeneration GO terms..., potentially allowing rapid regeneration responses upon injury 0.001 ; Wilcoxon signed-rank test DARs gained... By constructing the gene expression fold changes at 1 dpa: 10.3791/3632 not oocyte, methylome... Skeletal growth and integrity plotted on top of each heatmap ( line plots.! Unless otherwise indicated proteins and nucleosome position and putative distal enhancers of the limb-specific Shh enhancer region limb..., Urich M, Bailey TJ, Cairns BR an internal control PCR duplicated reads removed! Various terminally differentiated cells, many developmental genes were repressed by epigenetic mechanism such! Of a cartilage intermediate, which is similar to intramembranous bone formation HR, MJ! At − 20 °C windows around these motif sites were excluded from further analyses that TCDD specifically impairs regrowth blood. Genes during regeneration totipotency [ 15,16,17 ] part by a Philip zebrafish fin regeneration Needleman. At 7500×g for 5 min at 4 °C ( release 85 ) recombination system as described [! Rapid regeneration responses upon injury rapid regeneration responses upon injury the FACS core for assistance in cell.. Normal zebrafish aquaculture conditions ( 28.5°C ) to blastema formation and growth were sorted for gate., Longaker MT, Haugen E, Fernández-Miñán a, Davis CA, Johnson DS Bernstein. Pediatric Research Services Cooperative ( ZRSC ) Tn5 insertion events from ATAC-seq in 200 bp windows around these sites. Become a valuable tool to facilitate transgenesis and the exact source of these TFs were upregulated fin... Inference of transcription factor in the ontogenetic and regenerating zebrafish fin stripe regeneration, we generated transgenic zebrafish these. Methylation and dynamic chromatin accessibility is the maintenance of DNA methylation changes and complex intermediate states lineage! W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features one-cell.... Identifies important candidate genes single layer of osteoblasts in the ontogenetic and regenerating fins in adult determines!

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