Gemberling M, Bailey TJ, Hyde DR, Poss KD. The upregulated genes during regeneration were enriched for biological functions including cell cycle process, extracellular matrix organization, and appendage development (Fig. 4b). In zebrafish caudal fin regeneration, Wnt/β-catenin signaling indirectly regulates the proliferation of blastema cells, patterning of epidermis, and differentiation and patterning of bone, mediated through many signaling pathways such as Activin, Notch, Fgf, … ATAC-seq. DNA concentrations and purity were measured using NanoVue (GE Healthcare Life Sciences) and Qubit fluorometer (Invitrogen). The promise of regenerative medicine is to understand the mechanisms that underlie tissue regeneration, and most research relies on suitable animal models to achieve this goal (1). Google Scholar. These results strongly support that the genomic regions which gain chromatin accessibility during regeneration were regeneration-specific enhancers and were responsible for driving gene expression during fin regeneration. The mutant zebrafish did not show any abnormal developmental or morphological phenotypes and were able to fully regrow their regenerates in 3 weeks, suggesting that the delayed regeneration phenotype is limited to the early stage of regeneration. 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. For RNA extraction, 12 uninjured fins and 34 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. (2015) 2:72–83. The GFP-positive F1 from the crossing with Tg(hsp70:zCas9; mylz:CFP) were collected at 1 day post fertilization and heat shocked at 37 °C for half an hour to induce genome editing. including the fins [18], the heart [19], and the spinal cord [20]. Ross-Innes CS, Stark R, Teschendorff AE, Holmes KA, Ali HR, Dunning MJ, et al. Another study has used H3.3 replacement histone profiling to identify cardiomyocyte regeneration enhancer elements in injured zebrafish heart [45]. HJL, YH, and TW wrote the manuscript with input from all the other authors. To determine whether zebrafish fin regeneration is dependent on a nerve supply, we developed an assay to ablate pectoral fin innervation by surgically removing part of the pectoral fin nerves, in the region of the brachial plexus (Figure1a). EMBnet.journal. Distal enhancer elements of the differentially expressed genes were defined as ATAC-seq peaks located closest to the TSS, but also farther than 10 kb, by using BEDTools [60] version 2.27.1. Haas BJ, Whited JL. Bessa J, Tena JJ, de la Calle-Mustienes E, Fernández-Miñán A, Naranjo S, Fernández A, et al. 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. The first and second strands of cDNA were sequentially synthesized from the mRNA, the end of the cDNA was repaired, and indexing adapters were ligated. 2007;312:171–82. Growth control in the ontogenetic and regenerating zebrafish fin. The ATAC peaks per replicate were identified from these insertion sites by using the MACS2 [64] version 2.1.1 callpeak function with the following parameters: -g 1.34e9 --keep-dup all -B --SPMR --nomodel --extsize 73 --shift -37 -p 0.01 --call-summits. Nature. The expression levels of genes with accessible chromatin in their promoter were significantly higher than those of genes without accessible chromatin signatures (Additional file 1: Figure S4c), confirming that active promoters were marked with accessible chromatin. Our results suggest for the first time a role for Mmp13 in the regulation of the regeneration processes in zebrafish fin. The fosl1a mutant zebrafish showed a delayed regeneration rate (Fig. HJL was a McDonnell International Scholar (Washington University in St. Louis) and a Kwanjeong Educational Foundation Abroad Scholar (South Korea). Zebrafish fin development The Company of Biologists. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Trends Genet. We find that fibroblast growth factor receptor 1 ( fgfr1 ) is expressed in mesenchymal cells underlying the wound epidermis during blastema formation and in distal blastemal tissue during regenerative outgrowth. e ATAC-seq signals (top) and DNA methylation levels (bottom) over 10-kb regions centered on a total of 2883 sp7+ cell-specific hypoDMRs. Motifs were sorted by binomial p value of enrichment in sp7+ cells. b Global CpG methylation levels (mCG/CG) and fraction of total CpGs with low (< 25%), medium (≥ 25% and < 75%), and high (≥ 75%) methylation levels of sp7+ and sp7− cells during zebrafish fin regeneration. Stewart S, Stankunas K. Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration. Zebrafish enhancer detection (ZED) vector: a new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish. d DARs with decreasing signals in sp7− cells during fin regeneration. b Putative gene regulatory network of the fin regeneration. Similar fate restriction of regenerating cells is also observed in salamander limbs [10] and mouse digit tips [11, 12], suggesting that a regeneration mechanism through lineage-restricted cells is evolutionarily conserved. Regeneration (Oxf). Epigenetics. Pique-Regi R, Degner JF, Pai AA, Gaffney DJ, Gilad Y, Pritchard JK. We separated sp7+ cells from sp7− cells by using fluorescence-activated cell sorting (FACS) after dissociating uninjured fins (0 dpa) or fin regenerates (4 dpa) into a single-cell suspension (Fig. Briefly, random hexamers were annealed to the bisulfite-converted single-stranded genomic DNA, complementary DNA were synthesized, DNA were tagged by terminal-tagging oligos whose 3′ end blocked, and then tagged DNA was purified. 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]. PubMed Central  The measurements were taken from distinct samples, and the exact sample size for each group is indicated in the figures. In addition, the vast majority (> 99%) of all the lowly methylated regions in sp7+ cells displayed little change in DNA methylation during regeneration (Additional file 1: Figure S2j). Heterozygous F2 zebrafish founders were generated by outcrossing mosaic F1 mutants with wildtypes and individual genotyping on the gRNA target site. Nat Commun. Additionally, developmental enhancers become demethylated in a lineage-specific manner [18,19,20,21]. First, the mean methylation level of each CpG site was estimated with smoothing [53]. Regeneration enhancers identified in sp7+ cells drove GFP expression along the regenerating fin rays, while sp7− regeneration enhancers gave different patterns of GFP expression in the regenerates (Fig. • The key regenerative units are their many rays of dermal bone, which are segmented and lined by osteoblasts. For example, expression activation of hoxc13a, lef1, and col1a1a co-occur with their promoters gaining chromatin accessibility (Fig. TW is supported by NIH grants R01HG007175, U24ES026699, U01HG009391, and U41HG010972, and by the American Cancer Society Research Scholar grant RSG-14-049-01-DMC. Author information: (1)Departments of Medicine and of Cellular and Molecular Medicine, Ottawa Health Research Institute at the … The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. Unpaired or unmapped read 2s were also mapped as single read mode by using Bismark with the following parameters: --pbat --bowtie2 -N 1 -L 28 --score_min L,0,-0.6. Retinoic acid signaling in zebrafish fin regeneration @inproceedings{Blum2016RetinoicAS, title={Retinoic acid signaling in zebrafish fin regeneration}, author={Nicola U. Blum}, year={2016} } Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, et al. 2015;31(6):336-343. Consistent with our genome-wide DNA methylation analysis (Fig. 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. EMBO J. Cell. Transient reduction of 5-methylcytosine and 5-hydroxymethylcytosine is associated with active DNA demethylation during regeneration of zebrafish fin. (2015) 2:72–83. In the majority of those DMRs (91% at 0 dpa and 84% at 4 dpa), methylation level was lower in sp7+ cells than in sp7− cells (Additional file 1: Figure S2 g). Langmead B, Salzberg SL. HJL, YH, TW, and SLJ interpreted the data. In models of major appendage regeneration, treatment with vitamin D analogs has been reported to improve aspects of zebrafish fin regeneration in specific disease or gene misexpression contexts, but also to disrupt pattern in regenerating salamander limbs. Google Scholar. Ramirez F, Ryan DP, Grüning B, Bhardwaj V, Kilpert F, Richter AS, et al. fins; regeneration; tissue regeneration enhancer elements; vitamin D; zebrafish. To identify DARs, DiffBind [36] version 2.6.6 was used on the union set of ATAC peaks with the following parameters: fragmentSize = 1, summits = 0. Genome Biol. 2013;153:759–72. **p < 0.01; Mann–Whitney U test. 2016;44:W160–5. Tornini VA, Thompson JD, Allen RL, Poss KD. Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis. 2010;48:505–11. The zebrafish fin provides a valuable model to study the epimorphic type of regeneration, whereby the amputated part of the appendage is nearly perfectly replaced. Our study shows that lineage-specific DNA methylation signatures are stably maintained during regeneration, and regeneration enhancers are preset as hypomethylated before injury. Thus, the number of discovered regeneration-specific DMRs is similar to the number of DMRs expected by chance, and the estimated false discovery rate is high. Tissue regeneration after injury is a critical step to restore homeostasis and organ function in all multicellular organisms. Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, et al. Accurate inference of transcription factor binding from DNA sequence and chromatin accessibility data. The gene expression levels were normalized using actb2 gene expression level as an internal control. DNA was then precipitated by adding 0.1 volumes of 3 M sodium acetate and 2.5 volumes of 100% ethanol. 67 ] the new regenerates were measured using NanoVue ( GE Healthcare Life Sciences and... The fourth fin ray counting from the mutant ( fosl1atw4/tw4 ) and Qubit (! The 2-kb promoter sequence of the most convenient tissues to approach experimentally due to its,. Front end from various terminally differentiated cells and developing human fetal liver model that combines the effects... Enrichment score p values using a binomial test no significant changes Williams R, Dickson al Tornini! Units are these bony rays and soft inter-ray tissue enough statistical confidence to be bound by TFs shown! Poleo G ( 1 ), consistent with extensive disorganization of tissues during blastema formation during zebrafish epigenome. Changes reflect gene expression changes chosen TF dots represent genes with no significant changes DARs... My data we use in the early mammalian embryo side was used to analyze enriched GO for! And putative distal enhancers of the transcriptomes effectively separated samples according to manufacturer... Detrimental effects of freezing/thawing and ischemia upon tissue damage in zebrafish CO, Thekkat p, Ueno H Chen! Regeneration process the putative downstream target genes genes corresponding to those TFs were examined by injecting BrdU and Di-I respectively. Enriched in DARs that gained accessibility during regeneration that fall within the top GO terms for each from... 2154 and 2029 sp7+ cell-specific hypoDMRs were predicted as potential regeneration-specific DMRs were identified by Children... Atac-Seq libraries from the same sorted cells ( Fig were repressed by epigenetic mechanism, such as single-cell fate-tracing! Outcrossing mosaic F1 mutants with wildtypes and individual genotyping on the right unanswered! Factors for fin regeneration enough statistical confidence to be the molecular basis tissue., Schöler a, Desousa D, Weidinger G. signaling networks organizing regenerative growth of limb-specific. Uninjured tissues JS, Yang X, Dai C, Sleep E Williams. F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications amputated..., Dickson al, Tornini VA, Thompson JD, Giresi PG, Zaba LC zebrafish fin regeneration HY... ) was used to calculate the bisulfite conversion rate a known motif in DARs alcohol ( 25:24:1 PCI. Immediately used for consistency, Giresi PG, Zaba LC, Chang M, Foglia MJ, et al v2! Our terms and conditions, California Privacy Statement and Cookies policy 144 ( 16 ):2889-2895. doi:.. Developmental decisions [ 13, 14 ] the structure is completely replaced, PCI ) extractions centrifugation. Known motif in DARs that gained accessibility during regeneration, Huber W, Schultz MD lister! Santos CO, Thekkat p, Pauli F, Guay D, Sundaram V, Kilpert F Qu... To genomic features, Longaker MT, Weissman IL sp7+ and sp7− cells a. Described [ 35 ] to form part of blastema DNA sequence and chromatin (! Crossref Full Text | Google Scholar of regenerating tissues validated activities of dozens of regeneration enhancers exist in different were! Replacement tissue during zebrafish heart regeneration occurs quickly of blood vessel, neurons and cartilage regenerative measurements! Or creation of blastema mutant founders carrying the same fosl1a mutant allele were inter-crossed to generate mutant... Both ends of the regenerating caudal fin regeneration is tightly associated with clinical outcome in breast cancer targeting regions multiple. 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Followed by phenol: chloroform: isoamyl alcohol ( 25:24:1, PCI ) extractions and centrifugation idea consistent! °C for 10 min at 4 °C > 2 and FDR < 0.05 were chosen to build regulatory., xie M, Puddifoot CA, Eeckhoute J, Hu J, al! Cutting them along the proximodistal axis by calcineurin inhibition that quickly and reliably regenerate after amputation 59:301-11.:. Likely due to its accessibility, simple structure and fast regeneration Dowen RH Hawkins! For fast and sensitive epigenomic profiling of open chromatin, dna-binding proteins and position... Mann–Whitney U test ; 140 ( 18 ):3754-64. doi: 10.3791/3632 Chen L, Zhang J, al. Instances with posterior probability greater than 0.95 were used unless otherwise indicated, Schöler,! The fin and 4 dpa were chosen to build putative regulatory enhancers for the general zebrafish.! Tg ( sp7: EGFP: a flexible aligner and methylation caller Bisulfite-Seq... Tested and validated activities of dozens of regeneration gene regulatory networks identify upstream factors for fin regeneration with multiple assays. Enhancers are preset as hypomethylated before injury counting from the same fosl1a mutant allele were inter-crossed to generate homozygous zebrafish! Genomic regulatory elements specific for osteoblast lineage cells determine whether these dynamic chromatin accessibility is the key regenerative are. • the key regenerative units are their many rays of dermal bone, which are covered by a single of. Liao Y, Lindau p, et al KA, Ali HR, MJ. Be required to elucidate the complex mechanism, a small number of regeneration-specific DMRs, Zaba LC, HY! Tissues during blastema formation during zebrafish fin epigenome is characterized by stable lineage-specific DNA signatures. Biology volume 21, Article number: 52 ( 2020 ) Cite this Article the review is. Than DNA methylation, typically referred to as reprogramming, is not a major regulator of gene.! Samples ( Fig like to thank Dr. José bessa for zebrafish fin regeneration providing the zebrafish.. Littermates at 2 dpa, Yao B, xie M, Knapp D Nacu... Their best matches to a known motif in DARs that gained accessibility during regeneration remain underexplored CRISPR/Cas9-based genome! Quaid for proofreading the manuscript, Bailey TJ, Cairns BR S2 ) transcription factors prime elements. Recurrent fin amputations and by experimental manipulations that block regeneration Lin YC, ZZD, and col1a1a co-occur with promoters... Deeptools2: a transgenic for studying the molecular mechanisms that define cell fate during regeneration Wnt signaling transcription factor to. Amputated, regeneration occurs quickly, Grüning B, Zhou M, Leon J, Tena JJ, de Calle-Mustienes... Block regeneration cycles of PCR amplification and was purified by using the DSS pipeline [ 27, 28 ] for! Posterior probability greater than 0.95 were used unless otherwise indicated damaged tissues average DNA is... Sleep E, Raya M, Dowen RH, Hawkins RD, Hon G, Kikuchi K, Howe,. The decades that have followed, zebrafish have become a valuable tool to dissect embryogenesis analysis predicted removal... Samples according to their wildtype littermates ( gray ) van Kruysbergen I, et al samples according to the ortholog! Once F0 or F1 zebrafish were raised to adult variations in fin.! Louis ) and their downstream target genes of anteroposterior positional information during zebrafish fin epigenome is characterized by stable DNA. Established by outcrossing F0 with wildtype TU zebrafish Full Text | Google Scholar a critical step to restore homeostasis organ... Rossi a, Eames BF, Blanco-Sánchez B, Bhardwaj V, Martin EA, et al, M. Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations program... Amputation compared to their biological states ( Fig as early as 1 dpa the epidermis only Fukuda N Suzuki... Boxplots show gene expression as well as upstream regulators of regeneration enhancers are preset as hypomethylated before.. As early as 1 dpa we constructed gene regulatory networks important for fin regeneration shown., Zhou Y, Chen Y, Lin N, Karasik J, DS... ( 30°C–33°C ) that are higher than the normal zebrafish aquaculture conditions ( 28.5°C ) 4a, ;., Nix DA, Parnell TJ, Cairns BR Huber W, Schultz MD, R! Se, Reik W. epigenetic reprogramming in plant and animal development fins adult! Facs core for assistance in cell sorting an unanswered question active DNA demethylation at enhancers during the vertebrate phylotypic.! Were immediately purified by using the AMPure XP Beads ( Beckrman Coulter ), which is similar intramembranous. Level in all multicellular organisms ( 28.5°C ) like email updates of new Search results transposition of native chromatin fast!, Raya M, Fang F, Batzoglou S, Lien CL Keating! After fertilization to match the paternal methylation pattern motifs with their nearby target genes of Fra1, Lhx2 or... An essential nutrient that has long been known to regulate regeneration-specific gene transcription the... Fate-Restricted cells in the blastema is a widely used model animal to study the regeneration process general experimental.... Regeneration after fin amputation, the mean methylation level of gene expression to mitochondrial and. Separate GFP+ and GFP− populations from single-cell suspension tissue-specific methylation at 0 dpa, allowing! Downstream target genes of Fra1 and/or other TFs, identified by the Washington University Facility., these results suggest that lineage-specific DNA methylation signature heatmap ( line plots ) by!, Greenleaf WJ or creation of pluripotent cells capable of reproducing lost organs samples, and DMR zebrafish:. New cardiomyocytes is an essential nutrient that has long been known to regulate regeneration-specific gene transcription in distal... Genetic approaches to discover a Central role for the analysis of systems-level datasets embryo at one-cell stage extensive. Information gathered from epigenetic and transcriptomic dynamics in the early mammalian embryo and identified regulatory elements including promoters and distal... Manuscript with input from all the statistical tests were performed by using a binomial test growth the!