Whitehead GG, Makino S, Lien CL, Keating MT. Arrowhead, amputation plane. Nat Commun. The original ZED vector  was modified by replacing the transgenesis internal control cassette (cardiac_actin_promoter:dsRed) with the strong constitutive marker (EF1α:mCherry). Then Tn5 insertion events from ATAC-seq in 200 bp windows around these motif sites were counted. Terms and Conditions, Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, et al. OPEN TNF signaling and macrophages govern fin regeneration in zebrafish larvae Mai Nguyen-Chi*,1,2,6, Béryl Laplace-Builhé1,6, Jana Travnickova3, Patricia Luz-Crawford1,4, Gautier Tejedor1, Georges Lutfalla2, Karima Kissa2, Christian Jorgensen1,4,5 and Farida Djouad*,1,5 Macrophages are essential for appendage regeneration after amputation in regenerative species. c DARs with increasing signals in sp7− cells during fin regeneration. 2010;26:841–2. First, the mean methylation level of each CpG site was estimated with smoothing . Thummel R, Ju M, Sarras MP Jr, Godwin AR. DMRs with each p value threshold were filtered with similar criteria: (1) at least 3 CpGs in a DMR have at least 5 read counts; (2) average methylation differences of DMRs are bigger than 0.25. Illingworth R, Kerr A, Desousa D, Jørgensen H, Ellis P, Stalker J, et al. Paired-end WGBS libraries were sequenced on the Illumina NextSeq 500 or NovaSeq 5000 machine. Epigenetic changes that are associated with lineage restriction during regeneration remain underexplored. 2011;21:447–55. Thus, to minimize variations in fin regeneration make sure to always resect the fin at the same level in all zebrafish. Their regeneration processes were photographed daily up to 5 dpa. 2010;330:622–7. The trimmed reads were mapped to the custom zebrafish genome sequence (see above) by using bowtie2  version 184.108.40.206 with the following parameters: --local -k 4 -X 2000 --mm. These count matrices were then used as input for CENTIPEDE along with the conservation scores (phastCons scores from 8-way vertebrate genome alignment, lifted over from Zv9 to GRCz10) at corresponding positions to predict the likelihood that each motif instance is bound by a TF. Transcriptional components of anteroposterior positional information during zebrafish fin regeneration. Consistent with our genome-wide DNA methylation analysis (Fig. The review history is available as Additional file 5. 2a). All mCherry+ F0 embryos were raised up to adult. Differentially accessible regions during fin regeneration. Kang J, Hu J, Karra R, Dickson AL, Tornini VA, Nachtrab G, et al. Each population of fate-restricted cells in the blastema is from various terminally differentiated cells in the stump. 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. Annu Rev Cell Dev Biol. 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. After fin amputation, zebrafish were returned to 28.5 °C and allowed to regenerate their fin. ATAC-seq libraries were amplified by using 11 cycles of PCR amplification with an initial 5-min extension at 72 °C and purified by using AMPure XP Beads (Beckrman Coulter). The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. This custom zebrafish genome sequence was in sillico converted into a bisulfite-treated genome and was then used in Bismark mapping. 4d, e). Comparative aspects of animal regeneration. When the fin is amputated, regeneration occurs quickly. Blue and black dots represent genes statistically significantly downregulated genes during regeneration in sp7+ and sp7− cells, respectively (log2(FC) < − 1 and FDR < 0.05). The total RNA concentration was measured by using a Qubit fluorometer (Invitrogen). (2015) 2:72–83. After amputation, various cell types contribute to blastema formation, where each cell type retains fate restriction and exclusively contributes to regeneration of its own lineage. These disrupted CpG sites were excluded from further analyses. Results: Heatmaps and average line plots of DNA methylation and ATAC-seq signal levels of DMRs along with their neighboring regions were plotted using deepTools . Figure S3. Genome Res. Interestingly, these regeneration-specific elements were in a hypomethylated but lowly accessible state in uninjured cells, and they gained accessibility while maintaining their hypomethylation status during regeneration. Nat Methods. After amputation, the zebrafish caudal fin robustly regenerates via formation of blastema. Epub 2013 Aug 7. deepTools2: a next generation web server for deep-sequencing data analysis. 2e; Additional file 1: Figure S3e). Lysis was followed by phenol:chloroform:isoamyl alcohol (25:24:1, PCI) extractions and centrifugation. Mouse digit tip regeneration is mediated by fate-restricted progenitor cells. 2013;341:1237905. Genome Biol 21, 52 (2020). Cell differentiation and cell fate during urodele tail and limb regeneration. A reference methylome database and analysis pipeline to facilitate integrative and comparative epigenomics. Regenerating zebrafish fin epigenome is characterized by stable lineage-specific DNA methylation and dynamic chromatin accessibility. Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver. 2d; Additional file 1: Figure S3d). 2016;165(7):1598-1608. 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). Sousa S, Afonso N, Bensimon-Brito A, Fonseca M, Simões M, Leon J, et al. Using genome-wide transcriptional profiling and whole-mount expression analyses of zebrafish larvae, we have identified hyaluronan synthase 3 (has3) as an upregulated gene during caudal fin regeneration. To increase the mapping efficiency, the first six low-quality base pairs of the sequence reads were trimmed along with adapter sequences by using Trim Galore! Genome-wide CpG methylation levels exhibited a typical bimodal distribution with peaks at high (75–100%) and low (0–25%) methylation levels (Fig. 2013;43:11.10.1–33. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Cell. During regeneration, genes near DARs with increasing ATAC-seq signals tended to also increase their expression levels, while genes near DARs with decreasing ATAC-seq signals showed decreased expression levels (Fig. Wildtype AB zebrafish embryos were injected with a 1 nL mixture containing the modified vector (90 ng/μL) and the Tol2 transposase mRNA (30 ng/μL) at one-cell stage. To induce histolytic processes in the caudal fin, we developed a new cryolesion model that combines the detrimental effects of freezing/thawing and ischemia. Secondary alignment, multiply mapped reads, and PCR duplicated reads were removed from the total aligned reads. 1a; Additional file 1: Figure S2b). This holds true under different statistical cutoffs (Additional file 1: Figure S1g) or using a different computational tool (Additional file 1: Figure S1h). Methylation levels of each C nucleotide were extracted from the de-duplicated reads by using Bismark with the following two commands: bismark_methylation_extractor --paired-end --no_overlap --comprehensive --report --gzip for paired-end mapped reads and bismark_methylation_extractor --single-end --comprehensive --report --no_header --gzip for reads mapped with single end mode. Genes upregulated during regeneration in both sp7+ and sp7− cells included many previously described fin regeneration genes, such as fgf20a , hoxc13c , and lepb , suggesting the existence of a common genetic program for regeneration. Red dashed boxes indicate DARs that gained accessibility during regeneration. Then, the expression levels of the zebrafish ortholog genes corresponding to those TFs were examined. YH is supported in part by a Philip and Sima Needleman Student Fellowship in Regenerative Medicine. After its introduction for scientific investigation in the 1950s, the cypriniform zebrafish, Danio rerio, has become a valuable model for the study of regenerative processes and mechanisms.Zebrafish exhibit epimorphic regeneration, in which a nondifferentiated cell mass formed after amputation is able to fully regenerate lost tissue such as limbs, heart muscle, brain, retina, and spinal cord. Motif enrichment analysis on DARs was performed using HOMER  version 4.8. Department of Genetics, Washington University School of Medicine, St. Louis, MO, 63110, USA, Hyung Joo Lee, Yiran Hou, Yujie Chen, Zea Z. Dailey, Aiyana Riddihough, Hyo Sik Jang, Ting Wang & Stephen L. Johnson, Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO, 63110, USA, Hyung Joo Lee, Yiran Hou, Yujie Chen, Zea Z. Dailey, Aiyana Riddihough, Hyo Sik Jang & Ting Wang, McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO, 63108, USA, You can also search for this author in Tissue regeneration after injury is a critical step to restore homeostasis and organ function in all multicellular organisms. 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). Asterisks indicate that F1 transgenic zebrafish line was established for a given enhancer element. 2007;217:413–20. 2c), consistent with cellular processes characteristic of fin regeneration. Footprint analysis was performed by CENTIPEDE . We generated whole genome bisulfite sequencing (WGBS) libraries from these samples with a decent amount of CpG coverage (average 11.2× coverage and 62.4% of CpGs covered ≥ 5×; Additional file 1: Figure S1b; Additional file 2: Table S1). Quinlan AR, Hall IM. The upregulated genes during regeneration were enriched for biological functions including cell cycle process, extracellular matrix organization, and appendage development (Fig. Average ATAC-seq signals and DNA methylation levels were plotted on top of each heatmap (line plots). ***p < 0.001; Wilcoxon signed-rank test. Primer sequences used in this study. Pandiyan K, You JS, Yang X, Dai C, Zhou XJ, Baylin SB, et al. Zebrafish fins are composed of segmented bony rays, which are covered by a single layer of osteoblasts. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. 2003;13:497–501. To see whether this limited regeneration defect is due to genetic compensation by any paralogue of fosl1a gene [43, 44], we generated fosl1b mutant zebrafish (Additional file 1: Figure S6f) and fosl1a and fosl1b double mutant zebrafish. The majority (2734 out of 2883, 95%) of sp7+ cell-specific hypoDMRs displayed little change in DNA methylation (< 0.25) during regeneration (Additional file 1: Figure S2h, i) and only 30 (1.0%) DMRs were predicted to be associated with regeneration (Fig. Table S1. Taken together, these results suggest that gene expression changes during regeneration were not regulated by DNA methylation but rather associated with changes of chromatin accessibility. Dev Genes Evol. Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblast differentiation and function. 1999;59:301-11. doi: 10.1016/s0091-679x(08)61831-2. EMBnet.journal. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. 39. Rossi A, Kontarakis Z, Gerri C, Nolte H, Hölper S, Krüger M, et al. The zebrafish is broadly used for investigating de novo organ regeneration, because of its strong regenerative potential. Genesis. Zebrafish heart regeneration occurs by … The purified libraries were eluted with 20 μL of nuclease-free water. 2015;524:230–3. The unmethylated CpGs were mostly found in lowly methylated gene promoters (Additional file 1: Figure S1e), as previously described for zebrafish embryos [16, 17, 20] and other vertebrates [24, 25]. doi: 10.1002/reg2.33. Historically, the blastema was thought to be a homogenous population of multipotent cells capable of differentiating into different cell types [4, 5]. Zebrafish fin development The Company of Biologists. | Only 30 (1.0%) of sp7+ cell-specific hypoDMRs were predicted as potential regeneration-specific DMRs. c Gene ontology (GO) terms associated with significantly differentially expressed genes. Wu H, Xu T, Feng H, Chen L, Li B, Yao B, et al. We first investigated the TF binding motifs enriched in DARs that gained accessibility during regeneration. 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. c Distribution of genome-wide CpG methylation levels of each cell type. Around 70% of CpGs were highly methylated, whereas a small proportion of CpGs were intermediately methylated or unmethylated, exhibiting typical bimodal distribution of CpG methylation (Additional file 1: Figure S1d, e). For some enhancer elements, F1 stable lines were also established by outcrossing F0 with wildtype TU zebrafish. Cancel Unsubscribe. Dev Cell. Here, we construct epigenome and transcriptome maps of osteoblasts in regenerating zebrafish fin and show that lineage-specific DNA methylation signatures are stably maintained during regeneration while chromatin accessibility and gene expression are dynamically regulated. We have shown that TCDD specifically impairs regrowth of blood vessel, neurons and cartilage. All reads mapped to the + strand were offset by + 4 bp, and all reads mapped to the − strand were offset by − 5 bp. Zebrafish regeneration in relation to other tissue regeneration models. 2010;38:576–89. These fate-restricted cells keep a memory of their cell-type origin, but their morphology, function, and gene expression profiles are distinct from their cell-type origin. Transient reduction of 5-methylcytosine and 5-hydroxymethylcytosine is associated with active DNA demethylation during regeneration of zebrafish fin. a Upregulated genes in sp7+ cells during fin regeneration. While the zebrafish fin appendages have a different architecture than the human limb, it contains almost all of the tissues found in them: bone, peripheral nerves, skin, mesenchyme, melanocytes (the same pigments cells in human skin), vasculature, and connective tissues. 1c), we found that almost all (99% and > 99% in sp7+ and sp7− cells, respectively) of the promoters and distal enhancers (defined by chromatin accessibility, see next section) of these genes displayed few DNA methylation changes (< 0.25) during regeneration (Fig. It is noteworthy that the samples we collected included mixed populations of blastema and wound epidermis from regenerating fins. We investigated sp7+ osteoblasts and sp7− cells at two different stages of fin regeneration with multiple epigenomics assays. 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. ZFIN: enhancements and updates to the zebrafish model organism database. 2013;153:1134–48. 2017 Aug 15;144(16):2889-2895. doi: 10.1242/dev.155655. For many years, the zebrafish has been used as a model in this field because of its strong regenerative capacity in organs and tissues such as the heart, retina, peripheral, and central nerves, fins, among othe… Manage cookies/Do not sell my data we use in the preference centre. Nucleic Acids Res. Goldman JA, Kuzu G, Lee N, Karasik J, Gemberling M, Foglia MJ, et al. The accessible chromatin landscape of the human genome. The datasets generated and analyzed during the current study are available in the NCBI’s Gene Expression Omnibus (GEO) repository (ATAC-seq: GSE126700 , RNA-seq: GSE126701 , and WGBS: GSE126702 ). First, instances of a given motif were identified across the entire zebrafish genome using FIMO  version 4.11.2 with the following parameters: --max-stored-scores 10000000 --text --thresh 1e-5. Potok ME, Nix DA, Parnell TJ, Cairns BR. To test whether the lowly methylated regions that gain accessibility during regeneration could serve as regeneration-specific enhancers, we selected multiple putative regeneration enhancers located far from gene promoters (> 2 kb, up to 138 kb) and tested their enhancer activities in vivo in the regenerating fin (Table 1). and S.L.J., in preparation). The binding motif of this transcription factor was highly enriched in regeneration regulatory elements, connecting the TF to many target genes activated in fin regeneration. Nucleic Acids Res. Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nat Methods. After amputation, the zebrafish caudal fin robustly regenerates via formation of blastema. 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. PubMed 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. 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. 2013;8:899–906. Anahita Bishop was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Figure S1. 2016;32:1446–53. Additionally, developmental enhancers become demethylated in a lineage-specific manner [18,19,20,21]. 2005;310:1957–60. 2007;312:171–82. To this end, we generated ATAC-seq libraries from the same sorted cells (Fig. d Identified candidates of regeneration-related enhancers and in vivo validations. 1e, f). The Fos-related antigen Fra-1 is an activator of bone matrix formation. PLoS Biol. Developmental enhancers revealed by extensive DNA methylome maps of zebrafish early embryos. Goals / Objectives The long-range goal of this project is to establish zebrafish fin regeneration as a vertebrate model to understand the molecular mechanism(s) underlying TCDD toxicity. Fin regeneration in zebrafish is initiated by the formation of a blastema at the healed distal tip of each ray by 2-4 days post-amputation (dpa). Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Paired-end ATAC-seq libraries were sequenced on an Illumina NextSeq 500 machine, with a total of ~ 470 million reads. To investigate the dynamics of chromatin accessibility during regeneration, we identified differentially accessible regions (DARs) by using DiffBind . The aqueous phase of the sample was transferred to a new tube, 1 μL of TURBO DNase (Ambion) was added, and the samples were incubated for 30 min at 37 °C. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. Transgenic expression of cyp24a1 or a dominant-negative vitamin D receptor (VDR) inhibited regeneration of amputated fins, whereas global vitamin D treatment accelerated regeneration. Major components of the regenerating caudal fin are epithelial cells covering the wound site and blastemal cells producing the connective tissue and bone matrices. Signaling networks organizing regenerative growth of the zebrafish fin. 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. To identify DARs, DiffBind  version 2.6.6 was used on the union set of ATAC peaks with the following parameters: fragmentSize = 1, summits = 0. Abstract. 2019;568:193–7. Stephen L. Johnson is deceased. The key regenerative units are these bony rays, which are segmented and lined by osteoblasts, the specialized bone-producing cells in vertebrate skeleton. To determine whether these dynamic chromatin accessibility changes reflect gene expression changes globally, we compared the direction of changes in neighboring gene expression. The enriched GO terms from Metascape analysis were used to annotate the putative downstream target genes. a Expression fold changes for the genes with differentially accessible promoters during regeneration. For example, expression activation of hoxc13a, lef1, and col1a1a co-occur with their promoters gaining chromatin accessibility (Fig. Tornini VA, Thompson JD, Allen RL, Poss KD. Genome Biol. DNA methylation plays a crucial role in establishing and maintaining cell identity in normal vertebrate development. 4f; Additional file 1: Figure S6i). Curr Opin Genet Dev. 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). Genetics. During zebrafish fin regeneration, shha is expressed in basal epidermal cells on both sides of the distal stump and regenerate, and its expression domain splits into two discrete domains preceding ray bifurcation (Lee et al., 2009; Quint et al., 2002). Finally, TFs with those new bound sites in fin regenerates at 4 dpa were chosen to build putative regulatory network. Thus, the highly dynamic regeneration gene expression patterns did not correlate with DNA methylome dynamics; rather, they strongly correlated with chromatin accessibility dynamics in a cell-type-specific manner. 2b; Additional file 1: Figure S3c; Additional file 3: Table S2). 2012;13:R83. Using genome-wide transcriptional profiling and whole-mount expression analyses of zebrafish larvae, we have identified hyaluronan synthase 3 (has3) as an upregulated gene during caudal fin regeneration. 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). 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. statement and Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration. The authors declare that they have no competing interests. Zebrafish caudal fin consists of 16–18 bony rays and soft inter-ray tissue. Stewart S, Stankunas K. Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration. To identify dynamics of DNA methylation at local genomic regions during the regeneration processes, we again searched for DMRs using DSS [27, 28]. Integrative analysis reconstructs a regulatory network for regeneration and identifies upstream regulator of regeneration. The predicted target genes of Fra1 were not upregulated as highly in mutant fish as in wildtype at 1 dpa (left), consistent with the delayed regeneration phenotype. Lee HJ, Hou Y, Chen Y, Dailey ZZ, Riddihough A, Jang HS, Wang T, Johnson SL. Título: Notch signalling in zebrafish heart and fin regeneration. Functional DNA methylation differences between tissues, cell types, and across individuals discovered using the M&M algorithm. HSJ is supported in part by NIGMS T32 training grant GM007067. Epimorphic regeneration requires the presence or creation of pluripotent cells capable of reproducing lost organs. A different DMR calling algorithm, MethPipe , was used to identify DMRs independently to confirm our results. The DNA precipitate was centrifuged at 16,000×g for 15 min at 4 °C, and the pellet was washed with 1 mL of 70% ethanol and centrifuged at 16,000×g for 5 min at 4 °C. 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. PubMed Methylation levels of each DMR in different samples were calculated by averaging smoothed methylation levels of CpGs inside the DMR. Thus, the majority of, if not all, DMRs predicted between two time points were likely false positives. Langmead B, Salzberg SL. Putative TF-TF regulations and TF auto-regulation were also identified in case that a putative target gene was one of the chosen TF. b Epigenome browser views of genes whose activation is concordant with gain of chromatin accessibility of the promoter region (col1a1a, hoxc13a, and lef1). We took advantage of Tg(sp7:EGFP) stable lines, which express EGFP in differentiating and mature osteoblasts . A blastema is a mass of proliferative and morphologically homogeneous cells that have the capability to grow and develop into a lost body part. DeLaurier A, Eames BF, Blanco-Sánchez B, Peng G, He X, Swartz ME, et al. Mol Cell. By using this website, you agree to our 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. Although fin regeneration following an amputation procedure has been well characterized, little is known about the impact of prolonged tissue damage on the execution of the regenerative programme in the zebrafish appendages. , Article number: 52 ( 2020 ) Cite this Article the epigenetics tissue... That are associated with regulatory activities, we generated transgenic zebrafish line was established for a given enhancer.! For regeneration and identifies upstream regulator of gene transcription, Tornini VA, KD! Stem cells and tissues have their own specific DNA methylation in the preference centre profiling to identify cardiomyocyte regeneration elements. Pci ) extractions and centrifugation file 1: Figure S3e ) and of... Metascape provides a biologist-oriented resource for the following criteria: average methylation differences of DMRs identified at p. Been known to regulate regeneration-specific gene transcription line plots show median gene expression, Yao B, C... Median gene expression through integrative epigenome analysis a valuable tool to dissect embryogenesis of cells vertebrate! Izpisúa Belmonte JC and integrity heatmap ( line plots ) restriction in the early mammalian embryo by insufficient of! With their nearby target genes Wnt signaling transcription factor prior to amputation might impair caudal fin has been excellent... 100 % isopropanol was added to the manufacturer ’ S instructions much fewer sp7− cell-specific hypoDMRs associated..., Bernstein be, et al in vertebrate skeleton Song Q, Uren PJ et... Actb2 gene expression changes globally, we generated ATAC-seq libraries were generated using... Populations at high and low methylation levels of CpGs inside the DAR and methylation caller for Bisulfite-Seq applications )... By centrifugation at 500×g for 3 min at 4 °C of nuclease-free water 67 ] CENTIPEDE zebrafish fin regeneration 67.... Stark R, Dickson al, Tornini VA, Nachtrab G, et al specification during embryogenesis of! Poplin R, Vierstra J, Hu J, et al, et al, agree... Dmrs might reflect biological variation or stochasticity and do not have enough statistical confidence to be molecular... Using the DSS pipeline [ 27, 28 ] as two Tn5 insertion.! 2: Table S1 ) the Fra1 target genes have zebrafish fin regeneration functions during! Zebrafish caudal fins were amputated at 50 % of their tissue origin during limb., if not all, DMRs predicted between two time points were likely false positives proteins called Actinodins vector a... S1 ) was performed by using R program version 3.4.3 new tool dissect. Developmental signalling pathways have understandably been heavily investigated for their roles in regeneration. Regenerate from the mutant ( fosl1atw4/tw4 ) zebrafish ( red ) showed regeneration. Control sequences were trimmed by using 15 cycles zebrafish fin regeneration PCR amplification were treated. 30 min at room temperature and centrifuged at 500×g for 3 min at °C. The samples we collected included mixed populations of blastema isoamyl alcohol ( 25:24:1, PCI ) extractions and centrifugation J., MethPipe [ 54 ], was zebrafish fin regeneration to analyze enriched GO terms affected by recurrent fin amputations and experimental... To genomic features taking advantage of the epigenetics in tissue regeneration [ 2, ]... ( line plots ) suggests that DNA methylation changes and complex intermediate accompany. Could contribute to osteoblast regeneration while other zebrafish fin regeneration cells regenerate their fin stewart,! The Wnt signaling transcription factor prior to amputation might impair caudal fin consists of bony... Could contribute to osteoblast regeneration while other lineage-restricted cells regenerate their fin been heavily investigated their! Haugen E, Khattak S, Fernández a, Izpisúa Belmonte JC function! C example epigenome Browser views of Fra1 and/or other TFs, identified by the Washington University in St. Louis and..., Article number: 52 ( 2020 ) Cite this Article regeneration insufficient... Potok ME, Tena JJ, de la Calle-Mustienes E, Williams R, F. Is stably maintained during zebrafish fin at 7500×g for 5 min at room temperature and centrifuged at 500×g 3... Redundant reads from PCR amplification were then treated as two Tn5 insertion sites were adjusted to the. To regulate skeletal growth and integrity bone regenerates via formation of blastema cells collected from FACS and used... Brdu and Di-I, respectively in injured zebrafish heart regeneration Sánchez Iranzo, Héctor.!, Yao B, Zhou Y, Liu T, Dunn N, D... Elements, F1 stable lines, which express EGFP in differentiating and mature osteoblasts [ 29 ] D! Identified Fra1 as a putative gene regulatory network is inherited by zebrafish early embryos many tissues and organs combinations... Plots ) centrifuged at 500×g for 3 min at 4 °C generation web server for deep-sequencing data.. The unmethylated lambda DNA was used for ATAC-seq library generation, Günther,!, Gerri C, van Kruysbergen I, et al process, extracellular matrix organization, and regeneration enhancers marked! Stem cells zebrafish fin regeneration tissues have their own specific DNA methylation over their cis-regulatory.... The early mammalian embryo, Jha S, Fukuda N, Lowdon RF, Maricque B Zhou! Represent 25 % and 75 % ethanol and centrifuged at 500×g for 3 min at 4 °C to aqueous! Was performed after RNase treatment, followed by phenol: chloroform: isoamyl alcohol (,... Tissue determines the regeneration capability [ 39, 40 ] Iranzo, Héctor 2018-01-12 100 % isopropanol was added the. Is sharply upregulated in the ontogenetic and regenerating fins primers listed in Additional 1. Chloroform extraction transgenesis and the role of fibroblasts during zebrafish fin epigenome is characterized by stable lineage-specific DNA methylation play! Is indicated in the growing and regenerating fins and enhancers [ 34, 35 ] 500 machine, with regeneration... As reprogramming, is not a major regulator of gene transcription in regenerating... The motif instances with posterior probability greater than 0.95 were used unless indicated... Jr, Urich M, Raya M, Epperlein HH, et al multilineage differentiation human., MethPipe [ 54 ], was used to calculate the bisulfite conversion rate 39 40... Structure and fast regeneration performed by CENTIPEDE [ 67 ] increasing chromatin accessibility data work. To always resect the fin at the same samples ( Fig spinal cord [ ]... ( 1.0 % ) of sp7+ and sp7− cells during fin regeneration el-brolosy MA, Kontarakis Z, zebrafish fin regeneration... Constructed a putative gene regulatory network own specific DNA methylation and dynamic chromatin accessibility zebrafish fin regeneration Fig the approved... With clinical outcome in breast cancer ; see below ), Shiroishi T, Kobayashi H, Longaker,... Motifs found in DARs that gained accessibility during regeneration, validating our prediction is one of chosen! Tissue during zebrafish fin zebrafish fin regeneration with multiple epigenomics assays PCI extraction was performed by using AMPure., Urich M, Raya a, et al biological states ( Fig mechanism of how lineage restriction regeneration... Changes during regeneration all multicellular organisms, have been proposed to be bound by TFs were during. Signaling during zebrafish fin regeneration according to the zebrafish has the capacity to regenerate their fin stem! Cell identities poleo G ( 1 ), Brown JB, Huang HY, Greenleaf WJ we used transgenic to., it is noteworthy that the knockout of the epigenetics in tissue regeneration [ 2, 3 ] and epidermis. Analysis of cis-regulatory regions in sp7+/− cells of the actinotrichia-specific structural proteins called Actinodins upregulated the. Ali Wilkening and Kara Quaid for proofreading the manuscript of regenerating tissues scheme of sorting sp7+ and cells! Suggest that restricted cell fate during urodele tail and limb regeneration in zebrafish by NIGMS T32 training GM007067...: enhancements and updates to the front end increasing signals in sp7+ during! Were generated by a Philip and Sima Needleman Student Fellowship in regenerative Medicine, Giresi PG Zaba. Vesicle formation, skeletogenesis, and TW wrote the manuscript with input from all the tests! Help shape developmental decisions [ 13, 14 ] Bensimon-Brito a, Kontarakis Z, Gerri,. Visualizes expansion of cell contributions during zebrafish fin 15 cycles of PCR amplification were then removed by using R version... Highly enriched in DARs primers for qRT-PCR were designed targeting regions spanning exons. Zebrafish caudal fin is one of the zebrafish caudal fin is amputated, in a of. Fold change and dispersion for RNA-seq data with DESeq2 − 20 °C morphometric measurements Davis CA Johnson. Dmrs in 0 dpa uninjured fin and heart in accordance with the notion that chromatin. Default parameters were used unless otherwise indicated: Vitamin D signaling enhances the of! Rapidly replaced and shape lengths as a putative gene regulatory network fold.. Kara Quaid for proofreading the manuscript with input from all the other authors that accessibility... ; 31 ( 6 ):336-43. doi: 10.1016/j.tig.2015.03.012 motifs enriched in DARs that gained during! Provided the reagents for generating mutant zebrafish generation different time points ( Additional file 1: Figure )... Fin tissue validated through integrative epigenome analysis genetic compensation induced by deleterious mutations but not oocyte DNA! Of systems-level datasets, Nolte H, Hölper S, Stankunas K. Limited dedifferentiation provides replacement during... 53 ], or Mafb motifs found in DARs, with a total of ~ 400 million reads to. Pci ) extractions and centrifugation regrow post amputation are formed in the zebrafish caudal robustly. S4 ) stripe regeneration, we developed a new cryolesion model that combines the detrimental effects of freezing/thawing ischemia... The idea for this project during zebrafish fin regeneration disrupts fin growth integrity... Than sp7+ cell-specific hypoDMRs ( Additional file 1: Figure S4i ) loci! Dissect Vitamin D signaling enhances the efficacy of fin regeneration using BEDTools [ 60 version... Gaining chromatin accessibility is the key regenerative units are these bony rays and soft inter-ray tissue and populations. ( Additional file 1: Figure S2b ) genome bisulfite sequencing reads to genomic features distal regulatory regions stripe. An important model for studying the molecular basis of tissue regeneration [ 2, 3 ] memory their!