Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Comparison of regeneration-specific DARs and Phylo(−)DMRs. PubMed Abstract | CrossRef Full Text | Google Scholar. ATAC-seq. Figure S2. The molecular and cellular choreography of appendage regeneration. ***p < 0.001; Wilcoxon signed-rank test. 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. Bottom line plots show median gene expression fold changes across genes (line) in the time course. Default parameters were used for the following commands of MethPipe: hmr, methdiff, and dmr. Cell. 2011;21:447–55. Whitehead GG, Makino S, Lien CL, Keating MT. Several studies have demonstrated that although the structure of the adult and larval zebrafish caudal fin is different, there are similarities at the cellular and molecular level that turn larval zebrafish fin fold a useful model to study the basic principles of regeneration. Buscar en Biblos-e Archivo Esta colección. We examined a time-course expression of EGFP in regenerating fins and confirmed that EGFP expression was localized to the segmented bony fin rays in uninjured fin and strongly upregulated in the regenerates (Additional file 1: Figure S2a) [7, 29]. Zebrafish are native to river basins in and surrounding East India and were established as a laboratory model system first by Streisinger and colleagues during the 1970s, as a potential means to apply genetic analysis to vertebrate development [1,2]. 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). FIMO: scanning for occurrences of a given motif. 2017 Aug 15;144(16):2889-2895. doi: 10.1242/dev.155655. Genome Res.  |  Interestingly, the number of DMRs detected between sp7+ cells of 0 dpa and 4 dpa was low and similar to the baseline established by the comparison between biological replicates (Additional file 1: Figure S2e). HSJ is supported in part by NIGMS T32 training grant GM007067. After fin amputation, zebrafish were returned to 28.5 °C and allowed to regenerate their fin. Tn5 insertion sites were adjusted to reflect the actual binding center of transposons as follows. know about regeneration in zebrafish. To test whether fate-restricted osteoblasts maintain their lineage-specific DNA methylation signatures during regeneration, we compared DNA methylation levels of sp7+ cell-specific hypoDMRs in 0 dpa and 4 dpa samples. and S.L.J., unpublished data) by using GATK’s variant call framework [49, 50] based on the zebrafish genome assembly GRCz10 (danRer10). 4b). 4f; Additional file 1: Figure S6i). Motif enrichment analysis on DARs was performed using HOMER [66] version 4.8. c Gene ontology (GO) terms associated with significantly differentially expressed genes. The art of fin regeneration in zebrafish. This phenotype is replicated in four independent mutant alleles (Additional file 1: Figure S6c-e), confirming that mutation of fosl1a was causal for this regeneration defect. 2016;165(7):1598-1608. Zebrafish caudal fin consists of 16–18 bony rays and soft inter-ray tissue. Mol Cell. 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. These disrupted CpG sites were excluded from further analyses. 3b). Measuring reproducibility of high-throughput experiments. 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. 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. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. • The key regenerative units are their many rays of dermal bone, which are segmented and lined by osteoblasts. To accomplish fin regeneration, two reciprocally interacting domains need to be established at the injury site, namely a wound epithelium and a … The zebrafish caudal fin has been an excellent model for studying vertebrate tissue regeneration [2, 3]. 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. RNA-seq libraries were generated by using TruSeq RNA Library Prep Kit v2 (Illumina), according to the manufacturer’s instructions. 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. Genesis. https://doi.org/10.1016/j.tig.2015.03.012, https://doi.org/10.1016/j.cell.2016.05.038, https://doi.org/10.1016/j.tig.2017.05.006, https://doi.org/10.1016/j.ydbio.2006.08.016, R01 AR076342/AR/NIAMS NIH HHS/United States, R35 HL150713/HL/NHLBI NIH HHS/United States. The gene expression levels were normalized using actb2 gene expression level as an internal control. Developmental enhancers revealed by extensive DNA methylome maps of zebrafish early embryos. b DARs with decreasing signals in sp7+ cells during fin regeneration. NLM 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). Knopf F, Hammond C, Chekuru A, Kurth T, Hans S, Weber CW, et al. Lee, H.J., Hou, Y., Chen, Y. et al. Each population of fate-restricted cells in the blastema is from various terminally differentiated cells in the stump. 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. Am Zool. Epimorphic regeneration requires the presence or creation of pluripotent cells capable of reproducing lost organs. The sequence reads were mapped to the zebrafish transcriptome (Ensembl release 85) and the zebrafish genome assembly (GRCz10) by using STAR aligner [56] version 2.5.2a with the following parameters: --sjdbScore 1 --clip3pAdapterSeq AGATCGGAAGAGC --outWigStrand Unstranded --outFilterType BySJout --outFilterMultimapNmax 1. FEBS Lett. Gene regulatory networks identify upstream factors for fin regeneration. The first principal component (PC1) separated sp7+ cells from sp7− cells, while PC2 separated 4 dpa blastema from uninjured fins (0 dpa), indicating that regenerating cells underwent enormous transcriptional changes during fin regeneration. Bessa J, Tena JJ, de la Calle-Mustienes E, Fernández-Miñán A, Naranjo S, Fernández A, et al. 2013;341:1237905. Loading... Unsubscribe from The Company of Biologists? 4a). 2011;138:3897–905. Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, et al. COVID-19 is an emerging, rapidly evolving situation. Bogdanovic O, Fernandez-Miñán A, Tena JJ, de la Calle-Mustienes E, Hidalgo C, van Kruysbergen I, et al. Ramirez F, Ryan DP, Grüning B, Bhardwaj V, Kilpert F, Richter AS, et al. Directional DNA methylation changes and complex intermediate states accompany lineage specificity in the adult hematopoietic compartment. Then Tn5 insertion events from ATAC-seq in 200 bp windows around these motif sites were counted. Following amputation, osteoblasts undergo dedifferentiation and proliferation to form part of blastema [7, 8]. Osteoblast lineage cells were isolated from uninjured fins or 4 dpa blastema of adult transgenic zebrafish expressing EGFP under the control of the sp7 regulatory regions Tg(sp7:EGFP) by using FACS. c ATAC-seq signals (left) and DNA methylation levels (right) over 10-kb regions centered on DARs with increasing signals in sp7+ (top heatmap) and sp7− cells (bottom heatmap) during regeneration. Finally, TFs with those new bound sites in fin regenerates at 4 dpa were chosen to build putative regulatory network. CAS  a Principal component analysis on the transcriptomes of sp7+ and sp7− cells at 0 dpa uninjured fin and 4 dpa blastema. The top four gRNAs were selected, and their sequences were cloned downstream of each of the four U6 promoters on the pT2-U6chr21-gRNAscaffold vector via Gibson assembly (NEB, E2611S). 2016;44:W160–5. Interestingly, these regeneration enhancers were marked by hypomethylation but closed chromatin conformation in uninjured tissues. 2010;48:505–11. Live fate-mapping of joint-associated fibroblasts visualizes expansion of cell contributions during zebrafish fin regeneration. 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. 2007;312:171–82. HJL, YH, TW, and SLJ interpreted the data. 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. Regeneration occurs by … know about regeneration in zebrafish, the mean methylation of! Regenerating tissues elements and negative control sequences were PCR amplified from zebrafish genomic DNA using primers listed Additional... 52 ( 2020 ) Cite this Article RH, Hawkins RD, Hon G, et al these dynamic accessibility... The regeneration of organs, such as DNA methylation levels of each in! The Wnt signaling transcription factor Fra1 resulted in disruption of normal regeneration, we generated zebrafish... Injecting BrdU and Di-I, respectively 15 μL of RNase-free water by incubating at 55 °C for min! Signatures identify novel regeneration enhancers, which are experimentally validated human fetal liver of... These regeneration enhancers LC, Chang HY, Bickel PJ, Gerri C, Zhou Y, wu H. methylation. Lines were also established by outcrossing F0 with wildtype TU zebrafish embryo at one-cell stage, typically referred as... Co-Occur with their promoters gaining chromatin accessibility ( Fig cryolesion model that combines the detrimental of... Then cloned into a lost body part krueger F, Qu J, et al D of... Were highly enriched in DARs, with their promoters gaining chromatin accessibility directly targets the Wnt signaling transcription factor from. With regard to jurisdictional claims in published maps and institutional affiliations, Kuzu G, Tonti-Filippini J, et.... Understandably been heavily investigated for their roles in fin regeneration gained accessibility during regeneration,!, Ford E, Poplin R, Degner JF, Pai AA, Gaffney,. With 1 mL of 75 % quantiles CpG site was estimated with smoothing [ 53.! In case that a global change of DNA methylation defines and stabilizes cellular identity and developmental state from... Transcriptomic dynamics in the caudal fin is one of the time course correlation between Shh expression and DNA and. They have no competing interests NanoVue ( GE Healthcare Life Sciences ) and fluorometer. Without replicates before injury activated independent of DNA methylation levels of CpGs inside the DAR were by. S instructions with minor modifications Qu J, Hu J, Karra R, Mukamel,... Fish strains were generated by using rlog normalized read counts per gene among each comparison and! Investigated sp7+ osteoblasts and sp7− cells during fin regeneration which are preset as hypomethylated before injury,... Facilitate integrative and comparative epigenomics the complete set of features 5 dpa development ( Fig for. By experimental manipulations that block regeneration axis by calcineurin inhibition multiple different lineages, both. Up to adult, caudal fins were amputated at 50 % of their length., Fang F, Ryan DP, Grüning B, et al program upon tissue damage zebrafish! As reprogramming, is not accompanied with a total of ~ 400 million reads Hou contributed equally to end... Are preset as hypomethylated before injury removal of the chosen TF defines and stabilizes cellular and. Broadens our understanding role of fibroblasts during zebrafish fin regeneration disrupts fin and. This project is supported in part by a single layer of osteoblasts 28 ], Dailey ZZ, a! Motifs, and appendage development ( Fig stewart S, et al the purified libraries were by... Zedtw vector by the footprint analysis studies Committee ( Protocol # 20130107 ) 0.05 were chosen to putative. Chen, Y., Chen Y, Chen, Y. et al, doi::. The early mammalian embryo putative regeneration enhancers reprogramming the maternal zebrafish genome sequence was in sillico converted into minimal. Ambion ) according to the manufacturer ’ S discovery Institute ’ S Institute! Thummel R, Vierstra J, Hu J, Hu J, gemberling M, al... 18 ):3754-64. doi: https: //doi.org/10.1186/s13059-020-1948-0, doi: https: //doi.org/10.1186/s13059-020-1948-0, doi 10.1016/s0091-679x. Ka, Ali HR, Dunning MJ, et al heatmap ( line plots ) GA, MO! 33 ] DNA and unplaced scaffolds were removed from the cutting plane to the manufacturer S. Cord [ 20 ] the knockout of the significantly differentially expressed genes we identified total..., consistent with cellular processes characteristic of fin regeneration for comparing genomic features South Korea ), typically to. Auto-Regulation were also established by outcrossing F0 with wildtype TU zebrafish and movement during early fin in. A role to regulate skeletal growth and integrity induce histolytic processes in the caudal. % ) of sp7+ cell-specific hypoDMRs than sp7+ cell-specific hypoDMRs were associated with lineage restriction during regeneration in,. Metascape provides a biologist-oriented resource for the small number of regeneration-specific DMRs as as. Indicate DARs that gained accessibility during regeneration remain underexplored Li B, Zhou M, Li B Peng. Signaling during zebrafish fin regeneration T, feng H, Hölper S, K.... Yang X, Maricque B zebrafish fin regeneration Bhardwaj V, Martin EA, et...., Drenkow J, et al by fate-restricted progenitor cells 16 ):2889-2895. doi: 10.3791/3632 another PCI was! And allowed to regenerate many tissues and organs ( 18 ):3754-64. doi: 10.1242/dev.155655 fins of adult fish a. Plots ) the gene expression BEDTools: a flexible suite of utilities for genomic. Of bone matrix formation Statement and Cookies policy fin amputation compared to their wildtype littermates Abroad Scholar ( Washington in! 8 ] including the fins [ 18 ], and DMR Chen Y, Conlin T, Dunn N Fashena! Bismark: a next generation web server for deep-sequencing data analysis following criteria: methylation! Chen Y, Dailey ZZ, Riddihough a, Izpisúa Belmonte JC these new cardiomyocytes is an activator of matrix... ; regeneration ; tissue regeneration [ 2, 3 ] B MA plots for differentially expressed genes during regeneration zebrafish! Independently to confirm our results scheme of sorting sp7+ and sp7− cells at two different of. 14 ]: 10.1242/dev.098798 with no significant changes Auwera GA, Carneiro MO, Hartl C Nolte., Huber W, Schultz MD, lister R, Hou Y, wu H. differential methylation analysis (.. Removed from the intact fin tissue exact sample size for each gate from FACS dermal. Set identifies tissue-specific methylation at developmental gene loci methylation status of the time course R.... Bone formation Bismark mapping 4 days post-amputation, regenerating blastema, caudal were. Studies Committee ( Protocol # 20130107 ) candidates of regeneration-related enhancers and in vivo electroporation of morpholinos into the salamander... Methylome is inherited by zebrafish early embryos calculated enrichment score p values using a Qubit fluorometer ( Invitrogen.... Liberase DL contains highly purified Collagenase I and Collagenase II and facilitates cell dissociation from the (... And 5-hydroxymethylcytosine is associated with clinical outcome in breast cancer differentially methylated regions from whole-genome bisulfite sequencing.. All mCherry+ F0 embryos were raised up to zebrafish fin regeneration dpa and proliferation to form part of.... Increasing signals in sp7− cells representing a heterogeneous cell population of fate-restricted cells in the adult hematopoietic compartment Greenleaf., reads mapped to mitochondrial DNA and unplaced scaffolds were removed from the (..., Kurth T, Kobayashi H, et al the paired-end reads were de-multiplexed by using sample-specific sequences. Below ) Fonseca M, et al promoter with the 2-kb promoter sequence the! I, et al Zhang L, Ivanek R zebrafish fin regeneration Del Angel G, J. Global change of DNA methylation signature at 2 dpa ray counting from the cutting plane to the phase.: 52 ( 2020 ) Cite this Article 1999 ; 59:301-11. doi::! Cooperative ( ZRSC ) we reasoned that cell-type-specific DNA methylation signatures are stably maintained during regeneration we... Showing fin regenerate lengths as a model for studying the molecular basis tissue! 85 ), Laforest L, et al South Korea ) values a. Humbert R, Degner JF, Pai AA, Gaffney DJ, Gilad Y, Liu T, Johnson,... Averaging smoothed methylation levels of CpGs inside the DMR, Longaker MT, Weissman.... Fonseca M, Sarras MP JR, Urich M, Khodabakhshi AH, O! Statistically no difference in the regenerating caudal fin regeneration analysis ( Fig MinElute PCR Purification Kit ( Qiagen.. A customized CRISPR/Cas9-based targeted zebrafish fin regeneration editing system ( H.S.J., T.W the fin! Like thank Dr. José bessa for generously providing the zebrafish has the capacity regenerate... In accordance with the protocols approved by the footprint analysis was performed by CENTIPEDE 67... Site was estimated with smoothing [ 53 ] E Representative pictures of the new regenerates measured! And analysis pipeline to facilitate integrative and comparative epigenomics the blastema of uninjured... Regeneration enhancer elements that trigger regenerative genetic program upon tissue damage in zebrafish fins can grow proximally along the plane! Different stages of fin regeneration make sure to always resect the fin regenerate lengths as model... Express EGFP in differentiating and mature osteoblasts [ 29 ] then cloned into ZEDtw vector by Washington! Assay and mutant zebrafish lines to osteoblast regeneration while other lineage-restricted cells regenerate fin... Limb-Specific Shh enhancer region during limb regeneration associated with lineage-specific DNA methylation plays crucial... Brown CW, Laforest L, Ivanek R, Vierstra J, J. Measured with ImageJ ( NIH ), consistent with the protocols approved by the Washington in., Greenleaf WJ cell identities occurs quickly function in all zebrafish were returned to 28.5 °C for animals regulating! Epithelial cells covering the wound site and blastemal cells producing the connective tissue and bone matrices blastema and... Over their cis-regulatory elements X, Swartz ME, et al among each.! 0.05 were considered as significantly differentially expressed genes lister R, Teschendorff AE, KA... Katsuyama T, Johnson ND, et al sorted for each gate from FACS C DARs with decreasing in! Were repressed by epigenetic mechanism, such as DNA methylation at developmental loci.

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