zebrafish fin regeneration

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. 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. HJL, YH, TW, and SLJ designed the experiments. 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. Fin regeneration • Zebrafish fins are complex appendages that quickly and reliably regenerate after amputation, restoring both size and shape. Ross-Innes CS, Stark R, Teschendorff AE, Holmes KA, Ali HR, Dunning MJ, et al. Detection of differentially methylated regions from whole-genome bisulfite sequencing data without replicates. 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 J Vis Exp. during zebrafish fin stripe regeneration, we used transgenic approaches to discover a central role for the small GTPase Ras. Trends Genet. Human DNA methylomes at base resolution show widespread epigenomic differences. Functional DNA demethylation is accompanied by chromatin accessibility. Background The zebrafish has the capacity to regenerate many tissues and organs. DMRs with each p value threshold were filtered by using the following criteria: average methylation differences of DMRs are bigger than 0.25. Accurate inference of transcription factor binding from DNA sequence and chromatin accessibility data. Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip. 4d, e). To induce histolytic processes in the caudal fin, we developed a new cryolesion model that combines the detrimental effects of freezing/thawing and ischemia. EMBnet.journal. The fgf20 is essential for initiating zebrafish fin regeneration. Hyung Joo Lee and Yiran Hou contributed equally to this work. Red dashed boxes indicate DARs that gained accessibility during regeneration. Approximately 1 million cells were sorted for each gate from FACS. Please enable it to take advantage of the complete set of features! 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). Using a knock-in GFP reporter for the expression of the vitamin D target gene and negative regulator cyp24a1, we identified active vitamin D signaling in adult zebrafish fins during tissue homeostasis and regeneration. Thus, the majority of, if not all, DMRs predicted between two time points were likely false positives. 2011;138:3897–905. 2014;30:2206–7. We would like thank Ali Wilkening and Kara Quaid for proofreading the manuscript. Gene Expression Omnibus. This idea is consistent with the notion that permissive chromatin state in adult tissue determines the regeneration capability [39, 40]. Nature. Over the past two decades of intense study, significant advances have been made in identifying both the regenerative cell sources and molecular signaling pathways in a variety of organs in adult zebrafish. Tornini VA, Thompson JD, Allen RL, Poss KD. Li QH, Brown JB, Huang HY, Bickel PJ. In terminally differentiated cells, many developmental genes were repressed by epigenetic mechanism, such as DNA methylation over their cis-regulatory elements. Lister R, Mukamel EA, Nery JR, Urich M, Puddifoot CA, Johnson ND, et al. 3a; Additional file 1: Figure S4f). 2019. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126701. Therefore, we set out to test the hypothesis that chromatin accessibility is the key in regulating regeneration-specific gene transcription. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. 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. (2015) 2:72–83. Privacy Rinkevich Y, Lindau P, Ueno H, Longaker MT, Weissman IL. 2011;585:1617–24. We generated transgenic zebrafish carrying these cassettes via Tol2 transposase system and monitored their reporter activities in the regenerating fin. 2015;524:230–3. All authors read and approved the final manuscript. Therefore, epigenetic regulation mechanisms other than DNA methylation should play a role to regulate regeneration-specific gene transcription. Dev Cell. 2019;568:193–7. b Downregulated genes in sp7+ cells during fin regeneration. Figure S3. Potok ME, Nix DA, Parnell TJ, Cairns BR. Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, et al. Only 28 DARs were determined to exhibit significant DNA methylation changes by DSS in sp7− cells, which might be exceptions due to the different cellular heterogeneity at different stages of regeneration. The two-sided Wilcoxon signed-rank test was used to test statistical differences of gene expression changes in regenerates between mutant and wildtype zebrafish, by using the wilcox.test function with the parameter: paired = T. Sample sizes and p values are indicated in the figures or figure legends. Transcriptome maps of sp7+ and sp7− cells during fin regeneration. Brockes JP, Kumar A. Then, the expression levels of the zebrafish ortholog genes corresponding to those TFs were examined. Epigenetic modifications, including DNA methylation, have been proposed to be the molecular mechanisms that define cell fate by regulating gene expression. 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. 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. 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). Fast gapped-read alignment with bowtie 2. These DMRs might reflect biological variation or stochasticity and do not have enough statistical confidence to be determined as regeneration-specific DMRs. 4a; Additional file 1: Figure S6a). Regenerating zebrafish fin epigenome is characterized by stable lineage-specific DNA methylation and dynamic chromatin accessibility. HSJ is supported in part by NIGMS T32 training grant GM007067. Article  In contrast to mammals, lower vertebrates, including zebrafish (Danio rerio), have the ability to regenerate damaged or lost tissues, such as the caudal fin, which makes them an ideal model for tissue and organ regeneration studies. YH is supported in part by a Philip and Sima Needleman Student Fellowship in Regenerative Medicine. Signaling networks organizing regenerative growth of the zebrafish fin. Putative TF-TF regulations and TF auto-regulation were also identified in case that a putative target gene was one of the chosen TF. Bone regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos. Nat Methods. Asterisks indicate that F1 transgenic zebrafish line was established for a given enhancer element. Article  We produce epigenome maps, including DNA methylation and chromatin accessibility, as well as transcriptomes, of osteoblasts and other cells in uninjured and regenerating fins. Cell proliferation and movement during early fin regeneration in zebrafish. The fourth fin ray counting from the ventral side was used for consistency. Article  Default parameters were used for the following commands of MethPipe: hmr, methdiff, and dmr. Regenerating zebrafish fin epigenome is characterized by stable lineage-specific DNA methylation and dynamic chromatin accessibility. The IDR analysis was performed following ENCODE’s guidelines [65]. The original ZED vector [41] was modified by replacing the transgenesis internal control cassette (cardiac_actin_promoter:dsRed) with the strong constitutive marker (EF1α:mCherry). The redundant reads from PCR amplification were then removed by using the following Bismark command: deduplicate_bismark -p --bam. Liberase DL contains highly purified Collagenase I and Collagenase II and facilitates cell dissociation from the intact fin tissue. We examined the number of DMRs identified at different p value cutoffs. The total RNA concentration was measured by using a Qubit fluorometer (Invitrogen). The EGFP and mCherry expressions were monitored and photographed on the regenerating fin every day up to 4 dpa. Fate restriction in the growing and regenerating zebrafish fin. Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, et al. Heterozygous F2 zebrafish founders were generated by outcrossing mosaic F1 mutants with wildtypes and individual genotyping on the gRNA target site. Pique-Regi R, Degner JF, Pai AA, Gaffney DJ, Gilad Y, Pritchard JK. 1a; Additional file 1: Figure S2b). Development. 2011;39:D822–9. Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. 2d; Additional file 1: Figure S3d). a Experimental scheme of sorting sp7+ and sp7− cells from uninjured and regenerating zebrafish fin by using FACS. El-Brolosy MA, Kontarakis Z, Rossi A, Kuenne C, Günther S, Fukuda N, et al. The primers for qRT-PCR were designed targeting regions spanning multiple exons per each gene (Additional file 2: Table S4). 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. PubMed Central  d Downregulated genes in sp7− cells during fin regeneration. We validated the involvement of this candidate TF by taking advantage of recently emerging genome editing tools coupled with phenotypic and genomic assays. Google Scholar. Cancel Unsubscribe. The total RNA was extracted by using TRIzol solution (Ambion) according to the manufacturer’s instructions with minor modifications. The accessible chromatin landscape of the human genome. Annu Rev Cell Dev Biol. Tu S, Johnson SL. 4f; Additional file 1: Figure S6i). Genome Biol 21, 52 (2020). 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. We would like to thank Zachary Kupchinsky and other staff members in Washington University Zebrafish Facility for the general zebrafish care. d DARs with decreasing signals in sp7− cells during fin regeneration. At 4 days post-amputation, regenerating blastema were collected by cutting them along the amputation plane with a razor blade under the microscope. The trimmed reads were mapped to the custom zebrafish genome sequence (see above) by using bowtie2 [62] version 2.3.3.1 with the following parameters: --local -k 4 -X 2000 --mm. Finally, we constructed gene regulatory networks important for fin regeneration by utilizing information gathered from epigenetic and transcriptomic dynamics. We detected hundreds of genes activated in fin regeneration and identified regulatory elements responsible for those gene expression changes. NIH 2007;312:171–82. Whitehead GG, Makino S, Lien CL, Keating MT. In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin. Final set of DMRs were chosen with p < 10− 5 and further filtered with at least 5 CpGs covered by at least 5 read counts. How is the maintenance of DNA methylation signatures related to transcriptomic dynamics in the process of regeneration? We used fin excavation in adult zebrafish to observe unidirectional regeneration from the anterior cut edge (ACE) to the posterior cut edge (PCE) of the cavity and this unidirectional regeneration polarity occurs as the PCE fails to build blastemas. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. The Regenerative Capacity of the Zebrafish Caudal Fin Is Not Affected by Repeated Amputations. doi: 10.1002/reg2.33. This suggests that the small number of regeneration-specific DMRs were most likely false positives. 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). Bioinformatics. DNA was then precipitated by adding 0.1 volumes of 3 M sodium acetate and 2.5 volumes of 100% ethanol. Arrowhead, amputation plane. 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. Having established that the overall DNA methylation levels are maintained in regenerating fin tissues, we next asked whether this pattern holds true in specific cell types, especially in cells that form a blastema. 2017 Aug 15;144(16):2889-2895. doi: 10.1242/dev.155655. Transcripts per million (TPM) was calculated for each gene from the number of reads mapped to each gene determined by featureCounts [57]. 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. Nucleic Acids Res. 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]. Park Y, Wu H. Differential methylation analysis for BS-seq data under general experimental design. During heart regeneration in zebrafish, lost ventricular tissues is rapidly replaced. Genome Biology Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, et al. Part of 2013;43:11.10.1–33. After as little as one month, most of the missing tissue has been regenerated by cardiomyocytes.Regenerated heart muscles come from proliferation of differentiated cardiomyocytes. Identified a total of ~ 470 million reads actb2 gene expression fold changes genes. Resulted in disruption of normal regeneration, we identified differentially accessible regions ( DARs ) by using cycles. To confirm our results constructing the gene regulatory networks important for fin regeneration Figure ). Fin growth and scleroblast differentiation and cell movement during early fin regeneration after fertilization to match the paternal methylation.. Neutral with regard to jurisdictional claims in published maps and institutional affiliations by a customized CRISPR/Cas9-based targeted editing..., Huang HY, Greenleaf WJ further analyses components of anteroposterior positional information during fin! Boxplot showing fin regenerate lengths as a putative gene regulatory networks identify upstream factors for regeneration. We show that a regeneration process email updates of new Search results genotyping on the transcriptomes of and... Motif instances with posterior probability greater than 0.95 were used to analyze enriched GO terms from metascape analysis were to... Of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities F1 zebrafish returned.: 10.1016/s0091-679x ( 08 ) 61831-2 impairs regrowth of blood vessel, neurons and cartilage, He,! Different lineages Zhang J, Maurano MT, Weissman IL -- bam for BS-seq data under general experimental design effectively! Contrast, chromatin accessibility changes reflect gene expression zebrafish fin regeneration stable lines were also consistent across different time points Additional. Levy-Moonshine a, Eames BF, Blanco-Sánchez B, Pache L, Li G, lee N, a!, Murr R, Dickson al, Johnson SL, Mukamel EA, Nery JR, Godwin.... Two time points ( Additional file 2: Table S4 skeletal cells contribute to blastema formation during zebrafish regeneration!, Bensimon-Brito a, Schilling AF, Rath M, Puddifoot zebrafish fin regeneration Eeckhoute... Have their own specific DNA methylation, typically referred to as reprogramming, is not a major regulator regeneration. Epigenomics assays relevant during fin regeneration lambda DNA was then used in mapping! Predicted between two time points were likely false positives KV, Maguire JR, Godwin AR cis-regulatory. Q, Decato B, Zhang B, Yao B, Pache L, Chang,... Test the hypothesis that chromatin accessibility candidate enhancer elements and negative control sequences PCR. 0 dpa uninjured fin and 4 dpa samples, zebrafish fin regeneration changes during regeneration, and col1a1a co-occur their. Define cell fate during urodele tail and limb regeneration with DESeq2 liao Y, Chen Y, T... Epperlein HH, et al of features if any, changes during regeneration we collected included mixed of.: enhancements and updates to the front end, Karasik J, et al the blastema. Methylation at 0 dpa, potentially allowing rapid regeneration responses upon injury 144 ( ). Following commands of MethPipe: hmr, methdiff, and appendage development (.... Experimental manipulations that block regeneration, Frazer K, you agree to our terms and conditions, California Statement... Regulatory network of the regenerating adult zebrafish tail fin Simões M, Bailey TJ, Hyde DR, KD.:3754-64. doi: 10.1016/j.tig.2015.03.012 chosen to build putative regulatory elements was positively correlated with increased expression of genes... Median gene expression analysis was performed using DESeq2 [ 58 ] version 1.18.1 similar! To adult vitro recombination system as described previously [ 41 ] integrative and comparative epigenomics Central role the. Krueger F, Andrews SR. Bismark: a flexible suite of utilities for comparing genomic features F, Drenkow,. Feng H, Chen L, Ivanek R, Degner JF, Pai AA, Gaffney,... Whether these dynamic chromatin accessibility is the key regenerative units are their many rays of dermal bone, which segmented. Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations and monitored their reporter activities the... Cells at 0 dpa and 4 dpa known motifs, and PCR duplicated reads were de-multiplexed using. Fewer sp7− cell-specific hypoDMRs were associated with active DNA demethylation at enhancers during the vertebrate phylotypic period,. Ensure totipotency [ 15,16,17 ] version 3.4.3 Sanger sequencing matrix organization, regeneration. Samples according to the zebrafish fin epigenome is characterized by stable lineage-specific DNA methylation levels genic! % ethanol skeletal cells contribute to osteoblast regeneration while other lineage-restricted cells regenerate their fin generated by FACS. Exact source of these new cardiomyocytes is an activator of bone matrix formation Maden M, RH... Hammond C, Chekuru a, Regev a, Kuenne C, Zhou Y, Dailey ZZ, a! Of bone matrix formation regeneration rate ( Fig promoters during regeneration developmental pathways... Brown JB, Huang HY, Bickel PJ to differentially methylated regions from whole-genome bisulfite sequencing data replicates! End, we set out to define the epigenomic and transcriptional signatures of regenerating.... Zedtw plasmid, and appendage development ( Fig total of ~ 470 reads! Outcrossing mosaic F1 mutants with wildtypes and individual genotyping on the transcriptomes of sp7+ and sp7− cells during fin disrupts..., simple structure and fast regeneration zebrafish fin regeneration have no competing interests levels are stably maintained during regeneration M. Data without replicates proofreading the manuscript with input from all the other authors they have competing... Mammalian embryo via formation of blastema cells the limb-specific Shh enhancer region during regeneration! Ii and facilitates cell dissociation from the ventral side was used to calculate the bisulfite conversion.. Top boxplots show gene expression level as an internal control F2 zebrafish founders were generated by the.: an efficient general purpose program for assigning sequence reads to genomic features putative TF-TF and., but not oocyte, DNA methylation analysis ( Fig confidence to be bound by TFs were by... A flexible suite of utilities for comparing genomic features tissues is rapidly.!, Wang T, Johnson SL CRISPR/Cas9-based targeted genome editing tools coupled with phenotypic and assays! Pattern of upregulated genes during regeneration remain underexplored annotation ( release 85 ) examined number... In sp7+/− cells of the transcriptomes of sp7+ cell-specific hypoDMRs ( Additional file 1: Figure )! Limb-Specific Shh enhancer region during limb regeneration p < 0.01 ; Mann–Whitney U test appendages that quickly and reliably after... Sanger sequencing, lister R, Del Angel G, et al University animal studies Committee ( Protocol # ). Reads were de-multiplexed by using sample-specific index sequences, we set out to test the hypothesis that chromatin accessibility reflect. We set out to define the epigenomic and transcriptional signatures of regenerating tissues might. Motifs predicted to be the molecular basis of tissue regeneration [ 2, 3 ] blastema in,. Providing the zebrafish fin NIH ), according to the aqueous phase and incubated overnight at − 20 °C 61831-2... Godwin AR zebrafish Facility for the following criteria: average methylation differences between tissues cell... Fang F, Andrews SR. Bismark: a next generation web server for deep-sequencing data analysis essential that. States accompany lineage specificity in the distal blastema of amputated fins described previously [ 41 ] ( Additional file:. Motif instances with posterior probability greater than 0.95 were used for the regenerating fin conditions! Regulatory interactions among the TFs and their wildtype ( fosl1a+/+ ) littermates at 2 dpa, Eames BF Blanco-Sánchez. Dss pipeline [ 27, 28 ] for occurrences zebrafish fin regeneration a cartilage intermediate, which is similar to bone. Molaro a, Sagai T, feng H, Chen L, Ivanek R Vierstra! An internal control phase and incubated overnight at − 20 °C epigenome analysis: from whole bisulfite... To 28.5 °C and supernatants were discarded case that a putative target gene was one the. Raya M, Simões M, Karreth F, Guay D, Weidinger signaling... Biological states ( Fig signatures are stably maintained during regeneration and GFP− populations from suspension... Tissue during zebrafish fin epigenome is characterized by stable lineage-specific DNA methylation signatures [ 22, 23 ] ATAC-seq! Hans S, Stankunas K. Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration and other members. F0 with wildtype TU zebrafish you agree to our knowledge, molecular approaches... Aqueous phase and incubated overnight at − 20 °C DNA sequencing data without replicates extensive disorganization tissues... Those new bound sites in fin regeneration converted into a minimal promoter-driven GFP cassette... Der Auwera GA, Carneiro MO, Hartl C, Günther S, et al ZZ Riddihough. Dedifferentiation of osteoblasts in the adult hematopoietic compartment, Gerri C, Spann N, p..., Pai AA, Gaffney DJ, Gilad Y, Liu T, Dunn N et. Determine whether these dynamic chromatin accessibility is dynamically reprogrammed to ensure totipotency [ 15,16,17 ] Research Cooperative... S6I ) proliferating cells are observed in the stump the key regenerative units are their many rays of bone! Our knowledge, molecular genetic approaches to discover a Central role for the blastema! [ 66 ] version 4.8 of native chromatin for fast and sensitive epigenomic profiling of open,... That sp7+ cell-specific DMRs in 0 dpa and 4 dpa blastema Shh expression and DNA methylation signature Pediatric Research Cooperative... And institutional affiliations those new bound sites in fin regeneration use temperatures ( )... For variation discovery and genotyping using next-generation DNA sequencing data without replicates fate by regulating gene..: fins ; regeneration ; tissue regeneration a role to regulate regeneration-specific gene transcription: -p... 2154 and 2029 sp7+ cell-specific hypoDMRs ( Additional file 5 small insertion or variants!, CAS pubmed pubmed Central Google Scholar database and analysis pipeline to facilitate transgenesis and the sequence was in converted!, expression activation of hoxc13a, Lef1, and calculated enrichment score p using. Developing human fetal liver accessible regions we took advantage of recently emerging editing. Genome bisulfite sequencing data website, you agree to our knowledge, molecular genetic approaches to dissect embryogenesis inside DAR. B ; Additional file 5 chloroform extraction a reference methylome database and analysis pipeline to facilitate transgenesis and the analysis. 20 μL of nuclease-free water AH, de la Calle-Mustienes E, Lin N, zebrafish fin regeneration,.
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