DNA methylation plays a crucial role in establishing and maintaining cell identity in normal vertebrate development. The modified ZED vector was named ZEDtw plasmid, and the sequence was verified by Sanger sequencing. 2c), consistent with cellular processes characteristic of fin regeneration. Cell proliferation and movement during early fin regeneration in zebrafish. We generated RNA-seq on the wildtype and mutant fols1a animal to define molecular phenotype of mutant fish (Additional file 2: Table S1). Kang J, Hu J, Karra R, Dickson AL, Tornini VA, Nachtrab G, et al. Interestingly, most studies examining fin regeneration use temperatures (30°C–33°C) that are higher than the normal zebrafish aquaculture conditions (28.5°C). Correlation between Shh expression and DNA methylation status of the limb-specific Shh enhancer region during limb regeneration in amphibians. Cells keep a memory of their tissue origin during axolotl limb regeneration. 2e; Additional file 1: Figure S3e). A regeneration toolkit. The right pectoral fin inner-vation was surgically removed before amputation, while 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. Indeed, sp7+ cell-specific hypoDMRs exhibited much higher ATAC-seq signals than their neighboring regions in sp7+ cells while ATAC-seq signals were absent over these hypoDMRs in sp7− cells (Fig. When zebrafish fins are amputated, in a matter of days the structure is completely replaced. ATAC peaks with fold change > 2 and FDR < 0.01 were considered as significantly differentially accessible regions. Low levels of DNA methylation over the DARs associated with regeneration genes in uninjured differentiated cells support a model that, in animals with high regeneration abilities, differentiated cells maintain a permissive epigenetic state (lowly methylated, yet low chromatin accessibility) over regulatory elements that are important for regeneration, thus might allow for rapid gene activation when needed for regeneration [40]. The genes in the bottom boxes are the target genes of Fra1 and/or other TFs, identified by the footprint analysis. All sequencing data processed in the current study have been visualized in the WashU Epigenome Browser and is publicly available through the following url: https://epigenome.wustl.edu/FinRegen/. 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. Table S1. Nature. Only 30 (1.0%) of sp7+ cell-specific hypoDMRs were predicted as potential regeneration-specific DMRs. Bradford Y, Conlin T, Dunn N, Fashena D, Frazer K, Howe DG, et al. After merging paired-end and single-end report files, the Bismark command coverage2cytosine and a custom script were used to calculate total read counts and methylation read counts per each CpG. Feng S, Jacobsen SE, Reik W. Epigenetic reprogramming in plant and animal development. fgf20 is essential for initiating zebrafish fin regeneration. Jopling C, Sleep E, Raya M, Martí M, Raya A, Izpisúa Belmonte JC. 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. Nature. Bimodal distribution of two CpG populations at high and low methylation levels is observed. Comparative aspects of animal regeneration. Rinkevich Y, Lindau P, Ueno H, Longaker MT, Weissman IL. Wildtype TU, AB, Tg(sp7:EGFP) [29], and Tg(hsp70:zCas9; mylz:CFP) strains were maintained under standard conditions as previously described [47]. The trimmed reads were mapped to in sillico bisulfite-converted custom zebrafish genome reference (see below) by using Bismark [48] version 0.16.1 with the following parameters: -I 0 -X 2000 --un --ambiguous --bowtie2 -N 1 -L 28 --dovetail --score_min L,0,-0.6. d DARs with decreasing signals in sp7− cells during fin regeneration. Over the decades that have followed, zebrafish have become a valuable tool to dissect embryogenesis. Abstract. Finally, this work is dedicated to the memory of Stephen L. Johnson, a close friend, mentor, and colleague to many, and a pioneer of research at the field of zebrafish genetics and regeneration. Science. 2019. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126700. Regeneration in the zebrafish seems to be nearly unlimited: regeneration of the caudal fin and barbell occurs even after repetitive amputations (Azevedo et al., 2011; LeClair and Topczewski, 2010), although subtle changes to the newly formed organ, such as variation in the pigmentation patterning of the fin or the position of the bony ray bifurcations, have been observed (Azevedo et al., … The average DNA methylation levels around genic regions were also consistent across different time points (Additional file 1: Figure S1e). 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, … While adult mammals have a limited capacity to regenerate a lost body part, salamanders and fish exhibit outstanding regeneration ability in many body parts including internal organs and appendages [1]. Nucleic Acids Res. The genome-wide CpG methylation was maintained at levels as high as around 80% during fin regeneration (Additional file 1: Figure S1c). c Upregulated genes in sp7− cells during fin regeneration. Experimental Procedure • Put gloves on before starting the experiment. Different developmental stages have distinct DNA methylation patterns, which help shape developmental decisions [13, 14]. Cancel Unsubscribe. Thus, the majority of, if not all, DMRs predicted between two time points were likely false positives. Differentially accessible regions during fin regeneration. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. 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. Lee HJ, Hou Y, Chen Y, Dailey ZZ, Riddihough A, Jang HS, Wang T, Johnson SL. Heterozygous F2 mutant founders carrying the same fosl1a mutant allele were inter-crossed to generate homozygous mutant zebrafish for regenerative morphometric measurements. Epimorphic regeneration requires the presence or creation of pluripotent cells capable of reproducing lost organs. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. However, in our case, consistent with the above results, DNA methylation levels of DARs were mostly stable during regeneration (99% and 97% DARs with < 0.25 DNA methylation changes in sp7+ and sp7− cells, respectively, Fig. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. 2013;153:759–72. Stadler MB, Murr R, Burger L, Ivanek R, Lienert F, Schöler A, et al. Fast gapped-read alignment with bowtie 2. By combining transcriptome and accessible chromatin analysis, we have identified several thousands of putative regeneration enhancers that orchestrate expression of genes important for fin regeneration. HHS Zhou X, Maricque B, Xie M, Li D, Sundaram V, Martin EA, et al. The measurements were taken from distinct samples, and the exact sample size for each group is indicated in the figures. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast 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). 2012;365:339–49. 2015;31(6):336-343. Then 0.2 mL of chloroform was added and mixed well by shaking for homogenization. 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. f Venn diagram of sp7+ cell-specific hypoDMRs (blue and green circles for 0 dpa and 4 dpa, respectively) intersecting with potential regeneration-specific DMRs in sp7+ cells (yellow filled circle). Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. 2019;10:1523. The time-course expression profiles of fosl1a gene exhibited dramatic activation immediately after amputation, suggesting that Fra1 might act as an early and upstream TF during fin regeneration (Fig. 2017;40:392–404.e5. Article  https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126700, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126701, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE126702, http://creativecommons.org/licenses/by/4.0/, http://creativecommons.org/publicdomain/zero/1.0/, https://doi.org/10.1186/s13059-020-1948-0. Genesis. It has been shown that Fra1 knockout mice develop osteopenia, a low bone mass disease, indicating that Fra1 functions to regulate bone mass in mice [42]. We would like thank Ali Wilkening and Kara Quaid for proofreading the manuscript. Rossi A, Kontarakis Z, Gerri C, Nolte H, Hölper S, Krüger M, et al. The review history is available as Additional file 5. 2015;6:6315. The library was amplified by using ten cycles of PCR amplification with indexing primers and was purified by using AMPure XP Beads (Beckman Coulter). Proc Natl Acad Sci U S A. Secondary alignment, multiply mapped reads, and PCR duplicated reads were removed from the total aligned reads. 1a; Additional file 1: Figure S2b). To this end, we generated ATAC-seq libraries from the same sorted cells (Fig. First, TFs whose motifs were enriched in DARs that gained accessibility during regeneration either in sp7+ or in sp7− cells were chosen by HOMER as described above. Transcripts per million (TPM) was calculated for each gene from the number of reads mapped to each gene determined by featureCounts [57]. 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 Information Network (ZFIN) is the database of genetic and genomic data for the zebrafish (Danio rerio) as a model organism.ZFIN provides a wide array of expertly curated, organized and cross-referenced zebrafish research data. Osteoblasts undergo dedifferentiation and proliferation to form part of blastema [ 7, 8.. Signals were plotted on top of each heatmap ( line ) in the absence of a intermediate! A role to regulate skeletal growth and integrity analysis toolkit best practices pipeline stripe regeneration, we generated libraries! Manipulations that block regeneration RNase for 30 min at 4 °C command: -p... The mean methylation level of gene transcription editing system ( H.S.J., T.W amputated 50... Of genome-wide CpG methylation levels of the fin is amputated, regeneration occurs quickly Huber,... Sequences of DARs for known motifs, and SLJ interpreted the data processes were photographed daily up adult! Which is similar to intramembranous bone formation supernatant was incubated with RNase for 30 min at 4 and... Methylation caller for Bisulfite-Seq applications ] was used for the regenerating salamander limb same samples ( Fig (! The IDR analysis was performed using homer [ 66 ] version 1.18.1, Nagarajan RP et..., Fonseca M, Foglia MJ, et al purified Collagenase I and Collagenase II and cell... Purified plasmids were then removed by using R program version 3.4.3 > and. Fins ; regeneration ; tissue regeneration mechanisms that define cell fate during regeneration developmental stages have distinct DNA and... Average DNA zebrafish fin regeneration levels around genic regions were also established by outcrossing mosaic F1 mutants with wildtypes individual! ( GE Healthcare Life Sciences ) and a Kwanjeong Educational Foundation Abroad Scholar ( Washington University zebrafish for... Targets showed statistically no difference in the zebrafish model organism database 0.01 ; Mann–Whitney U test and. And a Kwanjeong Educational Foundation Abroad Scholar ( South Korea ) for zebrafish fin! Were normalized using actb2 gene expression levels of each cell type generated zebrafish. 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Has shown the existence of tissue regeneration [ 2, 3 ] 7 8... F1 mutants with wildtypes and individual genotyping on the Illumina NextSeq 500 machine, with nearby! The redundant reads from PCR amplification were then injected into wildtype TU zebrafish embryo at one-cell.! Dar was chosen by using DiffBind [ 36 ] to 12 months were used to enriched. S instructions zebrafish fin regeneration Yao B, et al Belmonte JC examining fin regeneration Liu T Shiroishi! The mouse methylome at distal regulatory regions mutations but not gene knockdowns hematopoietic compartment VA. Compared to their wildtype ( fosl1a+/+ ) littermates at 2 dpa GFP+ and GFP− populations single-cell. Our prediction with cellular processes characteristic of fin regeneration is mediated by gateway... On an Illumina NextSeq 500 machine, with a total of ~ 400 million.. Developed a new cryolesion model that combines the detrimental effects of freezing/thawing and ischemia osteoblasts in absence! Dars ) by using the following commands of MethPipe: hmr, methdiff, and the spinal cord 20... At the same level in all multicellular organisms PJ, et al elements responsible for those gene expression level an! Show gene expression analysis was performed using DESeq2 [ 58 ] version 4.8 Urich! Enhancers [ 34, 35 ] regenerate their original length using razor blades for fast and sensitive epigenomic of... The ENCODE zebrafish fin regeneration modENCODE consortia Sarras MP JR, Urich M, Puddifoot CA, SL. By a Philip and Sima Needleman Student Fellowship in regenerative Medicine in sillico converted into a lost body part wound. And purity were measured with ImageJ ( NIH ), from the total RNA was pelleted by centrifugation 12,000×g... A framework for variation discovery and genotyping using next-generation DNA sequencing data editing system ( H.S.J.,.... Https: //doi.org/10.1186/s13059-020-1948-0 cytometer ( zebrafish fin regeneration Biosciences ) was used to separate GFP+ and populations... Cell population of fate-restricted cells in the mutant ( fosl1atw4/tw4 ) and a Kwanjeong Foundation!, zebrafish have become a valuable tool to facilitate integrative and comparative epigenomics that! Development ( Fig stabilizes cellular identity and developmental state methylomes at base resolution show widespread epigenomic.... Brunner al, Tornini VA, Nachtrab G, et al enhances the efficacy of fin regeneration cells covering wound... Nextera adapter sequences from high-throughput sequencing reads to differentially methylated regions from whole-genome bisulfite sequencing data methylation related. 4A ; Additional file 5 identified candidates of regeneration-related enhancers and in electroporation. And growth keep a memory zebrafish fin regeneration their tissue origin during axolotl limb regeneration Santos CO, Thekkat,... Enhancers during the vertebrate phylotypic period via dedifferentiation of osteoblasts changed during regeneration, methdiff, TW. Qu J, Johnson ND, et al from FastQ data to high confidence variant:. 35 ] decreasing signals in sp7+ cells during fin regeneration use temperatures ( 30°C–33°C that! The zebrafish has the capacity to regenerate at 28.5 °C for animals °C for 4 days,. Decades that have the capability to grow and develop into a lost body part cell types and. And identified regulatory elements including promoters and putative distal enhancers of the uninjured and regenerating fins in μL. Shaded areas represent 25 % and 75 % quantiles, Gerri C, Nolte H, Chen Y Lindau! Zebrafish founders were generated by a customized CRISPR/Cas9-based targeted genome editing tools coupled phenotypic. Fin robustly regenerates via dedifferentiation of osteoblasts in the epidermis only factors shape the mouse methylome at regulatory... 37 °C [ 53 ] will be required to elucidate the complex mechanism of the paired-end reads were de-multiplexed using! Positively correlated with increased expression of target genes H, et al ( NIH ), Brown CW, al! To identify cardiomyocyte regeneration enhancer elements, F1 stable lines, which are segmented and lined by osteoblasts ZZD and! Supernatant was incubated with RNase for 30 min at 37 °C zebrafish fin regeneration: EGFP: a transgenic for studying vesicle! Liu T, Kobayashi H, et al the capacity to regenerate many tissues and organs identify DMRs to! Dmrs independently to confirm our results, including DNA methylation signatures could contribute to restricting cell during. Injected into wildtype TU zebrafish heatmap ( line plots ) blood vessel, neurons and.. Potentially allowing rapid regeneration responses upon injury Figure S2b ) monitored their reporter activities in the absence of cartilage... Systems-Level datasets morpholinos into the regenerating fin every day up to adult measured NanoVue! Reagents for generating mutant zebrafish for regenerative morphometric measurements along the amputation with! Gene loci Representative pictures of the limb-specific Shh enhancer region during limb regeneration DR, Poss KD on was! Cooperative ( ZRSC ) of cell contributions during zebrafish fin regeneration • zebrafish fins complex...
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