==== Front eLife Elife eLife eLife 2050-084X eLife Sciences Publications, Ltd 32142407 51808-80-4776/eLife.51217 Research Article Developmental Biology Stem Cells and Regenerative Medicine Eya2 promotes cell cycle progression by regulating DNA damage response during vertebrate limb regeneration Sousounis Konstantinos 12 Bryant Donald M 1 Martinez Fernandez Jose 1 Eddy Samuel S 3 Tsai Stephanie L http://orcid.org/0000-0001-7549-341814 Gundberg Gregory C 12 Han Jihee 1 Courtemanche Katharine 1 Levin Michael http://orcid.org/0000-0001-7292-808425 Whited Jessica L https://dawn42@example.org 1 Department of Stem Cell and Regenerative Biology, Harvard UniversityCambridgeUnited States 2 The Allen Discovery Center at Tufts UniversityMedfordUnited States 3 Department of Orthopedic SurgeryBostonUnited States 4 Department of Molecular and Cellular Biology, Harvard UniversityCambridgeUnited States 5 Department of Biology, Tufts UniversityMedfordUnited States 6 The Harvard Stem Cell InstituteCambridgeUnited States 7 The Broad Institute of MIT and HarvardCambridgeUnited States Voss Stephen Randal Reviewing EditorUniversity of Kentucky Stainier Didier YR Senior EditorMax Planck Institute for Heart and Lung ResearchGermany 06 3 2020 2020 9 e5121720 8 2019 05 3 2020 © 2020, Sousounis et al2020Sousounis et alhttp://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.How salamanders accomplish progenitor cell proliferation while faithfully maintaining genomic integrity and regenerative potential remains elusive. Here we found an innate DNA damage response mechanism that is evident during blastema proliferation (early- to late-bud) and studied its role during tissue regeneration by ablating the function of one of its components, Eyes absent 2. In eya2 mutant axolotls, we found that DNA damage signaling through the H2AX histone variant was deregulated, especially within the proliferating progenitors during limb regeneration. Ultimately, cell cycle progression was impaired at the G1/S and G2/M transitions and regeneration rate was reduced. Similar data were acquired using acute pharmacological inhibition of the Eya2 phosphatase activity and the DNA damage checkpoint kinases Chk1 and Chk2 in wild-type axolotls. Together, our data indicate that highly-regenerative animals employ a robust DNA damage response pathway which involves regulation of H2AX phosphorylation via Eya2 to facilitate proper cell cycle progression upon injury. axolotlregenerationprogenitor cellsDNA damage responselimb regenerationResearch organism Otherhttp://dx.doi.org/10.13039/100003212Sara Elizabeth O'Brien TrustPostdoctoral fellowshipSousounis Konstantinos http://dx.doi.org/10.13039/100000002National Institutes of HealthK99EY029361Sousounis Konstantinos http://dx.doi.org/10.13039/100000952Paul G. Allen Family FoundationAllen Discovery Center at TuftsLevin Michael Whited Jessica L http://dx.doi.org/10.13039/100000002National Institutes of Health1DP2HD087953Whited Jessica L http://dx.doi.org/10.13039/100000002National Institutes of Health1R01HD095494Whited Jessica L http://dx.doi.org/10.13039/100008035Harvard Stem Cell InstituteHIPMartinez Fernandez Jose http://dx.doi.org/10.13039/100000011Howard Hughes Medical InstituteGilliam FellowshipBryant Donald M The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Author impact statementExperimental manipulation of a core DNA damage response factor and cell-cycle checkpoint regulators reveals a key role for these processes in the progenitor cells that fuel limb regeneration. ==== Body Introduction DNA damage response (DDR) is an important process in ensuring cellular function. Genotoxic stress can cause senescence and apoptosis in proliferating cells or carcinogenesis when oncogenes are affected (Zeman and Cimprich, 2014; Vitale et al., 2017). Upon DNA damage, sensor proteins initiate a DDR that eventually guides DNA repair enzymes to resolve the lesion (Huen and Chen, 2008). The histone variant H2AX is considered a major DNA damage mediator protein because it is used as a scaffold for protein-protein interactions based on its amino acid modifications. H2AX phosphorylated at the 139 serine, commonly known as gamma-H2AX (γ-H2AX), is widely used as a DNA damage marker because it is found aggregated near lesions shortly after they occur (Huen and Chen, 2008; Yuan et al., 2010; Rogakou et al., 1998). Acting in concert to ensure genomic stability, cell cycle checkpoints ordinarily prevent cells from progressing before resolving DNA damage. Cell cycle arrest through activation of DNA damage checkpoints via Chk1 and Chk2 is an important part of the DDR mechanism (Zhou and Elledge, 2000; Bartek and Lukas, 2003; Smith et al., 2010). DNA repair resolves the DDR and protein modifications are reversed so normal cellular function can be resumed (Shaltiel et al., 2015). However, DDR does not always lead to DNA repair; molecular safeguards mediate critical decisions as to whether the amount of DNA damage is amendable, or whether apoptosis should be triggered instead (Cook et al., 2009). These molecular switches, such as p53, are known tumor suppressors and, when deregulated, apoptosis is circumvented, and carcinogenesis could occur (Lakin and Jackson, 1999). For instance, in vitro cultured human stem cells experience DNA damage and chromosomal rearrangements that change their cellular fate and render them carcinogenic and unsuitable for use in clinical applications in regenerative medicine (Vitale et al., 2017). On the other hand, amphibians can faithfully regenerate body parts throughout life, often even when repeatedly challenged (Bryant et al., 2017a; Bryant et al., 2017b; Sousounis et al., 2015; Eguchi et al., 2011). Replicative stress and reactive oxygen species generated during the process could challenge regeneration, at the cellular level by inhibiting cell cycle progression or at the functional level by losing the ability to form a proper structure (Sousounis et al., 2014; Al Haj Baddar et al., 2018; Ferreira et al., 2016). How do amphibians regulate genotoxic stress and DDR while ensuring tissue regeneration? Previous studies have shown that during early phases of regeneration, the activity of p53 is reduced to allow for progenitor cell cycle re-entry (Yun et al., 2013), but it is later activated to enable differentiation (Villiard et al., 2007). Since p53 is a major regulator of DDR, amphibians might be de-sensitized to DNA damage allowing for rapid progression through the cell cycle checkpoints. Their massive genome could also facilitate this process; it has been previously shown that DNA damage is more likely to occur in areas that do not encompass coding regions and regulatory elements, like retrotransposons, which are significantly abundant in amphibian genomes (Poetsch et al., 2018; Smith et al., 2019; Nowoshilow et al., 2018; Elewa et al., 2017). A completely opposite (but not mutually exclusive) strategy would entail the initiation of a DDR together with the onset of the regeneration process to ensure rapid DNA repair and cell cycle progression. Studies that have repeatedly initiated the regeneration process, for example when newt lenses were removed and successfully regenerated 19 times over 18 years, support this hypothesis since the structure and function of the genome would remain intact over many rounds of cell division (Sousounis et al., 2015; Eguchi et al., 2011; Sousounis et al., 2014a). In addition, high-throughput gene expression datasets acquired from regenerative tissues show up-regulation of DNA repair genes across multiple organs and animal species, indicating that a proper DDR might be an innate part of the regenerative mechanism (Sousounis et al., 2014; Darnet et al., 2019; Barghouth et al., 2019; Shiroor et al., 2019; Bedelbaeva et al., 2010). This mechanism could occur in conjunction with clearing senescent cells from regenerating tissues, which would allow only resilient proliferating progenitors to be present in the blastema (Yun et al., 2015). Here, we address these two hypotheses in the context of axolotl limb regeneration. We first investigated the relationship between DDR and tissue regeneration. We found an elevated DDR following limb amputation, which was accompanied by reduced relative γ-H2AX levels and baseline amounts of DNA damage. Then we asked whether DDR is necessary for limb regeneration. To answer this question, we genetically targeted a DDR- and H2AX-associated gene, eyes absent 2 (eya2). Eya2 regulates H2AX phosphorylation status, mediates decisions regarding DNA repair (Cook et al., 2009), and has been shown to be up-regulated during regenerative processes (Stewart et al., 2013; Sousounis et al., 2014b). We generated Eya2 mutant axolotls and found that their regenerative capability was significantly reduced and their γ-H2AX levels were significantly elevated, especially in cells that have entered the cell cycle. Interestingly, we did not detect accumulation of DNA damage, indicating that our model specifically de-regulated the DDR process. RNAseq analysis of eya2-/- tissues and pharmacological inhibition of Eya2's phosphatase activity were also used to corroborate our findings. In addition, we uncovered parallel evidence that DDR is required for successful regeneration as inhibiting a separate component of this process, the proper control of the DNA damage checkpoints by Chk1 and Chk2, led to severe regenerative impairment. This study reveals that DDR is an innate part of the regeneration process and identifies Eya2 as a regulator of DDR and cell cycle progression during progenitor cell proliferation. How amphibians regulate DDR and allow for cellular proliferation while maintaining cell fate could reveal important clues for improving the use of human stem cells in regenerative medicine. Results Axolotl limb regeneration is associated with elevated DNA damage response and low genotoxic stress in progenitor cells To identify the presence of a DNA damage response (DDR) following limb amputation, we analyzed transcriptomic profiles from previously published expression datasets acquired by RNAseq and microarrays that included a complete time course of regeneration (Stewart et al., 2013; Voss et al., 2015). We analyzed 190 genes associated with DDR and DNA repair from both datasets (Supplementary file 1). We found expression of DDR-related genes to be mostly up-regulated during regeneration (Figure 1a, Green). Genes also appeared up-regulated irrespective of their molecular role during DDR (Figure 1—figure supplement 1a). We validated the expression of 17 genes using qPCR and found that 11 (65%) of them were up-regulated during regeneration (Figure 1—figure supplement 2). We then focused on genes that have tentative functions in promoting DNA repair, as these genes may regulate DDR for proper cell cycle entry and regeneration (see Materials and methods, Supplementary file 1). The genes in this subcategory also appeared up-regulated during regeneration (+DDR, Figure 1b). Overall, our transcriptomic and qPCR analysis of DNA repair/damage genes revealed that DDR was evident as early as the early-bud blastema (~5 dpa) and peaked at the late-bud blastema (~14 dpa). To associate this response with the levels of actual DNA damage, we performed alkaline single cell gel electrophoresis (comet assay) and found that there are no differences between intact and 14 dpa blastema cells, whereas cells treated with UV showed more DNA damage (Figure 1c and Figure 1—figure supplement 1b). To corroborate these data, we focused on the DNA damage mediator histone H2AX. First, we analyzed the axolotl h2afx sequence using two strategies: phylogenetic tree construction (Figure 1d) and multiple sequence alignment (Figure 1—figure supplement 1c). We found that axolotl H2AX has a conserved carboxyl tail containing the S139, also known as γ-H2AX when phosphorylated, and Y142 (Cook et al., 2009; Yuan et al., 2010; Figure 1—figure supplement 1c, green box). We validated antibodies detecting these phosphorylation sites in vitro using AL1 axolotl fibroblast cells following UV treatment (Figure 1—figure supplement 1d–e). RNAseq and qPCR also revealed that h2afx expression was significantly up-regulated during regeneration (Figure 1b and Figure 1e). We then used western blot to determine the phosphorylation state of H2AX in axolotl tissues. Using samples acquired across different time points during limb regeneration, we found that the total amount of H2AX and pY142-H2AX were elevated compared to GAPDH starting at 5 dpa, corroborating the presence of a DDR in the early- and late-bud blastema (Materials and methods, Figure 1—figure supplement 3). To analyze this process in greater depth, we focused on blastema samples at 14 dpa, the time that DDR was found to be the most elevated (Figure 1f). We again found that the total amount of H2AX protein was increased during regeneration (Figure 1g), following the same trend as its mRNA levels (h2afx, Figure 1b and Figure 1e). The total levels of γ-H2AX protein were also increased (Figure 1h); however, the relative ratio of γ-H2AX to total H2AX protein was decreased during regeneration (Figure 1i). Overall, these data indicated that blastema cells may require more γ-H2AX to rapidly correct replication errors during proliferation but kept a low γ-H2AX/H2AX ratio to reduce potential genotoxic stress signals that could hinder cell cycle progression (Rogakou et al., 1998; Fernandez-Capetillo et al., 2004). Interestingly, we identified that the relative pY142-H2AX levels were increased at 14 dpa (Figure 1j and Figure 1k), which suggested that H2AX regulation was important during regeneration and a potential target to study in association with DDR and genome integrity. Together, these results suggest that DDR is evident during early-bud blastema and peaks at late-bud blastema, thereby preventing accumulation of DNA damage during regeneration. This may enable high levels of cellular proliferation despite local environmental genotoxic stressors. To further test this hypothesis, we aimed to genetically ablate one of the DDR components and identify whether challenging this process would have an impact on the regenerative capacity. Figure 1. DNA damage response is elevated during limb regeneration and is associated with regulation of H2AX phosphorylation status. (a) Regulation of DNA damage response (DDR) genes in two high-throughput gene expression datasets. The majority of these genes appeared up-regulated during limb regeneration (green area). See Materials and methods for regulation categories. (b) Relative gene expression changes of DNA damage response genes (+DDR) and h2afx during regeneration compared to intact. (c) Categories represent percent of cells in each comet assay stage score with stage 0 cells having no detectable DNA damage and stage four having the most. There is no statistical difference between intact (cyan) and blastema (orange) cells for the presence of DNA damage. Blastema cells treated with UV prior to comet assay were used as control and depict statistical significance compared to untreated blastema cells at stage 0 with less percent of cells (Two-way ANOVA with Bonferroni's multiple comparisons test: p-value<0.0001) and at stage three with more percent of cells (p-value<0.01). The data indicates that there is no DNA damage accumulation during regeneration. (d) Phylogenetic tree analysis of axolotl H2AX sequence with other vertebrates. C: Canis, B: Bos, H: Human, Mu: Mus, Ma: Macaca, R: Rattus. Scale Bar: 0.01. (e) Validation of axolotl h2afx expression during limb regeneration using qPCR. (f) Western blot for H2AX and its phosphorylation status during regeneration. Quantification on (g, h, i, j, k). (g) Total H2AX levels in intact and 14 dpa blastema tissue normalized by the total protein input. There is up-regulation of H2AX total protein during regeneration (Unpaired t test, p-value<0.001). (h) Total γ-H2AX levels in intact and 14 dpa blastema tissue normalized by the total protein input. There is up-regulation of γ-H2AX total protein during regeneration (Unpaired t test, p-value<0.01). (i) Relative γ-H2AX levels in intact and 14 dpa blastema tissue normalized by the respective H2AX levels. There is down-regulation of relative γ-H2AX levels during regeneration (Unpaired t test, p-value<0.05). (j) Relative pS139/Y142-H2AX levels in intact and 14 dpa blastema tissue normalized by the respective H2AX levels. There is up-regulation of relative pS139/Y142-H2AX levels during regeneration (Unpaired t test, p-value<0.05). (k) Relative pY142-H2AX levels in intact and 14 dpa blastema tissue normalized by the respective H2AX levels. There is up-regulation of relative pY142-H2AX levels during regeneration (Unpaired t test, p-value<0.01). These data indicate that there is dynamic regulation of H2AX phosphorylation status during regeneration. ns denotes p-value>0.05, * denotes p-value<0.05, ** denotes p-value<0.01, *** denotes p-value<0.001, **** denotes p-value<0.0001. Vertical bars on plots represent standard error of the mean (SEM). Figure 1—figure supplement 1. DDR pathways, comet assay staging, H2AX multiple sequence alignment and validation of H2AX antibodies. (a) Relative expression of genes related to DDR separated by function. Data are related to Supplementary file 1. On average, genes related to DDR are up-regulated during regeneration. (b) Representative images of comets at different stages from the comet assay. Scale Bar: 25 μm. At stage 0, there is no DNA damage present in the cells hence electrophoresis did not pull the DNA from the nuclear matrix and it appears as a compact round ball. As cells acquire more DNA damage, more DNA is getting pulled from the nuclear matrix, generating a 'comet' where the more DNA appears at the tail, the more DNA damage these cells have acquired. At stage 4, most of the DNA is in the tail. (c) H2AX sequence alignment from multiple organisms. Red boxes indicate minor differences between organisms mainly found in non-mammals. Yellow box indicates differences at the C-terminus of the H2AX. Green box indicates similarities in the last four amino acids, which are critical for regulating H2AX function through the phosphorylation of the serine (S139) and tyrosine (Y142). (d) In vitro culture of axolotl AL1 cells treated with UV and stained for CST-5438 antibody that detects mono or dual phosphorylated H2AX at S139 and Y142. Scale Bars: 23 μm. Note that the signal intensifies after UV treatment, indicating that the antibody recognizes the axolotl H2AX forms. (e) In vitro culture of axolotl AL1 cells treated with UV and stained for Ab11174 (similar results obtained with Ab81299) antibody that detects γ-H2AX (mono S139-H2AX phosphorylation). Scale Bars: 23 μm. Note that the signal intensifies following UV treatment, indicating that the antibody is able to recognize the axolotl γ-H2AX. Figure 1—figure supplement 2. Validation of DDR gene expression profiles using qPCR. Graphs show expression of ercc8, brcc3, ogg1, rif1, ung, xpc, xrcc6, msh2, rnf169, brca1, parp1, xrc, mms19, rad51, fanca, foxm1, and dhx9 relative to ef1a control at 0. 3. 7. 14 and 21 dpa. Red boxes indicate that expression values have a significant ANOVA p value (p-value<0.05). Comparisons shown on graphs represent significance between 0 dpa and the respective timepoint. Data showed that the majority of the DDR genes tested were significantly up-regulated during regeneration. * denotes p-value<0.05, ** denotes p-value<0.01, *** denotes p-value<0.001, **** denotes p-value<0.0001. Vertical bars on plots represent SEM. Figure 1—figure supplement 3. H2AX phosphorylation status at different timepoints during limb regeneration. Samples are derived from intact or blastema tissue following protein extraction. Lanes represent separate biological replicates. This experiment allowed us to assess differences in H2AX protein amounts across timepoints using a non-histone control (here, GAPDH). However, the method has increased the unintended bands of higher molecular weight which may represent histone complexes of different sizes. In red boxes the bands used for the quantification are indicated. The size of those bands were the same as what was derived using a histone extraction protocol (see Figures 1f and 4a) which overall generated more specific bands with our antibodies but without the ability to blot for non-histone controls. The data showed that the total H2AX and pY142-H2AX proteins were up-regulated during regeneration which validated the western blot experiment with samples acquired using the histone extraction protocol (Figure 1f). Eya2 is widely expressed in early-bud blastema and is associated with DNA repair and cell cycle during regeneration Since pY142-H2AX levels were found to be elevated during regeneration (Figure 1k and Figure 1—figure supplement 3), we focused on the Eyes absent (Eya) protein family, which are known regulators of H2AX phosphorylation status and initiators of DNA repair through their phosphatase domain (Cook et al., 2009). We first investigated the spatial and temporal expression of individual members upon limb amputation. We found that eya2 is the most highly-expressed gene of its family and is also highly up-regulated post-amputation (Figure 2a, eya2) (Stewart et al., 2013). We additionally performed qPCR and verified its expression profile during regeneration (Figure 2b). Phylogenetic tree analysis using the entire Eya family further validated homology to other vertebrate orthologs of Eya2 (Figure 2c). In order to associate Eya2 with DNA repair during limb regeneration, we performed a co-expression correlation analysis using RNAseq transcriptomic data from limb/blastema samples at 0, 3, 5, 7, 10, 14, 21, and 28 days following amputation (Supplementary file 2). Genes positively associated (R2 >0.8, slope >0) and negatively associated (R2 >0.8, slope <0) with eya2 expression were used for Gene Ontology Enrichment Analysis (Figure 2—figure supplement 1a and b, respectively). Using this method, we found that the expression profiles of genes involved in DNA repair and cell cycle were positively correlated with eya2 expression, suggesting Eya2's involvement in these processes during limb regeneration (Figure 2d, blue bar). Conversely, mature muscle genes were negatively correlated with eya2 expression, consistent with eya2's role during early muscle development in other vertebrates (Heanue et al., 1999; Figure 2d, red bar). To test whether the axolotl Eya2 protein physically interacts with an H2AX-containing complex in cells, we performed an immunoprecipitation experiment using a tagged version of the axolotl Eya2, and we detected the phosphorylated form of H2AX in the precipitate (Figure 2e). Taken together, these data suggest that Eya2 plays a role in cell cycle and DNA repair regulation during regeneration and that Eya2 interacts with phosphorylated H2AX. Figure 2. Eya2 is up-regulated during limb regeneration, expressed by limb progenitors and associated with cell cycle and DNA repair. (a) RNA-seq derived gene expression of the eya gene family during different time points of axolotl limb regeneration. Note that eya2 is the most expressed gene of the eya gene family and appears up-regulated upon amputation. (b) Validation of eya2 gene expression profile using qPCR. We found that normalized eya2/ef1a levels are significantly up-regulated following limb amputation (One-way ANOVA: p-value<0.0001, with Bonferroni's multiple comparisons test: 0 dpa vs 7 dpa: p-value<0.001, 0 dpa vs 14 dpa: p-value<0.0001, 0 dpa vs 21 dpa: p-value<0.01), indicating that Eya2 may play an important role during limb regeneration. (c) Validation of Eya2 homology with other vertebrate Eya2 proteins using phylogenetic tree analysis. (d) Gene Ontology Enrichment Analysis (FDR < 0.05) of genes found to be positively (R2 >0.8, slope >0, blue bars) or negatively (R2 >0.8, slope <0, red bars) associated with eya2 expression. These data suggest that Eya2 is associated with DNA repair and cell cycle during limb regeneration. (e) Co-immunoprecipitation (Co-IP) experiment using the axolotl Eya2 protein fused with a FLAG tag (AxEya2-FLAG) or mCherry control. Immunoblot against the FLAG tag revealed the axolotl Eya2 protein in the samples transfected with the Eya2-FLAG plasmid and not in the control, as expected (blot for FLAG). Immunoblot against the phosphorylated form of H2AX revealed a band in the precipitate derived from the AxEya2-FLAG IP sample and not in the mCherry control (blot for pS139/Y142-H2AX). I: Sample before IP, L: Flow-through after IP, IP: Pulled-down proteins. (f) RNA in situ hybridization for eya2 (red puncta) in 5 dpa blastema section, Scale Bar: 50 μm. Eya2 appears to be expressed by most cells in the wound epidermis and blastema. (g–g''') Time course RNA in situ hybridization for eya2 (red puncta) and prrx1 (blue puncta) during regeneration. Scale Bars: 250 μm. (g) Intact limb. (g'). 7 dpa early blastema. (g'') 14 dpa mid-late blastema. (g''') 21 dpa palette stage. (h–h''') High magnification inserts of (g–g''') images. Scale Bars: 25 μm. (h) Intact limb. Eya2 is expressed by muscle cells (red arrow) and prrx1+ periosteal cells (yellow) which are known cartilage progenitors. (h') 14 dpa blastema. Eya2 is robustly expressed in the regenerating muscle (red arrow) and blastema cells positive for prrx1 (yellow arrow) (h'') 21 dpa regenerate. Eya2 is expressed in the regenerating muscle (red arrows) while expression in the prrx1+ population is getting diminished (yellow arrow). (h''') 21 dpa regenerate-proximal area. Eya2 is expressed in the newly regenerated prrx1+ periosteal cells (yellow arrow), while there is no expression in fully developed muscle. ns denotes p-value>0.05, * denotes p-value<0.05, ** denotes p-value<0.01, *** denotes p-value<0.001, **** denotes p-value<0.0001. Vertical bars on plots represent standard error of the mean (SEM). Figure 2—figure supplement 1. Gene Ontology Enrichment Analysis of eya2 co-expression correlation analysis during limb regeneration. (a) Biological processes positively correlated with eya2 expression. These data represent functions of genes found to be expressed in a similar manner as eya2 indicating that eya2 may regulate, be part of or be regulated by these processes. What the result suggests is that the eya2 expression profile is correlated with expression profiles for genes that annotate to cell cycle (blue) and DNA repair (red) GO terms. (b) Biological processes negatively correlated with eya2 expression. These data represent functions of genes found to be expressed when eya2 expression is missing or down-regulated. Note that functions related to mature muscle (red) are strongly negatively correlated which is in support of the role of Eya2 during early muscle development. Figure 2—figure supplement 2. Eya2 lineage trace analysis. (a) The axolotl eya2 locus. Scale Bar: 3000 bp. As expected, the axolotl eya2 locus has the same number of exons as the human eya2 locus but considerably longer introns. (b) Exon 1 was targeted with a specific CRISPR/Cas9 complex. Red area denotes the target sequence. Scale Bar: 25 bp. (c) The axolotl eya2 locus following knock-in of the bait plasmid containing the full ORF of the eya2 gene and cre recombinase separated by the self-cleaving viral P2A peptide. The Cre recombinase will be expressed under the eya2 endogenous promoter and permanently label eya2-expressing cells with mCherry following removal of the loxp-GFP-stop-loxp sequence in Cre reporter animals. (d) Fluorescent images from limbs of F0 mosaic eya2 knock-in Cre reporter animals denoting lineage trace with mCherry. Since the knock-in event was targeted at the single-cell stage embryo, any cell expressing eya2 during development would be permanently labeled with mCherry expression. Note that all three major limb lineages, muscle, skin and connective tissue cells are able to express eya2. (e) The resulting mCherry fluorescence in regenerates when limbs in (d) are amputated. Scale Bar: 1 mm. To identify which cells express eya2 during regeneration, we performed RNA in situ hybridization and found eya2 mRNA detectable in the majority of blastema cells and wound epidermis at 5 dpa (red puncta, Figure 2f). These cells are known to encompass limb progenitors of all lineages including skin, muscle, connective tissue, and cartilage (Whited et al., 2013; Currie et al., 2016; Gerber et al., 2018; Leigh et al., 2018). Indeed, time-course double in situ hybridization of eya2 and prrx1, a connective tissue progenitor marker (Gerber et al., 2018), further validated eya2 expression in the connective tissue lineage. We found that eya2 and prrx1 co-localized in connective tissue progenitors throughout regeneration, and we further observed eya2 expression in muscle as well. In intact tissue, eya2 and prrx1 were present in periosteal cells, which are known to participate in cartilage regeneration (McCusker et al., 2016), while eya2 alone was present in the muscle (Figure 2g and h). In the early-bud blastema (7 dpa), eya2 and prrx1 showed expression throughout the blastema with prrx1 being very robust (Figure 2g'). In the mid- to late-bud blastema (14 dpa), eya2 and prrx1 started to compartmentalize, with eya2 having strong expression in the developing muscle which appeared to be contiguous with the mature fibers in the stump, and prrx1 aggregating in the rest of the blastema (Figure 2g''). Interestingly, eya2 and prrx1 double-positive cells were clearly present in the boundary between high-eya2 and high-prrx1 expressing areas (Figure 2h'). This suggested that these areas contained early connective tissue progenitor cells that still expressed eya2 and, as these cells mature, they become prrx1-only positive in the center of chondrogenesis (Balic et al., 2009). This phenomenon was more evident in palette stage limbs (21 dpa) where muscle/cartilage boundaries were more defined (Figure 2g''' and h''). We also identified that newly-formed areas containing connective tissue progenitor cells were strongly positive for both eya2 and prrx1 (Figure 2g''' and h'''). These data suggested that the majority of early-stage limb progenitors express eya2 during limb regeneration. To further strengthen these findings, we performed a lineage tracing analysis by generating knock-in axolotls expressing the Cre recombinase under the endogenous eya2 promoter in the previously described Cre reporter background (Figure 2—figure supplement 2a–c; Fei et al., 2017). Using this method, we showed that muscle, skin, connective tissue and cartilage were labeled during limb development and these labels were sustained during regeneration (Figure 2—figure supplement 2d–e). Taken together, eya2 was found up-regulated during regeneration, expressed by most of early-bud blastema cells, and highly correlated with DNA repair genes, all of which strongly suggests that Eya2 activity is part of the DDR initiated upon limb amputation. Eya2 mutant axolotls have reduced rate of regeneration and slower cell cycle progression To determine whether DDR is important for limb regeneration, we targeted the eya2 locus with a specific CRISPR/Cas9 ribonucleoprotein complex and generated eya2 mutant axolotls (eya2-/-) (Figure 3—figure supplement 1a–b). Based on the genotyping, the Eya2 mutant protein is predicted to be severely truncated and lacking both the phosphatase and transactivation domains (Figure 3—figure supplement 1a). Eya2-/- axolotls developed normal limbs with muscle, bone, and nerves (Figure 3—figure supplement 2a–d). The eya2-/- animals could be easily identified by the presence of an intra-peritoneal edema which was also correlated with malformed kidneys (Figure 3—figure supplement 2e, red box). To determine the effects of ablating the function of Eya2 during limb regeneration, we imaged blastemas and quantified their length over time (Figure 3a). The data indicated that eya2-/- axolotls have reduced blastema length compared to eya2+/- and eya2+/+ at multiple time points (Figure 3b and c). We did not observe haploinsufficiency or major differences between eya2+/+ and eya2+/-, so all subsequent comparisons were focused on eya2-/- and eya2+/+ genotypes. At 30 dpa, a pre-selected time point which captures regeneration before the end of the growth process, we collected the regenerated limbs and visualized their skeletons using alcian blue and alizarin red S stains (Figure 3d). To determine the size of the regenerate at 30 dpa, we measured their zeugopod length relative to animal size and found that eya2-/- animals regenerated smaller limbs, suggesting that the rate of regeneration was impaired (Figure 3e, see also Figure 3—figure supplement 2c–d for their size before amputation). We then analyzed their skeletal pattern for completion and found that none of the eya2-/- animals (n = 0/18 limbs from nine animals) had a fully-patterned skeleton compared to 17% of eya2+/+ (n = 3/18 limbs from nine animals, 3 × 2 Contingency Table: p-value<0.05, Figure 3f). These data indicated that eya2-/- axolotls regenerated limbs more slowly, impacting the volume of the regenerate as well as the phase of the regenerative process, compared to eya2+/+ siblings. Figure 3. CRISPR/Cas9-targeted eya2 mutant axolotls regenerate slower and have stalled cell cycle. (a) Experimental design to test limb regeneration rates in eya2-/- axolotls. Bright field imaging was conducted at 6, 10, and 13 dpa to determine blastema length overtime. Final limb collection was performed at 30 dpa, time that some control axolotls had all four digits regenerated. 30 dpa limb were processed to visualize their skeleton using alcian blue and alizarin red S. (b) Representative images of blastema at different time points during regeneration. Arrows point at the amputation site. Scale Bar: 0.5 mm. Note that eya2-/- axolotls have a smaller blastema even at 6 dpa. (c) Quantification of blastema length from (b). Eya2-/- axolotls have smaller blastema compared to eya2+/+ and eya2+/- (Repeated measures two-way ANOVA, interaction: p-value<0.05, time: p-value<0.0001, genotype <0.01; Bonferroni's multiple comparisons tests: 13 dpa eya2+/+ vs 13 dpa eya2-/-: p-value<0.01 and 13 dpa eya2+/- vs 13 dpa eya2-/-: p-value<0.0001). (d) Representative images of limb regenerates stained with alcian blue and alizarin red S stains to visualize skeletal elements. 30 dpa regenerates. Red arrows point to the beginning and the end of the zeugopod. Scale Bar: 1 mm. Note the relative smaller limb regenerates of the eya2-/- axolotls. (e), Quantification of zeugopod length from (d). Eya2-/- 30 dpa regenerates are significantly smaller than those from eya2+/+ and eya2+/- axolotls (One-way ANOVA with Bonferroni's multiple comparisons tests: eya2+/+ vs eya2-/-: p-value<0.05, eya2+/- vs eya2-/-: p-value<0.05). (f) Analysis of skeletal pattern from (d). Skeletal elements were quantified to determine whether or not the full limb pattern is regenerated. Eya2-/- animals did not have any limb that had successfully regenerated all skeletal elements (Fisher Exact Probability Test on a 2 × 3 table: p-value<0.05). (g) Experimental design to test cellular proliferation during regeneration. EdU was injected at 4 dpa and blastema tissues were harvested 18 hr later and processed for immunofluorescence (IF). (h) Representative images of blastema sections at 5 dpa stained with pS10-H3, EdU and DAPI. Scale Bar: 100 μm. (i) Quantification of percent +EdU cells in the whole blastema. There are less blastema cells entering the cell cycle in eya2-/- axolotls (Kruskal-Wallis test with Dunn's multiple comparisons test: eya2+/+ vs eya2-/-: p-value<0.05, eya2+/- vs eya2-/-: p-value<0.001). (j) Quantification of percent +pH3 cells in the whole blastema. There are less blastema cells actively dividing in eya2-/- axolotls (One-way ANOVA with Bonferroni's multiple comparisons test: eya2+/+ vs eya2-/-: p-value<0.01, eya2+/- vs eya2-/-: p-value<0.001). (k) Quantification of percent +pH3 cells in the +EdU population. There are less blastema cells that entered the cell cycle at the time of EdU pulse and actively dividing at the time of collection in eya2-/- axolotls (One-way ANOVA with Bonferroni's multiple comparisons test: eya2+/+ vs eya2-/-: p-value<0.05, eya2+/- vs eya2-/-: p-value<0.05). ns denotes p-value>0.05, * denotes p-value<0.05, ** denotes p-value<0.01, *** denotes p-value<0.001, **** denotes p-value<0.0001. Vertical bars on plots represent SEM. Figure 3—figure supplement 1. Eya2 mutant animal genotyping. (a) Eya2 Exon 3 was targeted with a specific CRISPR/Cas9 complex. Red area denotes the target sequence. Sequences indicate specific indels found in our population of animals used in the experiments. Scale Bar: 25 bp. Cartoons illustrate the differences between the Eya2 proteins and their domains predicted by the genotyping for each identified indel. The orange rectangle is the phosphatase domain and the yellow eclipse is the transactivation (TA) domain of the wild-type Eya2 protein. The green and blue rectangles illustrate extra amino acid sequences expected to be translated following the CRISPR/Cas9 target site which have no known predicted function and share no similarity to the wild-type Eya2 sequence. Scale Bar: 25aa. (b) Genotypes, observed indels, observed frequencies and total number of animals used in the experiments. Figure 3—figure supplement 2. Eya2 mutant animal characterization. (a) Cross sections of intact limbs from all genotypes stained with βIII-tubulin (green, neural axons), phalloidin (red, muscle) and DAPI (blue, bone margins). Scale Bar: 400 μm (b), Quantification of the bone, muscle and axon area relative to DAPI area or section area. Note that all eya2 genotypes have normal limbs with axons, muscle and bone (Two-way ANOVA: Interaction, Row Factor, Column Factor: p-value>0.05, with Bonferroni's multiple comparisons test: p-value>0.05 for all comparisons). (c) Representative images of alcian blue and alizarin red S from intact limbs across all eya2 genotypes. Red arrows show zeugopod length. Scale Bar: 1 mm. (d) Quantification of zeugopod length from (c). There is no difference among genotypes in the zeugopod length of intact limbs (One-way ANOVA: p-value=0.4839, with Bonferroni's multiple comparisons test: eya2-/- vs eya2+/+: p-value>0.05, eya2-/- vs eya2+/-: p-value>0.05). (e) Cross-sections of whole axolotls at a level where kidneys are shown (red box). All eya2-/- animals showed deformed kidneys (n = 7) versus eya2+/+ (n = 3) and eya2+/- (n = 3). Scale Bar: 1 mm. Dorsal is top, ventral is bottom. (f) Relative animal length at 0 dpa. Eya2 mutant animals appear to grow normally (One-way ANOVA: p-value=0.1758, with Bonferroni's multiple comparisons test: eya2-/- vs eya2+/+: p-value>0.05, eya2-/- vs eya2+/-: p-value>0.05). (g) Relative animal length at 30 dpa. Eya2 mutant animals appear to have grown slightly bigger than eya2+/+ and eya2+/- siblings (One-way ANOVA: p-value=0.0255, with Bonferroni's multiple comparisons test: eya2-/- vs eya2+/+: p-value<0.05, eya2-/- vs eya2+/-: p-value=0.5179, eya2-/+ vs eya2+/+: p-value=0.3724). ns denotes p-value>0.05, * denotes p-value<0.05, ** denotes p-value<0.01, *** denotes p-value<0.001, **** denotes p-value<0.0001. Vertical bars on plots represent SEM. Figure 3—figure supplement 3. Cell cycle regulation in 5 dpa blastemas across eya2 genotypes and at different time points during limb regeneration. (a) Cell cycle regulation in 5 dpa blastemas among eya2 genotypes. Cyclin-dependent kinase 2 (CDK2), G2/mitotic specific cyclin-B1 (CCNB1), phospho Tyr-15 Cyclin-dependent kinase 1 (p-CDC2) and GAPDH were assessed using western blot. Quantification indicates decreased levels of CDK2/GAPDH (One-way ANOVA: p-value=0.0136, with Bonferroni's multiple comparisons test: eya2-/- vs eya2+/+: p-value<0.05), increased levels of CCNB1/GAPDH (One-way ANOVA: p-value=0.0051, with Bonferroni's multiple comparisons test: eya2-/- vs eya2+/+: p-value<0.01) and no change in p-CDC2/GAPDH levels (One-way ANOVA: p-value=0.6513, with Bonferroni's multiple comparisons test: eya2-/- vs eya2+/+: p-value>0.05). The data validated that eya2-/- blastema cells proliferate slower and were stalled at the G2 phase as indicated by the increased levels of CCNB1. (b) Cell cycle regulation at different time points during regeneration in wild-type animals. CDK2, CCNB1 and p-CDC2 levels appear to decline relative to GAPDH at 1-3 dpa following amputation but to elevate, as expected, later during regeneration when blastema proliferation occurs (5-21 dpa). We then tested whether eya2-/- animals had an overall slower growth rate and found that even though all genotypes had approximately the same size animals at the time of amputation (Figure 3—figure supplement 2f), eya2-/- animals were slightly longer at 30 dpa (Figure 3—figure supplement 2g). These data indicated that the slower growth of the eya2-/- blastema was due to impairment of the regenerative process itself. We then investigated whether the slower regeneration rate was associated with proliferation deficiencies in limb progenitors. To test this possibility, we performed an 18 hr pulse with the nucleotide analogue EdU and subsequent detection as well as an immunofluorescence assay with the mitotic marker phosphoS10-Histone H3 (pH3) on 5 dpa blastema sections (Figure 3g and h). At this time point we had previously observed that eya2 was widely expressed throughout the blastema (Figure 2f). Eya2-/- blastemas had reduced rates of cell cycle entry indicated by a decrease in EdU+ cells (Figure 3i) and decreased mitotic activity as evidenced by a decrease in pH3+ blastema cells (Figure 3j). To identify whether the rate-limiting factor was S phase entry, which in turn limits the number of cells at the G2 and M phases, or whether there were roadblocks at later phases, we quantified double-positive EdU and pH3 cells. Interestingly, we found a reduced percentage of double EdU+/pH3+ cells in the eya2-/- blastemas, which indicated that cell cycle progression was also stalled at the G2/M phase (Figure 3k). To further investigate the proliferation deficits observed at the cellular level, we performed western blot analysis of proteins known for their role during the cell cycle (Figure 3—figure supplement 3a). We found that the amount of the Cyclin-dependent kinase 2 (CDK2) was reduced in the 5 dpa eya2-/- blastemas whereas the G2/M-specific Cyclin B1 was increased (CCNB1, Figure 3—figure supplement 3a). Given that CDK2 is not degraded at any phase of the cell cycle, its reduced presence indicated that the cell cycle was slower without Eya2 (Vermeulen et al., 2003; Malumbres and Barbacid, 2009). Interestingly, CDK2 has been shown to stall the cell cycle at the G1 phase when down-regulated which strengthen our EdU data showing G1/S arrest in Eya2 mutant blastemas (Neganova et al., 2011). Cyclin B1 protein levels normally increase during the G2 phase and decline following mitosis (Gong and Ferrell, 2010; Gong et al., 2007). The elevated levels of Cyclin B1 in eya2-/- blastemas strongly suggest that the cell cycle is stalled at the G2/M DNA damage checkpoint corroborating our pH3/EdU data (Figure 3k). Taken together, these data show that ablation of Eya2 transactivation and phosphatase domains has a regeneration phenotype associated with slower cell cycle progression at the two major checkpoint transitions, G1/S and G2/M. Eya2-/- axolotls have increased levels of γ-H2AX specifically associated with cycling blastema cells G1/S and G2/M checkpoints are activated upon cellular stress including DNA damage (Zhou and Elledge, 2000); thus, we analyzed the phosphorylation status of H2AX as described earlier (Figure 4a). We found that 5 dpa blastemas from eya2-/- animals had increased levels of relative γ-H2AX compared to those from eya2+/+ siblings (Figure 4b), suggesting that the activated cell cycle checkpoints may be associated with increased levels of genotoxic stress. In contrast, eya2+/- blastemas showed increased levels of pY142-H2AX, an outcome that correlated with the reduced Eya2 activity in the heterozygous background (Figure 4c and d). Since pY142-H2AX is a direct target of the Eya2 phosphatase domain, the data also indicate that the induced mutations in the eya2 locus do in fact generate dysfunctional Eya2 mutant protein. However, we did not detect significant changes at pY142-H2AX between eya2-/- and eya2+/+ (Figure 4c and d). The discrepancy between eya2-/- and eya2+/- in the pY142-H2AX levels might be due to compensatory mechanisms that prevents persistently high levels of bi-phosphorylated H2AX, a known cause of cell death (Cook et al., 2009). This possibility is supported by TUNEL staining in which we could not detect blastema cells undergoing apoptosis in eya2-/- animals (Figure 4—figure supplement 1a). We then focused on understanding why eya2-/- axolotls have increased γ-H2AX levels. To investigate this, we performed immunohistochemistry against γ-H2AX on tissue sections and identified a robust stain at the skin and wound epidermis (Figure 4—figure supplement 1a'). Its high fluorescence intensity throughout the skin did not allow us to detect the characteristic nuclear γ-H2AX foci (Nakamura et al., 2010; Figure 4e and f). We then focused on blastema cells in which we were able to detect and quantify nuclear γ-H2AX foci per area in EdU+ and EdU- cells (Figure 4g). Our analysis revealed that EdU+ cells had more γ-H2AX foci per nuclear area than EdU- cells, irrespective of genotype (Figure 4h, Two-way ANOVA, Row factor p-value<0.0001, F(1,30) = 62.39, DF = 1). These data support our initial findings that during limb regeneration total γ-H2AX levels were elevated to alleviate replication stress during blastema cell proliferation (Figure 1h, Figure 1—figure supplement 3). Notably, our data revealed that EdU+ eya2-/- blastema cells had significantly more γ-H2AX foci per area compared to those of eya2+/+ (Figure 4h). This indicated that when blastema cells without Eya2 activity enter S-phase, they acquire more hallmarks of genotoxic stress, which also corroborated our earlier data showing cell cycle arrest at the G2/M checkpoint (Figure 3k, Figure 3—figure supplement 3a). We further identified that the increased presence of γ-H2AX foci was due to more cells having a high number of foci per area and fewer cells having low number of foci (Figure 4i). We also found that EdU+ cells were bigger in size compared to EdU- cells across all genotypes (Figure 4—figure supplement 1b, two-way ANOVA, Row Factor p-value=0.004, F(1,30)=16.13, DF = 1); however, differences in the number of γ-H2AX foci per area of the nucleus within the EdU+ population were not due to variations in the cell size (Figure 4—figure supplement 1b–c). In addition, when foci from all blastema cells were taken into consideration, there was no difference across genotypes; thus, the effect was found to be specific to cycling blastema cells (Figure 4—figure supplement 1d). Since eya2-/- blastema cells showed increased levels of the DNA damage marker γ-H2AX they might have also accumulated DNA damage. To test this, we performed comet assays on blastema cells from all three genotypes and found that ablation of Eya2's transactivation and phosphatase domains does not affect the amount of DNA damage; hence, this phenotype appears specific to how DDR is regulated (Figure 4—figure supplement 1e). Taken together, these data revealed that functionally ablating one of the components of DDR, Eya2, reduced regeneration rate, which was associated with a genotoxic stress-like phenotype including slower cell cycle dynamics and increased levels of γ-H2AX in proliferating blastema cells. Figure 4. Proliferating eya2-/- blastema cells have increased γ-H2AX levels. (a) Western blot for H2AX and its phosphorylation status in 5 dpa blastema tissue. Note the robust up-regulation of γ-H2AX in eya2-/- tissues and the up-regulation of pS139/Y142-H2AX signal in eya2+/- tissues. (b,c,d) Quantification of (a). (b) Relative γ-H2AX levels across genotypes. Eya2-/- blastema have significantly more relative γ-H2AX levels, indicating higher genotoxic stress signals (One-way ANOVA with Bonferroni's multiple comparisons test: eya2+/+ vs eya2-/-: p-value<0.01, eya2+/- vs eya2-/-: p-value<0.01). (c) Relative pS139/Y142-H2AX levels across genotypes. Eya2+/+ blastema have significant less relative pS139/Y142-H2AX signal than eya2+/- and eya2-/- blastema (One-way ANOVA with Bonferroni's multiple comparisons test: eya2+/+ vs eya2+/-: p-value<0.01, eya2+/+ vs eya2-/-: p-value<0.05). (d) Relative pY142-H2AX levels across genotypes. Eya2+/- blastema have significantly more relative pY142-H2AX levels than eya2+/+ and eya2-/- (One-way ANOVA with Bonferroni's multiple comparisons test: eya2+/- vs eya2+/+: p-value<0.01, eya2+/- vs eya2-/-: p-value<0.05). (e) Representative images of γ-H2AX immunofluorescence on 5 dpa blastema sections at low magnification. Note the robust γ-H2AX signal intensity in eya2-/- skin. Scale Bar: 150 μm. (f) Quantification γ-H2AX intensity across the skin in each genotype from (e). Eya2-/- axolotls have significantly more γ-H2AX in their skin compared to eya2+/- and eya2+/+ (Two-way ANOVA interaction: p-value<0.0001, genotype: p-value<0.0001). (g) Representative images of blastema sections stained for DAPI (blue), γ-H2AX (green) and EdU (red). Stars (*) point to nuclear γ-H2AX foci, white lines denote the boundaries of the nucleus. Scale Bar: 10 μm. (h) Quantification of γ-H2AX foci per area of nucleus across different genotypes and proliferation status from (g). Blastema cells that have entered the cell cycle have significantly more γ-H2AX foci per area of nucleus irrespective of genotype (Two-way ANOVA: EdU: p-value<0.0001). EdU+ eya2-/- blastema cells have significantly increased numbers of γ-H2AX foci per area of nucleus compared to those from eya2+/+ and eya2+/- blastema (Two-way ANOVA interaction: p-value<0.001, with Bonferroni's multiple comparisons test: EdU+ eya2-/- vs EdU+ eya2+/+: p-value<0.01, EdU+ eya2-/- vs EdU+ eya2+/-: p-value<0.01). (i) Percent of cells in the +EdU blastema population in different categories of γ-H2AX foci concentrations. The data indicate that the same fraction of cells have γ-H2AX foci irrespective of genotype (Two-way ANOVA with Bonferroni's multiple comparisons test: for γ-H2AX zero concentration: eya2+/+ vs eya2+/-: p-value>0.05, eya2+/+ vs eya2-/-: p-value>0.05, eya2+/- vs eya2-/-: p-value>0.05) but eya2-/-blastema cells (red bars) have more cells with high and less cells with low γ-H2AX concentration (Two-way ANOVA with Bonferroni's multiple comparisons test: for low γ-H2AX concentration: eya2-/- vs eya2+/+: p-value<0.01, for high γ-H2AX concentration: eya2-/- vs eya2+/+: p-value<0.01, eya2-/- vs eya2+/-: p-value<0.05). ns denotes p-value>0.05, * denotes p-value<0.05, ** denotes p-value<0.01, *** denotes p-value<0.001, **** denotes p-value<0.0001. Vertical bars on plots represent SEM. Figure 4—figure supplement 1. TUNEL assay on Eya2 mutant blastema sections, DNA damage quantification in 5 dpa blastema cells and parameters associated with Figure 4h. (a) 5 dpa blastema sections from eya2+/+ and eya2-/- animals stained for TUNEL assay (red), γ-H2AX (green) and DAPI (blue). Scale Bar 150 μm. Eya2-/- blastema cells do not undergo apoptosis (0%, n = 3). (a') Higher magnification of red box insert from (a). Image shows γ-H2AX staining in green throughout all layers of the skin. Interestingly, the staining pattern in the skin is not nuclear (for γ-H2AX foci in blastema cells see Figure 4). Scale Bar: 10 μm. (b) Blastema cell size across different eya2 genotypes and proliferation status. EdU+ blastema cell nucleus is bigger in size than EdU- (Two-way ANOVA: EdU Factor: p-value<0.001). There is no difference in nucleus size among eya2 genotypes (Bonferroni's multiple comparisons test: EdU+ eya2-/- vs EdU+ eya2+/+: p-value>0.05, EdU+ eya2-/- vs EdU+ eya2+/-: p-value>0.05). (c) Average nucleus cell size across genotypes irrespective of EdU status. Blastema cell nucleus size appears the same among all eya2 genotypes (One-way ANOVA with Bonferroni's multiple comparisons test: eya2-/- vs eya2+/-: p-value>0.05, eya2-/- vs eya2+/+: p-value>0.05). (d) Average γ-H2AX foci per DAPI area across genotypes. There is no difference in γ-H2AX foci when cells are analyzed irrespective of their EdU status (One-way ANOVA with Bonferroni's multiple comparisons test: eya2-/- vs eya2+/-: p-value>0.05, eya2-/- vs eya2+/+: p-value>0.05). (e) Comet assay on 5 dpa blastema cells across eya2 genotypes. No differences were found among genotypes in the amount of DNA damage present in their genome (Two-way ANOVA: Interaction and Column Factor p-value>0.05, with Bonferroni's multiple comparisons tests: p-value>0.05 for all comparisons. ns denotes p-value>0.05. Vertical bars on plots represent SEM. RNAseq analysis of eya2-/- blastema during limb regeneration validates cell cycle arrest, H2AX de-regulation and elevated DDR In order to better understand the connection between Eya2, DDR and regeneration, we performed RNAseq in intact and blastema tissues at different timepoints upon amputation from all three eya2 genotypes. Computational analysis revealed 4461 genes to be differentially regulated (q < 0.05) between genotypes (Supplementary file 3). From these, most of them appeared to be down-regulated in eya2-/- compared to eya2+/+ tissues. GO analysis revealed terms previously found to be enriched in the dataset positively-associated with eya2 expression (Figure 5—figure supplement 1 vs Figure 2—figure supplement 1, Supplementary file 2 vs Supplementary file 3). This indicated that perturbating the normal activity of Eya2 disrupted its already predicted functions during regeneration. For instance, GO terms associated with cell cycle, like anaphase-promoting complex-dependent catabolic process, were found significantly different among the genotypes (Figure 5—figure supplement 1a). Focusing on the expression of genes associated with cell cycle progression, we found them significantly down-regulated in eya2-/- tissues, especially at the late-bud blastema (14 dpa, Figure 5a). Gene network analysis showed similar results, with genes associated with DNA replication to be enriched in the dataset found down-regulated in eya2-/- tissues (Figure 5—figure supplement 1b, purple nodes). The most over-represented terms were genes associated with metabolic processes and translation (Figure 5—figure supplement 1b, yellow and green nodes and Supplementary file 3), which previous studies have implicated as a sign of stress (Paschen et al., 2007; Liu and Qian, 2014; Lindqvist et al., 2018). To test whether this was at least in part related to genotoxic stress, we analyzed the expression of genes associated with DDR and found that they were up-regulated in eya2-/- blastemas versus eya2+/- and eya2+/+ (Figure 5b and Supplementary file 1). These data indicate an elevated DNA damage response in eya2-/- mutant blastema cells, which corroborates our previous molecular and histological data showing decreased cell cycle progression and elevated γ-H2AX levels (Figure 4h). On the other hand, the most over-represented terms on the dataset with genes up-regulated in eya2-/- tissues were regulation of biological quality,
{'source': 'PubMedDataset'}