Elevated DNA damage without signs of aging in the short-sleeping Mexican cavefish
- Department of Biology, Texas A&M University, United States
- Stowers Institute for Medical Research, United States
- Harriet Wilkes Honors College, Florida Atlantic University, United States
- Department of Genomics, University of Missouri, United States
- Faculty of Life Science and the Multidisciplinary Brain Research Center, Bar Illan University, Israel
Figures
Figure 1 with 1 supplement
Cavefish harbor increased neuronal DNA damage and gut reactive oxygen species (ROS).
(A, B) Representative images of cells stained with DAPI and γH2AX in the rhombencephalon of surface fish (A) and cavefish (B). Scale bar = 5 μm. (C) Mean γH2AX fluorescence across three regions of surface fish and cavefish brains. (rhomb: rhombencephalon; mes: mesencephalon; tele: telencephalon) (Mixed-effects analysis: F1, 68 = 32.08, p< 0.0001; surface n = 29, cave n = 41). (D) Representative image of larval gut showing regions in false color (re: rectum). (E, F) Representative images of surface fish and cavefish guts stained with dihydroethidium (DHE) marking ROS. Scale bar = 500 μm. (G) Mean DHE fluorescence across four regions of surface fish and cavefish guts (two-way repeated measures ANOVA: F1, 35 = 48.36, p<0.0001, surface n = 19, cave n = 20). All error bars represent S.E.M.
Figure 1—video 1
DNA damage in the cavefish brain.
Whole brain immunofluorescence images of surface fish (top) and cavefish (bottom). Immunostaining for γH2AX (green) and DAPI (blue).
Figure 2 with 1 supplement
Cavefish lack a sleep response to UV-B-induced DNA damage.
(A) The 24 hr sleep profiles of surface fish exposed to 30 or 60 s of UV-B light compared to controls. (B) Average sleep amount in surface fish in the 3 hr following UV-B exposure (one-way ANOVA: F2, 202 = 18.75, p< 0.0001). (C) Average bout length in surface fish in the 3 hr following UV-B exposure (one-way ANOVA: F2, 201 = 8.301, p=0.0003). (D) Bout number in the 3 hr following UV-B exposure (one-way ANOVA: F2, 201 = 11.5, p<0.0001). (E) The 24 hr sleep profiles of cavefish exposed to 30 or 60 s of UV-B light compared to controls. (F) Average sleep amount in cavefish in the 3 hr following UV-B exposure. (G) Average bout length in cavefish in the 3 hr following UV-B exposure. (H) Bout the number of cavefish in the 3 hr following UV-B exposure. (ZT = Zeitgeber time). All treatments performed at ZT0. Surface: Ctrl n = 65, 30s n = 70, 60s n = 70; Cave: Ctrl n = 41, 30s n = 34, 60s n = 29. All error bars represent S.E.M.
Figure 2—figure supplement 1
Response in sleep pressure to UV-B-induced DNA damage.
(A) The 24 hr P(wake) profiles of surface fish exposed to 30 or 60 s of UV-B light compared to controls. (B) P(wake) in surface fish in the 3 hr following UV-B exposure (one-way ANOVA: F2, 202 = 21.08, p<0.0001). (C) The 24 hr P(doze) profiles of surface fish exposed to 30 or 60 s of UV-B light compared to controls. (D) P(doze) in surface fish in the 3 hr following UV-B exposure (one-way ANOVA: F2, 202 = 14.09, p<0.0001). (E) The 24 hr P(wake) profiles of cavefish exposed to 30 or 60 s of UV-B light compared to controls. (F) P(wake) in cavefish in the 3 hr following UV-B exposure. (G) The 24 hr P(doze) profiles of cavefish exposed to 30 or 60 s of UV-B light compared to controls. (H) P(doze) in cavefish in the 3 hr following UV-B exposure. (ZT = Zeitgeber time). All treatments were performed at ZT0. Surface: Ctrl n = 65, 30s n = 70, 60s n = 70; Cave: Ctrl n = 41, 30s n = 34, 60s n = 29. All error bars represent S.E.M.
Figure 3 with 2 supplements
Transcriptional responses to UV-B-induced DNA damage in surface fish and cavefish.
(A) Schematic of experimental design. (B) Multidimensional scaling plot depicting the variances in principal component space between the processed sequencing samples. PC1 two-way ANOVA: (Treatment) F1,10=6.388, p=0.03, (Population) F1,10=4970, p<0.0001, (Interaction) F1,10=17.56, p=0.0019. PC2 two-way ANOVA: (Treatment) F1,10=465.0, p<0.0001, (Population) F1,10=0.4969, p=0.497, (Interaction) F1,10=18.56, p=0.0015 (C) Bi-directional volcano plot of changes in gene expression in surface and cave larvae after exposure to DNA-damaging UV-B radiation. (D) Heat map of gene expression in DNA repair genes which responded significantly in UV-B-exposed surface fish.
Figure 3—figure supplement 1
Transcriptional responses to UV-B-induced DNA damage in surface fish and cavefish.
(A) Volcano plot of gene expression in surface fish in response to UV-B radiation, with significant genes highlighted in red (adjusted p<0.05). (B) Volcano plot of gene expression in cavefish in response to UV-B radiation, with significant genes highlighted in red (adjusted p<0.05). (C) Most highly enriched Gene Ontology (GO) terms in a gene set enrichment analysis of surface fish exposed to UV-B radiation. (D) Most highly enriched GO terms in a gene set enrichment analysis of cavefish exposed to UV-B radiation.
Figure 3—figure supplement 2
DNA repair gene expression in surface fish and cavefish.
(A,D) Adult fins were collected at Zeitgeber or Circadian time 8. cpdp (A) and ddb2 (D) mRNA levels were determined by qPCR. Expression of cpdp (one-way ANOVA: F=11.99, p=0.0039) and ddb2 (one-way ANOVA: F=0.7576, p=0.4997) was compared among all populations. ns p≥0.05, **p<0.01. (B,E) RNA sequencing data obtained from cpdp (B) and ddb2 (E) gene expression levels in 30 dpf juvenile fish. (TPM: transcript per million). (C,F) Fish larvae aged 5 dpf were collected at different Zeitgeber or Circadian times. cpdp (C) and ddb2 (F) mRNA levels were determined by qPCR.
Figure 4 with 1 supplement
Pachón cavefish-derived cells exhibit a lower UV-induced DNA damage response and repair compared to surface fish.
(A, B) Immunostaining of cyclobutene pyrimidine dimer (CPD) shows a similar DNA damage level induced by UV in surface fish and Pachón cavefish embryonic fibroblasts. White circles indicate the nuclear area by DAPI staining. Orange indicates CPD. Scale bar, 40 μm. P-values were determined by two-way ANOVA: F=0.09703, p=0.7586. ns p=0.6404, ****p<0.0001. (C, D) Western blot of γH2AX indicates a diminished DNA damage response in Pachón cavefish embryonic fibroblasts compared to surface fish cells. (E, F) Flow cytometry images and quantification for host cell reactivation assays in surface fish and Pachón cavefish embryonic fibroblasts. Red line sets the green fluorescent protein (GFP) positive signal threshold. P-values were determined by unpaired t-test. (G) Representative GFP images for host cell reactivation assays in surface fish and Pachón cavefish embryonic fibroblasts. Scale bars, 500 μm.
-
Figure 4—source data 1
Original gels for data presented in Figure 4.
- https://cdn.elifesciences.org/articles/99191/elife-99191-fig4-data1-v1.zip
-
Figure 4—source data 2
Original western blots for data presented in Figure 4.
- https://cdn.elifesciences.org/articles/99191/elife-99191-fig4-data2-v1.zip
Figure 4—figure supplement 1
Pachón cavefish-derived cells exhibit a lower UV-induced DNA damage response and repair compared to surface fish.
(A) Bright field images of surface fish and Pachón cavefish embryonic fibroblasts. Scale bar, 200 μm. (B) Immunostaining of vimentin in surface fish and Pachón cavefish embryonic fibroblasts. Yellow and blue show vimentin and DAPI staining, respectively. Scale bar, 100 μm. (C) Heat map of differentially expressed genes among different cell types. The number below the heat map indicates independent biological replicates (n=13). Red and blue indicate upregulated and downregulated genes, respectively. (SEF: surface fish embryonic fibroblast; PEF: Pachón cavefish embryonic fibroblast; MEF: mouse embryonic fibroblast; iPSC: induced pluripotent stem cell; mESC: mouse embryonic fibroblast; SFL: surface fish liver-derived cell; CFL: Pachón cavefish liver-derived cell). (D) Western blot of γH2AX indicates a diminished DNA damage response in Pachón cavefish liver-derived cells compared to surface fish liver-derived cells.
-
Figure 4—figure supplement 1—source data 1
Original gels for data presented in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/99191/elife-99191-fig4-figsupp1-data1-v1.zip
-
Figure 4—figure supplement 1—source data 2
Original western blots for data presented in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/99191/elife-99191-fig4-figsupp1-data2-v1.zip
Figure 5 with 2 supplements
Transcriptional response to aging is diminished in cavefish across tissues.
(A) Multidimensional scaling plot plotting the distances in principal component space between the brain samples. PC1 two-way ANOVA: (Treatment) F1,11=4.209, p=0.0648, (Population) F1,11=2133, p<0.0001, (Interaction) F1,11=0.029, p=0.867 PC2 two-way ANOVA: (Treatment) F1,11=16.83, p=0.0018, (Population) F1,11=0.0002, p<0.99, (Interaction) F1,11=19.37, p=0.0011 (B) Number of differentially expressed genes in the aged condition across tissues. (C) Bi-directional volcano plot depicting differences in gene expression between young and aged brains. (D) Dot plot visualizing the top 8 activated and suppressed gene ontology terms in aged cavefish brains resulting from gene set enrichment (GSE) analysis.
Figure 5—figure supplement 1
Aging-induced changes in gene expression.
Multi-dimensional scaling plots of gene expression in young and aged surface fish and cavefish samples, in the gut (A) PC1 two-way ANOVA: (Treatment) F1,11=10.41, p=0.0081, (Population) F1,11=264, p<0.0001, (Interaction) F1,11=3.135, p=0.1043. PC2 two-way ANOVA: (Treatment) F1,11=8.257, p=0.0151, (Population) F1,11=0.594, p=0.4571, (Interaction) F1,11=7.113, p=0.0219. Heart (B) PC1 two-way ANOVA: (Treatment) F1,12=0.0302, p=0.8649, (Population) F1,12=0.8455, p=0.376, (Interaction) F1,12=0.9535, p=0.3481. PC2 two-way ANOVA: (Treatment) F1,12=7.354, p=0.0189, (Population) F1,12=0.7584, p=0.0175, (Interaction) F1,12=0.3345, p=0.5737. Liver (C) PC1 two-way ANOVA: (Treatment) F1,12=1.067, p=0.3219, (Population) F1,12=4.521, p=0.0549, (Interaction) F1,12=0.1329, p=0.7217. PC2 two-way ANOVA: (Treatment) F1,12=2.039, p=0.1788, (Population) F1,12=6.986, p=0.0214, (Interaction) F1,12=0.0419, p=0.8413. Muscle (D) PC1 two-way ANOVA: (Treatment) F1,12=0.5895, p=0.4574, (Population) F1,12=0.7313, p=0.4092, (Interaction) F1,12=0.01396, p=0.9079. PC2 two-way ANOVA: (Treatment) F1,12=0.15, p=0.7053, (Population) F1,12=89.42, p<0.0001, (Interaction) F1,12=0.009, p=0.9251.
Figure 5—figure supplement 2
Gene set enrichment (GSEA) analyses of aging surface fish and cavefish tissues.
Top Gene Ontology (GO) terms resulting from GSEA analysis in aged surface fish (left) and cavefish (right) tissues. Gut (A, B), heart (C, D), liver (E, F), and muscle (G, H). Top eight results in each direction are shown; if than eight terms were enriched, all results are shown.
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/99191/elife-99191-mdarchecklist1-v1.docx
-
Supplementary file 1
UV-B Treatment gene expression raw data.
- https://cdn.elifesciences.org/articles/99191/elife-99191-supp1-v1.xlsx
-
Supplementary file 2
Aging gene expression raw data.
- https://cdn.elifesciences.org/articles/99191/elife-99191-supp2-v1.xlsx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Elevated DNA damage without signs of aging in the short-sleeping Mexican cavefish
eLife 13:RP99191.
https://doi.org/10.7554/eLife.99191.3
