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( a ) Scheme for cell type-level analysis of the aging African turquoise killifish brain. Analyses were performed using the annotated snRNA-seq and snATAC-seq datasets. Changes in cell type proportion with aging were validated using RNAscope in-situ hybridization. ( b-c ) Annotated cell type abundances in the snRNA-seq brain atlas for the GRZ and ZMZ-1001 strains ( b ) and in the snATAC-seq brain atlas for the GRZ strain ( c ). ( d-e ) Cell type proportion change analysis with aging across sexes based on our snRNA-seq ( d ) and snATAC-seq ( e ) datasets using scProportionTest. Left-shifted cell types are more abundant in young brains and right-shifted cell types are more abundant in old brains. Data is colored according to significance, with significant changes corresponding to FDR < 5% based on scProportionTest statistics. Note consistent increases in microglia proportion with aging across comparisons. ( f ) Representative maximum z-projected images using 4-plex RNAscope for each of the GRZ conditions’ cell-type markers: apoeb in yellow , mpz in green , <t>s100b</t> in magenta , map2 in red and DAPI is in blue. See Extended Data Figure 5a for representative images from the ZMZ-1001 strain. Scale bars are 250mm in length. ( g ) Quantification of cell abundances in African turquoise killifish brain samples using top markers for in-situ hybridization by RNAscope. Values for each animal are reported as the average QuPath inferred values obtained across 3 Z-planes. Note that for this sample set, the middle-aged time point for GRZ corresponds to 13-week-old (rather than 10-week-old) fish. The effect of age and sex were evaluated using ANOVA for normally distributed data (GRZ astrocytes, microglia and neurons; ZMZ-1001 astrocytes, neurons, and oligodendrocytes) and non-parametric ART-ANOVA for non-normally distributed data (GRZ oligodendrocytes, ZMZ-1001 microglia). Normality of ANOVA residuals was determined using a Shapiro-Wilkes test, with p < 0.05 indicating violation of the normality assumption. Y: Young (6-7-week-old), M: middle-aged (13-week-old), O: Old (16-week-old), G: Geriatric (26-week-old). F: Female, M: Male.
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( a ) Scheme for cell type-level analysis of the aging African turquoise killifish brain. Analyses were performed using the annotated snRNA-seq and snATAC-seq datasets. Changes in cell type proportion with aging were validated using RNAscope in-situ hybridization. ( b-c ) Annotated cell type abundances in the snRNA-seq brain atlas for the GRZ and ZMZ-1001 strains ( b ) and in the snATAC-seq brain atlas for the GRZ strain ( c ). ( d-e ) Cell type proportion change analysis with aging across sexes based on our snRNA-seq ( d ) and snATAC-seq ( e ) datasets using scProportionTest. Left-shifted cell types are more abundant in young brains and right-shifted cell types are more abundant in old brains. Data is colored according to significance, with significant changes corresponding to FDR < 5% based on scProportionTest statistics. Note consistent increases in microglia proportion with aging across comparisons. ( f ) Representative maximum z-projected images using 4-plex RNAscope for each of the GRZ conditions’ cell-type markers: apoeb in yellow , mpz in green , <t>s100b</t> in magenta , map2 in red and DAPI is in blue. See Extended Data Figure 5a for representative images from the ZMZ-1001 strain. Scale bars are 250mm in length. ( g ) Quantification of cell abundances in African turquoise killifish brain samples using top markers for in-situ hybridization by RNAscope. Values for each animal are reported as the average QuPath inferred values obtained across 3 Z-planes. Note that for this sample set, the middle-aged time point for GRZ corresponds to 13-week-old (rather than 10-week-old) fish. The effect of age and sex were evaluated using ANOVA for normally distributed data (GRZ astrocytes, microglia and neurons; ZMZ-1001 astrocytes, neurons, and oligodendrocytes) and non-parametric ART-ANOVA for non-normally distributed data (GRZ oligodendrocytes, ZMZ-1001 microglia). Normality of ANOVA residuals was determined using a Shapiro-Wilkes test, with p < 0.05 indicating violation of the normality assumption. Y: Young (6-7-week-old), M: middle-aged (13-week-old), O: Old (16-week-old), G: Geriatric (26-week-old). F: Female, M: Male.
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( a ) Scheme for cell type-level analysis of the aging African turquoise killifish brain. Analyses were performed using the annotated snRNA-seq and snATAC-seq datasets. Changes in cell type proportion with aging were validated using RNAscope in-situ hybridization. ( b-c ) Annotated cell type abundances in the snRNA-seq brain atlas for the GRZ and ZMZ-1001 strains ( b ) and in the snATAC-seq brain atlas for the GRZ strain ( c ). ( d-e ) Cell type proportion change analysis with aging across sexes based on our snRNA-seq ( d ) and snATAC-seq ( e ) datasets using scProportionTest. Left-shifted cell types are more abundant in young brains and right-shifted cell types are more abundant in old brains. Data is colored according to significance, with significant changes corresponding to FDR < 5% based on scProportionTest statistics. Note consistent increases in microglia proportion with aging across comparisons. ( f ) Representative maximum z-projected images using 4-plex RNAscope for each of the GRZ conditions’ cell-type markers: apoeb in yellow , mpz in green , <t>s100b</t> in magenta , map2 in red and DAPI is in blue. See Extended Data Figure 5a for representative images from the ZMZ-1001 strain. Scale bars are 250mm in length. ( g ) Quantification of cell abundances in African turquoise killifish brain samples using top markers for in-situ hybridization by RNAscope. Values for each animal are reported as the average QuPath inferred values obtained across 3 Z-planes. Note that for this sample set, the middle-aged time point for GRZ corresponds to 13-week-old (rather than 10-week-old) fish. The effect of age and sex were evaluated using ANOVA for normally distributed data (GRZ astrocytes, microglia and neurons; ZMZ-1001 astrocytes, neurons, and oligodendrocytes) and non-parametric ART-ANOVA for non-normally distributed data (GRZ oligodendrocytes, ZMZ-1001 microglia). Normality of ANOVA residuals was determined using a Shapiro-Wilkes test, with p < 0.05 indicating violation of the normality assumption. Y: Young (6-7-week-old), M: middle-aged (13-week-old), O: Old (16-week-old), G: Geriatric (26-week-old). F: Female, M: Male.
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( a ) Scheme for cell type-level analysis of the aging African turquoise killifish brain. Analyses were performed using the annotated snRNA-seq and snATAC-seq datasets. Changes in cell type proportion with aging were validated using RNAscope in-situ hybridization. ( b-c ) Annotated cell type abundances in the snRNA-seq brain atlas for the GRZ and ZMZ-1001 strains ( b ) and in the snATAC-seq brain atlas for the GRZ strain ( c ). ( d-e ) Cell type proportion change analysis with aging across sexes based on our snRNA-seq ( d ) and snATAC-seq ( e ) datasets using scProportionTest. Left-shifted cell types are more abundant in young brains and right-shifted cell types are more abundant in old brains. Data is colored according to significance, with significant changes corresponding to FDR < 5% based on scProportionTest statistics. Note consistent increases in microglia proportion with aging across comparisons. ( f ) Representative maximum z-projected images using 4-plex RNAscope for each of the GRZ conditions’ cell-type markers: apoeb in yellow , mpz in green , <t>s100b</t> in magenta , map2 in red and DAPI is in blue. See Extended Data Figure 5a for representative images from the ZMZ-1001 strain. Scale bars are 250mm in length. ( g ) Quantification of cell abundances in African turquoise killifish brain samples using top markers for in-situ hybridization by RNAscope. Values for each animal are reported as the average QuPath inferred values obtained across 3 Z-planes. Note that for this sample set, the middle-aged time point for GRZ corresponds to 13-week-old (rather than 10-week-old) fish. The effect of age and sex were evaluated using ANOVA for normally distributed data (GRZ astrocytes, microglia and neurons; ZMZ-1001 astrocytes, neurons, and oligodendrocytes) and non-parametric ART-ANOVA for non-normally distributed data (GRZ oligodendrocytes, ZMZ-1001 microglia). Normality of ANOVA residuals was determined using a Shapiro-Wilkes test, with p < 0.05 indicating violation of the normality assumption. Y: Young (6-7-week-old), M: middle-aged (13-week-old), O: Old (16-week-old), G: Geriatric (26-week-old). F: Female, M: Male.
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( a ) Scheme for cell type-level analysis of the aging African turquoise killifish brain. Analyses were performed using the annotated snRNA-seq and snATAC-seq datasets. Changes in cell type proportion with aging were validated using RNAscope in-situ hybridization. ( b-c ) Annotated cell type abundances in the snRNA-seq brain atlas for the GRZ and ZMZ-1001 strains ( b ) and in the snATAC-seq brain atlas for the GRZ strain ( c ). ( d-e ) Cell type proportion change analysis with aging across sexes based on our snRNA-seq ( d ) and snATAC-seq ( e ) datasets using scProportionTest. Left-shifted cell types are more abundant in young brains and right-shifted cell types are more abundant in old brains. Data is colored according to significance, with significant changes corresponding to FDR < 5% based on scProportionTest statistics. Note consistent increases in microglia proportion with aging across comparisons. ( f ) Representative maximum z-projected images using 4-plex RNAscope for each of the GRZ conditions’ cell-type markers: apoeb in yellow , mpz in green , <t>s100b</t> in magenta , map2 in red and DAPI is in blue. See Extended Data Figure 5a for representative images from the ZMZ-1001 strain. Scale bars are 250mm in length. ( g ) Quantification of cell abundances in African turquoise killifish brain samples using top markers for in-situ hybridization by RNAscope. Values for each animal are reported as the average QuPath inferred values obtained across 3 Z-planes. Note that for this sample set, the middle-aged time point for GRZ corresponds to 13-week-old (rather than 10-week-old) fish. The effect of age and sex were evaluated using ANOVA for normally distributed data (GRZ astrocytes, microglia and neurons; ZMZ-1001 astrocytes, neurons, and oligodendrocytes) and non-parametric ART-ANOVA for non-normally distributed data (GRZ oligodendrocytes, ZMZ-1001 microglia). Normality of ANOVA residuals was determined using a Shapiro-Wilkes test, with p < 0.05 indicating violation of the normality assumption. Y: Young (6-7-week-old), M: middle-aged (13-week-old), O: Old (16-week-old), G: Geriatric (26-week-old). F: Female, M: Male.
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( a ) Scheme for cell type-level analysis of the aging African turquoise killifish brain. Analyses were performed using the annotated snRNA-seq and snATAC-seq datasets. Changes in cell type proportion with aging were validated using RNAscope in-situ hybridization. ( b-c ) Annotated cell type abundances in the snRNA-seq brain atlas for the GRZ and ZMZ-1001 strains ( b ) and in the snATAC-seq brain atlas for the GRZ strain ( c ). ( d-e ) Cell type proportion change analysis with aging across sexes based on our snRNA-seq ( d ) and snATAC-seq ( e ) datasets using scProportionTest. Left-shifted cell types are more abundant in young brains and right-shifted cell types are more abundant in old brains. Data is colored according to significance, with significant changes corresponding to FDR < 5% based on scProportionTest statistics. Note consistent increases in microglia proportion with aging across comparisons. ( f ) Representative maximum z-projected images using 4-plex RNAscope for each of the GRZ conditions’ cell-type markers: apoeb in yellow , mpz in green , <t>s100b</t> in magenta , map2 in red and DAPI is in blue. See Extended Data Figure 5a for representative images from the ZMZ-1001 strain. Scale bars are 250mm in length. ( g ) Quantification of cell abundances in African turquoise killifish brain samples using top markers for in-situ hybridization by RNAscope. Values for each animal are reported as the average QuPath inferred values obtained across 3 Z-planes. Note that for this sample set, the middle-aged time point for GRZ corresponds to 13-week-old (rather than 10-week-old) fish. The effect of age and sex were evaluated using ANOVA for normally distributed data (GRZ astrocytes, microglia and neurons; ZMZ-1001 astrocytes, neurons, and oligodendrocytes) and non-parametric ART-ANOVA for non-normally distributed data (GRZ oligodendrocytes, ZMZ-1001 microglia). Normality of ANOVA residuals was determined using a Shapiro-Wilkes test, with p < 0.05 indicating violation of the normality assumption. Y: Young (6-7-week-old), M: middle-aged (13-week-old), O: Old (16-week-old), G: Geriatric (26-week-old). F: Female, M: Male.
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( a ) Scheme for cell type-level analysis of the aging African turquoise killifish brain. Analyses were performed using the annotated snRNA-seq and snATAC-seq datasets. Changes in cell type proportion with aging were validated using RNAscope in-situ hybridization. ( b-c ) Annotated cell type abundances in the snRNA-seq brain atlas for the GRZ and ZMZ-1001 strains ( b ) and in the snATAC-seq brain atlas for the GRZ strain ( c ). ( d-e ) Cell type proportion change analysis with aging across sexes based on our snRNA-seq ( d ) and snATAC-seq ( e ) datasets using scProportionTest. Left-shifted cell types are more abundant in young brains and right-shifted cell types are more abundant in old brains. Data is colored according to significance, with significant changes corresponding to FDR < 5% based on scProportionTest statistics. Note consistent increases in microglia proportion with aging across comparisons. ( f ) Representative maximum z-projected images using 4-plex RNAscope for each of the GRZ conditions’ cell-type markers: apoeb in yellow , mpz in green , s100b in magenta , map2 in red and DAPI is in blue. See Extended Data Figure 5a for representative images from the ZMZ-1001 strain. Scale bars are 250mm in length. ( g ) Quantification of cell abundances in African turquoise killifish brain samples using top markers for in-situ hybridization by RNAscope. Values for each animal are reported as the average QuPath inferred values obtained across 3 Z-planes. Note that for this sample set, the middle-aged time point for GRZ corresponds to 13-week-old (rather than 10-week-old) fish. The effect of age and sex were evaluated using ANOVA for normally distributed data (GRZ astrocytes, microglia and neurons; ZMZ-1001 astrocytes, neurons, and oligodendrocytes) and non-parametric ART-ANOVA for non-normally distributed data (GRZ oligodendrocytes, ZMZ-1001 microglia). Normality of ANOVA residuals was determined using a Shapiro-Wilkes test, with p < 0.05 indicating violation of the normality assumption. Y: Young (6-7-week-old), M: middle-aged (13-week-old), O: Old (16-week-old), G: Geriatric (26-week-old). F: Female, M: Male.

Journal: bioRxiv

Article Title: A multi-omic atlas in the African turquoise killifish reveals increased glucocorticoid signaling as a hallmark of brain aging

doi: 10.64898/2026.04.09.717549

Figure Lengend Snippet: ( a ) Scheme for cell type-level analysis of the aging African turquoise killifish brain. Analyses were performed using the annotated snRNA-seq and snATAC-seq datasets. Changes in cell type proportion with aging were validated using RNAscope in-situ hybridization. ( b-c ) Annotated cell type abundances in the snRNA-seq brain atlas for the GRZ and ZMZ-1001 strains ( b ) and in the snATAC-seq brain atlas for the GRZ strain ( c ). ( d-e ) Cell type proportion change analysis with aging across sexes based on our snRNA-seq ( d ) and snATAC-seq ( e ) datasets using scProportionTest. Left-shifted cell types are more abundant in young brains and right-shifted cell types are more abundant in old brains. Data is colored according to significance, with significant changes corresponding to FDR < 5% based on scProportionTest statistics. Note consistent increases in microglia proportion with aging across comparisons. ( f ) Representative maximum z-projected images using 4-plex RNAscope for each of the GRZ conditions’ cell-type markers: apoeb in yellow , mpz in green , s100b in magenta , map2 in red and DAPI is in blue. See Extended Data Figure 5a for representative images from the ZMZ-1001 strain. Scale bars are 250mm in length. ( g ) Quantification of cell abundances in African turquoise killifish brain samples using top markers for in-situ hybridization by RNAscope. Values for each animal are reported as the average QuPath inferred values obtained across 3 Z-planes. Note that for this sample set, the middle-aged time point for GRZ corresponds to 13-week-old (rather than 10-week-old) fish. The effect of age and sex were evaluated using ANOVA for normally distributed data (GRZ astrocytes, microglia and neurons; ZMZ-1001 astrocytes, neurons, and oligodendrocytes) and non-parametric ART-ANOVA for non-normally distributed data (GRZ oligodendrocytes, ZMZ-1001 microglia). Normality of ANOVA residuals was determined using a Shapiro-Wilkes test, with p < 0.05 indicating violation of the normality assumption. Y: Young (6-7-week-old), M: middle-aged (13-week-old), O: Old (16-week-old), G: Geriatric (26-week-old). F: Female, M: Male.

Article Snippet: To develop the fluorescent signal, TSA Vivid Fluorophore 520 (Advanced Cell Diagnostics, 323272) was used to mark apoeb , TSA Vivid Fluorophore 570 (Advanced Cell Diagnostics, 323271) was used to mark mpz , Opal 620 (Akoya Biosciences, FP1495001KT) was used to mark s100b , and Opal 690 (Akoya Biosciences, FP1497001KT) was used to mark map2 .

Techniques: RNAscope, In Situ Hybridization