Figures and data
A model for engulfment by vertebrate brain myeloid cells.
a, Schematic for the SP-oScarlet killifish line, which features expression and secretion of a red fluorescent protein, oScarlet, from non-immune cells of the killifish brain using CRISPR knockin at the ELAVL3 locus. P2A, self-cleaving peptide; UTR, untranslated region. The SP-oScarlet line is maintained in the heterozygous state. Art by Rogelio Barajas (killifish). b, Representative images of brain sections from young adult (59 days) female heterozygous SP-oScarlet and young adult (59 days) female wildtype killifish, highlighting oScarlet protein signal (native fluorescence plus anti-mCherry immunofluorescence staining) in the SP-oScarlet brain (representative of n = 13 SP-oScarlet fish over three experiments and n = 3 wildtype fish in one experiment). Scale bars = 5 μm. c, Representative image of a young adult (59 days) female heterozygous SP-oScarlet killifish brain section, highlighting oScarlet protein signal (native fluorescence plus anti-mCherry immunofluorescence staining) in a cell that does not express oScarlet RNA (representative of n = 10 fish, 59-63 days females, over two experiments). Scale bar = 5 μm. d, (Left) 3D image of a cleared (modified Adipo-Clear method) young adult (50 days) male heterozygous SP-oScarlet killifish midbrain/hindbrain, highlighting punctate oScarlet protein signal (native fluorescence plus anti-RFP immunofluorescence staining) (n = 1 fish). (Right) Schematic for single-cell RNA sequencing of cells with and without oScarlet protein signal in young adult (49 days) and old (134 days) male heterozygous SP-oScarlet killifish brains (n = 4 fish, pooled, per age group, in one experiment). For data from old fish and for an independent experiment in old fish, see Fig. 4. e, (Left) Fluorescence-activated cell sorting (FACS) plot for young adult pooled brains from experiment in d, highlighting oScarletLOW and oScarletHIGH cells. Each dot represents one cell. (Right) Image of an oScarletHIGH cell isolated from a middle-aged (76 days) male heterozygous SP-oScarlet killifish brain by FACS, highlighting oScarlet protein signal (native fluorescence only) (n = 1 fish). Scale bar = 2 μm. f, Uniform Manifold Approximation and Projection (UMAP) of 11,503 oScarletLOW and 9,680 oScarletHIGH high-quality young adult cells from experiment in d. Each dot represents one cell. g, UMAP as in f colored according to manually annotated cell types, labeled in legend. Each dot represents one cell. h, (Left) UMAP as in f colored according to log-normalized CSF1R (LOC107387189) expression. Each dot represents one cell. (Right) Representative images of young adult (59-63 days) female heterozygous SP-oScarlet killifish brain sections, highlighting oScarlet protein signal (native fluorescence plus anti-mCherry immunofluorescence staining) in cells expressing CSF1R RNA (representative of n = 10 fish, 59-63 days females, over two experiments). Scale bars = 5 μm. i, UMAP as in f colored according to log-normalized ELAVL3 (LOC107378364) expression. Each dot represents one cell.
The killifish brain includes a population of macrophages with transcriptional similarities to mammalian monocyte-derived macrophages.
a, Dotplot highlighting expression of selected genes (CSF1R (LOC107387189), APOEB (LOC10737395), HLA-DPB1 (LOC107372638), CD74 (LOC107375728), C1QC (LOC107385068), C1QA (LOC107385067), MRC1 (LOC107383768), MARCO (LOC107390282), TMEM119 (LOC107393987), F13A1 (LOC107377057), and CLDN5 (LOC107394244)) in young adult oScarletLOW and oScarletHIGH cells from experiment in 1d. The color of each circle corresponds to average log-normalized expression among cells with detectable expression of the respective gene. The size of each circle corresponds to percentage of cells in each population with detectable expression of the gene. b, Violin plots for sum log-normalized expression of genes in Gene Ontology: Biological Process terms “endocytosis” (GO:0006897), “positive regulation of endocytosis” (GO:0045807), “lysosome” (GO:0005764), “positive regulation of lysosome organization” (GO:1905673), and “vacuolar acidification” (GO: 0007035) in young adult oScarletLOW and oScarletHIGH cells from experiment in 1d. Each dot represents one cell. Lines: medians; p-values, unpaired Wilcoxon rank-sum test at per-cell level (fish were pooled for single cell RNA-sequencing experiments to obtain sufficient numbers of oScarletHIGH cells). c, Principal component analysis (PCA) plots (principal components 1 vs. 2) of wildtype young adult mouse microglia, border-associated macrophages (BAMs), and monocyte-derived macrophages (MDMs) (Barr et al., 202575; 3,230 high-quality cells from clusters manually annotated by Barr et al.), young adult oScarletHIGH cells from experiment in 1d, and wildtype young adult killifish forebrain macrophages (Ayana et al., 202468; 176 high-quality cells from APOEB+ IBA1+ LCP1+ cluster) using marker genes of microglia, BAMs, and MDMs from the Barr et al. dataset, or all killifish homologs of these genes. Each dot represents one cell. Percentages on axes represent the percentage of variance explained by each respective principal component. Art by Rogelio Barajas. d, UMAPs of wildtype young adult mouse brain macrophages (Barr et al., 202575; 3,488 high-quality cells from clusters manually annotated by Barr et al.), young adult oScarletHIGH cells from experiment in 1d, and wildtype young adult killifish forebrain macrophages (Ayana et al., 202468) colored according to log-normalized Tmem119 (or killifish homolog TMEM119 (LOC107393987)) or CD74 (or killifish homolog CD74 (LOC107375728)). Each dot represents one cell.
Macrophages in the killifish brain are endocytic and present at brain borders.
a, (Left) Schematic for injection of dextran-Oregon Green 488 (70 kDa) into the brains of young adult to middle-aged (63-91 days) female heterozygous SP-oScarlet killifish (n = 7 fish over two experiments). (Center) Representative images of middle-aged (91 days) female heterozygous SP-oScarlet killifish brain sections highlighting co-occurrence of injected dextran signal, oScarlet protein signal (native fluorescence plus anti-mCherry immunofluorescence staining), and APOEB RNA in the same cells. Scale bars = 10 μm. (Right) Quantification of mean dextran fluorescence intensity and mean APOEB RNA fluorescence intensity in oScarletLOW and oScarletHIGH cells (n = 6 successfully injected fish, 63-91 days females, with 5-6 regions of interest over 1-2 sagittal brain sections per fish, over two experiments). Four images (one image per fish for each of four fish) of regions of interest with a visually apparent very high density of cells with high APOEB RNA expression, which could plausibly reflect an injection-related injury, were excluded from quantifications. The excluded images are listed in Supplementary Table 4. Each triangle represents one fish. Mean +/-standard error of mean; p-values, paired Wilcoxon rank-sum test at per-animal level. b, (Left) Representative images of young adult to middle-aged (74-101 days) male and female wildtype killifish brain sections, highlighting cells with high APOEB RNA expression that do and do not colocalize with vasculature as labeled by perfused sulfo-NHS-biotin (representative of n = 6 fish, 3 males, 74-101 days, and 3 females, 74-101 days, over two experiments). Scale bars = 50 μm (leftmost image), 5 μm (all others). Dashed lines: brain outside border. (In bottom right image) Filled arrows: example of cell with high APOEB RNA expression that does not colocalize with vasculature or other brain borders. Unfilled arrows: examples of cells with high APOEB RNA expression that are found near brain borders. (Right) Quantification of proportion of cells with high APOEB RNA expression among all cells and among cells colocalizing with vasculature, as defined by centroid-centroid distance to nearest sulfo-NHS-biotin-positive cell, in forebrain, midbrain, and hindbrain regions imaged (n = 6 fish, 74-101 days, with 1-2 regions of interest per brain region over 1-2 sagittal brain sections per fish, over two experiments). Regions of interest imaged generally avoided brain outside borders due to very high brightness of sulfo-NHS-biotin signal at some parts of brain outside borders, which could bias quantification. Each square (male) or triangle (female) represents one fish.
Killifish brain macrophages change transcriptionally with age in our model.
a, UMAP of 6,489 oScarletHIGH high-quality cells from old (134 days) male heterozygous SP-oScarlet brains (n = 4 fish, pooled, in one experiment), as well as 9,680 young adult oScarletHIGH high-quality cells from experiment in 1d (same experiment). b, Dotplot highlighting expression of selected homologs of mammalian brain macrophage marker genes (CSF1R (LOC107387189), APOEB (LOC10737395), HLA-DPB1 (LOC107372638), C1QC (LOC107385068), and MRC1 (LOC107383768)) in old and young adult oScarletHIGH cells from experiment in 1d. The color of each circle corresponds to average log-normalized expression among cells with detectable expression of the respective gene. The size of each circle corresponds to percentage of cells in each population with detectable expression of the gene. c, Violin plots for sum log-normalized expression of genes in Gene Ontology: Biological Process terms “cytoplasmic translation” (GO:0002181) and “vacuolar acidification” (GO: 0007035) in young adult and old oScarletHIGH cells from experiment in 1d. Each dot represents one cell. Lines: medians; p-values, unpaired Wilcoxon rank-sum test at per-cell level. d, Top Gene Ontology: Biological Process terms upregulated in old (left) and young adult (right) oScarletHIGH cells from experiment in 1d, as ranked by adjusted p-value (Fisher’s exact test, Benjamini-Hochberg false discovery rate (FDR)-corrected). Size of each circle corresponds to number of distinct human homologs among differentially expressed genes from each term. Full lists of differentially expressed genes and upregulated terms in Supplementary Tables 9-11. e, Schematic for single-cell RNA sequencing of oScarletHIGH cells from old (130 days) male heterozygous SP-oScarlet killifish brains and all Live cells from old (122 days) male wildtype killifish brains (n = 3 fish per genotype, pooled in one experiment; for an independent experiment involving old SP-oScarlet fish, see 1d). f, PCA plot (principal components 1 vs. 2) of oScarletHIGH and wildtype old killifish brain macrophages from experiment in e using all killifish homologs of marker genes of microglia, BAMs, and MDMs (Barr et al., 202575). Each dot represents one cell. Percentages on axes represent the percentage of variance explained by each respective principal component. g, UMAP of 12,575 old oScarletHIGH and old wildtype macrophages, as defined by APOEB+ IBA1+ LCP1+ clusters among all cells from experiment in e (resolution = 0.2). Each dot represents one cell. h, Dotplot highlighting expression of selected homologs of mammalian brain macrophage marker genes (CSF1R (LOC107387189), APOEB (LOC10737395), HLA-DPB1 (LOC107372638), C1QC (LOC107385068), and MRC1 (LOC107383768)) in old oScarletHIGH and old wildtype macrophages from experiment in e. The color of each circle corresponds to average log-normalized expression among cells with detectable expression of the respective gene. The size of each circle corresponds to percentage of cells in each population with detectable expression of the gene. i, Violin plot for sum log-normalized expression of genes in Gene Ontology: Biological Process term “cytoplasmic translation” (GO:0002181) in old oScarletHIGH and old wildtype macrophages from experiment in e. Each dot represents one cell. Lines: medians; p-values, unpaired Wilcoxon rank-sum test at per-cell level.
Engulfment capacity of killifish brain macrophages declines with age in our model.
a, (Left) Representative flow cytometry histograms from young adult (41 days, above) and old (160 days, below) male heterozygous SP-oScarlet killifish brains, highlighting mean oScarlet fluorescence intensity in oScarletHIGH cells. (Right) Quantification of mean oScarlet fluorescence intensity among young adult and old oScarletHIGH cells (n = 6 fish per age per sex, over two experiments per sex; young adult males: 41-63 days; old males: 148-160 days; young adult females: 57-63 days; old females: 161-167 days). Each square (male) or triangle (female) represents one fish. Mean +/- standard error of mean; p-values, unpaired Wilcoxon rank-sum test at per-animal level. b, Schematic for ex vivo engulfment assays performed on young adult and old heterozygous SP-oScarlet killifish brains (for ovalbumin-Alexa Fluor® 647: n = 6 fish per age and sex, over two experiments per sex; young adult males: 41-63 days; old males: 148-160 days; young adult females: 57-63 days; old females: 161-167 days; for dextran-Oregon Green 488 (70 kDa): n = 4 male fish per age in one experiment; young adult: 56 days; old: 156 days). c, (Left) Representative flow cytometry histograms from young adult (41 days) male heterozygous SP-oScarlet killifish brains, highlighting mean ovalbumin fluorescence intensity in oScarletLOW and oScarletHIGH cells. (Right) Quantification of mean ovalbumin fluorescence intensity among young adult oScarletLOW and oScarletHIGH cells (same experiments as in a). Each square (male) or triangle (female) represents one fish. Mean +/- standard error of mean; p-values, paired Wilcoxon rank-sum test at per-animal level. d, (Left) Representative flow cytometry histograms from young adult (41 days) (above) and old (160 days) (below) male heterozygous SP-oScarlet killifish brains, highlighting mean ovalbumin fluorescence intensity in oScarletHIGH cells. (Right) Quantification of mean ovalbumin fluorescence intensity among young adult and old oScarletHIGH cells (same experiments as in a). Each square (male) or triangle (female) represents one fish. Mean +/- standard error of mean; p-values, unpaired Wilcoxon rank-sum test at per-animal level. e, (Left) Representative flow cytometry histograms from young adult (56 days) male heterozygous SP-oScarlet killifish brains, highlighting mean dextran fluorescence intensity in oScarletLOW and oScarletHIGH cells. (Right) Quantification of mean dextran fluorescence intensity among young adult oScarletLOW and oScarletHIGH cells. Each square represents one fish. Mean +/- standard error of mean; p-values, paired Wilcoxon rank-sum test at per-animal level. f, (Left) Representative flow cytometry histograms from young adult (56 days) (above) and old (156 days) (below) male heterozygous SP-oScarlet killifish brains, highlighting mean dextran fluorescence intensity in oScarletHIGH cells. Each dot represents one cell. (Right) Quantification of mean dextran fluorescence intensity among young adult and old oScarletHIGH cells (same experiment as in e). Each square represents one fish. Mean +/-standard error of mean; p-values, unpaired Wilcoxon rank-sum test at per-animal level.
a, (Left) Schematic for PCR genotyping of a middle-aged (78 days) male heterozygous SP-oScarlet killifish, and DNA gel result (representative of n = 2 fish, 78 days males, in one experiment). (Right) In silico translated sequence of sequenced SP-oScarlet insertion (n = 1 fish, 78 days male). b, Representation of killifish brain highlighting approximate brain regions as referred to in this study. Art by Massimo Demma (adapted with permission from D’Angelo et al., 2013169). c, (Above) Representative image of a young adult (59 days) female heterozygous SP-oScarlet killifish brain section, highlighting ELAVL3 transcripts and oScarlet transcripts in the periventricular gray zone, a neuron-dense region of the killifish midbrain. Scale bar = 5 μm. (Below) Per-cell quantification of number of detected ELAVL3 transcripts and number of detected oScarlet transcripts (1,498 cells from n = 4 fish, 59 days females, with 1 region of interest in 1 sagittal brain section per fish, in one experiment). d, Same image as in 1b (left), highlighting examples of oScarletLOW and oScarletHIGH cells as detected in situ. (Below) Per-cell quantification of mean oScarlet fluorescence intensity and number of detected oScarlet transcripts from experiment in 1c (25,622 cells from n = 10 fish, 59-63 days females, with 3-6 regions of interest over 1-2 sagittal brain sections per fish, over two experiments). Each dot represents one cell. oScarletHIGH cells that have 0 detected oScarlet RNA transcripts are highlighted in red. e, FACS plots highlighting gating scheme used to isolate Live cells (as depicted in 1e) from dissociated young adult male SP-oScarlet brains from experiment in 1d. f, FACS plots highlighting oScarletHIGH cells notably detected in old (130 days old) male heterozygous SP-oScarlet but not in old (122 days old) male wildtype brains (n = 3 fish per genotype, pooled in one experiment). Each dot represents one cell.
a, Heatmap of expression of top 5 marker genes per cell type, as ranked by area under curve (Presto), in every young adult cell from experiment in 1d. Full list of cell type marker genes in Supplementary Table 8. b, Violin plots from experiment in 1d highlighting expression of APOEB (LOC10737395), IBA1 (LOC107378674), LCP1 (LOC107389591), and CSF1R (LOC107387189) in each cell type. Each dot represents one cell. Lines: medians.
a, Representative image of a young adult (59 days) female heterozygous SP-oScarlet killifish brain section, highlighting oScarlet protein signal colocalized with the late endosome and lysosome marker LAMP1 in an SP-oScarlet brain (representative of n = 8 fish, 59-63 days females, over two experiments). Scale bar = 2 μm. b, Principal component analysis (PCA) plots (principal components 1 vs. 3) of wildtype young adult mouse brain macrophages (Barr et al., 202575), young adult oScarletHIGH cells from experiment in 1d, and wildtype young adult killifish forebrain macrophages (Ayana et al., 202468) using marker genes of microglia, BAMs, and MDMs from the Barr et al. dataset, or all killifish homologs of these genes. Each dot represents one cell. Percentages on axes represent the percentage of variance explained by each respective principal component.
a, UMAPs of wildtype young adult mouse brain macrophages (Barr et al., 202575), young adult oScarletHIGH cells from experiment in 1d, and wildtype young adult killifish forebrain macrophages (Ayana et al., 202468) colored according to log-normalized Cldn5 (or killifish homolog CLDN5 (LOC107394244)), Mrc1 (or killifish homolog MRC1 (LOC107383768)), F13a1 (or killifish homolog F13A1 (LOC107377057)), Csf1r (or killifish homolog CSF1R (LOC107381415)), or Apoe (or killifish homolog APOEB (LOC107379395)) expression. Each dot represents one cell.
a, Heatmaps of expression of killifish homologs of strongest microglia, BAM, and MDM marker genes (Barr et al., 202575) in young adult oScarletHIGH cells from experiment in 1d.
a, Heatmaps of expression of killifish homologs of strongest microglia, BAM, and MDM marker genes (Barr et al., 202575) in wildtype young adult killifish forebrain macrophages (Ayana et al., 202468).
a, Quantification of mean oScarlet fluorescence intensity and mean APOEB RNA fluorescence intensity in dextran-negative and dextran-positive cells (n = 6 successfully injected fish, 63-91 days females, with 5-6 regions of interest over 1-2 sagittal brain sections per fish, over two experiments). Four images (one image per fish for each of four fish) of regions of interest with a visually apparent very high density of cells with high APOEB RNA expression, which could plausibly reflect an injection-related injury, were excluded from quantifications. The excluded images are listed in Supplementary Table 4. Each triangle represents one fish. Mean +/-standard error of mean; p-values, paired Wilcoxon rank-sum test at per-animal level. b, Quantification from experiment in 3a of proportion of dextran-positive cells per fish (n = 7 fish, 63-91 days females, with 5-6 regions of interest over 1-2 sagittal brain sections per fish, over two experiments), highlighting in grey one fish in which dextran was not observed to circulate throughout the brain (also confirmed by visual inspection of sections). This unsuccessfully injected fish (63 days, female) was excluded from subsequent quantifications. Each triangle represents one fish. c, Representative image of brain sections from the young adult (63 days) female heterozygous SP-oScarlet killifish unsuccessfully injected with dextran (n = 1 fish). Scale bars = 10 μm.
a, Violin plots for sum log-normalized expression of genes in Gene Ontology: Biological Process terms “endocytosis” (GO:0006897), “positive regulation of endocytosis” (GO:0045807), “lysosome” (GO:0005764), and “positive regulation of lysosome organization” (GO:1905673) in young adult and old oScarletHIGH cells from experiment in 1d. Each dot represents one cell. Lines: medians; p-values, unpaired Wilcoxon rank-sum test at per-cell level (fish were pooled for single cell RNA-sequencing experiments to obtain sufficient numbers of oScarletHIGH cells). b, Violin plots for sum log-normalized expression of genes in Gene Ontology: Biological Process terms “inflammatory response” (GO:0006954) and “positive regulation of inflammatory response” (GO:0050729) in young adult and old oScarletHIGH cells from experiment in 1d. Each dot represents one cell. Lines: medians; p-values, unpaired Wilcoxon rank-sum test at per-cell level. c, PCA plot (principal components 1 vs. 3) of oScarletHIGH and wildtype old killifish brain macrophages from experiment in 4e using all killifish homologs of marker genes of microglia, BAMs, and MDMs (Barr et al., 202575). Each dot represents one cell. Percentages on axes represent the percentage of variance explained by each respective principal component. d, Top Gene Ontology: Biological Process terms upregulated in old oScarletHIGH macrophages (above) and in old wildtype macrophages (below) from experiment in 4e, as ranked by adjusted p-value (Fisher’s exact test, Benjamini-Hochberg FDR-corrected). Size of each circle corresponds to number of distinct human homologs among differentially expressed genes from each term. Full lists of differentially expressed genes and upregulated terms in Supplementary Tables 12-14. e, Violin plots for sum log-normalized expression of genes in Gene Ontology: Biological Process terms “endocytosis” (GO:0006897), “positive regulation of endocytosis” (GO:0045807), “lysosome” (GO:0005764), “positive regulation of lysosome organization” (GO:1905673), and “vacuolar acidification” (GO: 0007035) in old oScarletHIGH and old wildtype macrophages from experiment in 4e. Each dot represents one cell. Lines: medians; p-values, unpaired Wilcoxon rank-sum test at per-cell level.
a, Representative flow cytometry plots from young adult (41 days, above) and old (160 days, below) male heterozygous SP-oScarlet killifish brains, highlighting oScarletHIGH cells. Each dot represents one cell. b, Plot of normalized ELAVL3 (LOC107378364) RNA counts across age from bulk RNA sequencing of whole killifish brains (Costa et al., 202655). Each square (male) or triangle (female) represents one fish. Dashed line: linear regression. r: Pearson correlation coefficient; p-value, two-sided t-test. c, (Left) Representative flow cytometry plots from young adult (41 days) male heterozygous SP-oScarlet killifish brains, highlighting ovalbumin+ cells as identified from all Live cells, with mean ovalbumin fluorescence intensity plotted against either FSC-A (above) or mean oScarlet fluorescence intensity (below). Each dot represents one cell. (Right) Quantifications of proportion of ovalbumin+ cells and mean ovalbumin fluorescence intensity of ovalbumin+ cells among young adult oScarletLOW and oScarletHIGH cells (same experiments as in 5a). Each square (male) or triangle (female) represents one fish. Mean +/-standard error of mean; p-values, paired Wilcoxon rank-sum test at per-animal level. d, (Left) Representative flow cytometry plots from young adult (41 days) (above) and old (160 days) (below) male heterozygous SP-oScarlet killifish brains, highlighting ovalbumin+ oScarletHIGH cells. Each dot represents one cell. (Right) Quantifications of proportion of ovalbumin+ cells and mean ovalbumin fluorescence intensity of ovalbumin+ cells among young adult and old oScarletHIGH cells (same experiments as in 5a). Each square (male) or triangle (female) represents one fish. Mean +/- standard error of mean; p-values, unpaired Wilcoxon rank-sum test at per-animal level. e, (Left) Representative flow cytometry plots from young adult (56 days) male heterozygous SP-oScarlet killifish brains, highlighting dextran+ cells as identified from all Live cells, with mean dextran fluorescence intensity plotted against either FSC-A (above) or mean oScarlet fluorescence intensity (below). Each dot represents one cell. (Right) Quantifications of proportion of dextran+ cells and mean dextran fluorescence intensity of dextran+ cells among young adult oScarletLOW and oScarletHIGH cells (same experiment as in 5e). Each square represents one fish. Mean +/- standard error of mean; p-values, paired Wilcoxon rank-sum test at per-animal level. f, (Left) Representative flow cytometry plots from young adult (56 days) (above) and old (156 days) (below) male heterozygous SP-oScarlet killifish brains, highlighting dextran+ oScarletHIGH cells. Each dot represents one cell. (Right) Quantifications of proportion of dextran+ cells and mean dextran fluorescence intensity of dextran+ cells among young adult and old oScarletHIGH cells (same experiment as in 5e). Each square represents one fish. Mean +/- standard error of mean; p-values, unpaired Wilcoxon ranksum test at per-animal level.
a, Same flow cytometry plot as in 5S1a (above), illustrating alternative gating schemes used in subsequent panels. b, Quantifications of mean ovalbumin fluorescence intensity, proportion of ovalbumin+ cells, and mean ovalbumin fluorescence intensity of ovalbumin+ cells among young adult and old oScarletHIGH cells (same experiments as in 5a) with a less stringent oScarletHIGH gate (lowered from 1000 to 500). Each square (male) or triangle (female) represents one fish. Mean +/-standard error of mean; p-values, unpaired Wilcoxon rank-sum test at per-animal level. c, Quantifications of mean ovalbumin fluorescence intensity, proportion of ovalbumin+ cells, and mean ovalbumin fluorescence intensity of ovalbumin+ cells among all young adult and old cells (same experiments as in 5a, no gating for oScarlet). Each square (male) or triangle (female) represents one fish. Mean +/- standard error of mean; p-values, unpaired Wilcoxon rank-sum test at per-animal level. d, Quantifications of mean dextran fluorescence intensity, proportion of dextran+ cells, and mean dextran fluorescence intensity of dextran+ cells among young adult and old oScarletHIGH cells (same experiment as in 5e) with a less stringent oScarletHIGH gate (lowered from 1000 to 500). Each square represents one fish. Mean +/- standard error of mean; p-values, unpaired Wilcoxon rank-sum test at per-animal level. e, Quantifications of mean dextran fluorescence intensity, proportion of dextran+ cells, and mean dextran fluorescence intensity of dextran+ cells among all young adult and old Live cells (same experiment as in 5e, no gating for oScarlet). Each square represents one fish. Mean +/-standard error of mean; p-values, unpaired Wilcoxon rank-sum test at per-animal level.
