Extended Data Fig. 5: GLUT4 knockout efficiency in vivo, proportion of newborn cells that become neurons, other cell types in the olfactory bulb, and GLUT4 expression with aging.
From: CRISPR–Cas9 screens reveal regulators of ageing in neural stem cells
a, Top panel: immunofluorescence images of SVZ sections from old (22 months old) female Cas9 mice, 5 weeks after injection with lentivirus expressing Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. One experiment; representative of 3 independent experiments. DAPI (nuclei, blue), GFAP (qNSCs/astrocytes, magenta), Ki67 (proliferative cells, green), S100a6 (NSCs, red), merge image. Bottom panel: immunofluorescent images of sections of the SVZ from the same mice as in top panel. DAPI (nuclei, blue), mCherry (lentivirus-infected cells, red), and GFAP (qNSCs/astrocytes, green). b, Immunofluorescence images of olfactory bulb (OB) sections from old (~20 months old) male Cas9 mice, 5 weeks after injection of lentivirus expressing control (unannotated genomic regions), Slc2a4 (GLUT4) or Vmn1r107 sgRNAs directly into the lateral ventricles. One experiment; representative of 3 independent experiments. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. EdU (newborn cells, green) and nuclei (DAPI, blue). Zoomed-out images with the dashed white squares representing the insets (top) and zoomed-in images as insets (bottom). c, Example image of mCherry (lentivirus-infected cells, magenta) and EdU (newborn cells, green) staining (left panel) and QuPath image quantification of the average number of EdU+ and mCherry+ cells in the olfactory bulbs, normalized to total EdU+ cells (right panel) in old Cas9 mice (18–23 months old) (right panel). Mean +/- SEM of the average number of cell counts for one mouse, from 3 serial sections taken at 100 µm intervals across the olfactory bulb pair (normalized to average of control), for n = 9 old mice (4 males, 5 females, 20–23 months) for control sgRNAs, n = 6 old mice (3 males, 3 females, 20 – 23 months) for Slc2a4 (GLUT4) sgRNAs, and n = 5 old Cas9 mice (3 males, 2 females, 18–22 months) for Vmn1r107 sgRNAs, over 3 independent experiments. Each dot represents one mouse. P-values: two-tailed Mann-Whitney test. d, Immunofluorescence images of olfactory bulb sections from old (20 months) male Cas9 mouse, 5 weeks after injection of lentivirus expressing control (unannotated genomic regions) or Vmn1r107 sgRNAs directly into the lateral ventricles. One experiment; representative of 3 independent experiments. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. EdU (newborn cells, green), mCherry (lentivirus-infected cells, magenta), NeuN (mature neurons, red), and DAPI (nuclei, blue). Dashed white squares: NeuN+ cells that are infected with lentivirus (expressing one of the sgRNAs). Insets: zoomed-in images. e, QuPath image quantification of NeuN mean fluorescent intensity in infected (mCherry+) newborn cells (EdU+) in the olfactory bulb of old Cas9 mice (18–23 months old) (mix of males and females, see Source Data), 5 weeks after injection of lentivirus expressing control (unannotated genomic regions), Slc2a4 (GLUT4), or Vmn1r107 sgRNAs directly into the lateral ventricles. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. Mean +/- SEM of the average NeuN fluorescence intensity of all enumerated cells in one mouse, from 3 serial sections taken at 100 µm intervals across the olfactory bulb pair, for n = 9 old Cas9 mice (4 males, 5 females, 20–23 months) for control sgRNAs, n = 6 old Cas9 mice (3 males, 3 females, 20–23 months) for Slc2a4 (GLUT4) sgRNAs, and n = 5 old Cas9 mice (3 males, 2 females, 18–22 months) for Vmn1r107 sgRNAs, over 3 independent experiments. Each lavender dot represents one mouse. P-values: two tailed Mann-Whitney test. Each grey dot represents a single cell NeuN fluorescence intensity, showing all cells across all samples for each respective treatment. f, Image quantification of the percentage of newborn cells (EdU+) that are also NeuN+, comparing cells with (mCherry+) or without (mCherry−), in control, Slc2a4, or Vmn1r107 sgRNA expression conditions. Mean +/− SEM of the average (%NeuN+) of all cell quantifications from a single mouse for n = 5 old Cas9 mice for each condition, over 3 independent experiments. Each dot represents one mouse. P-values: two-tailed Mann-Whitney test. g, Immunofluorescence images of olfactory bulb sections from old (20 months old) male Cas9 mouse, 5 weeks after injection of lentivirus expressing Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. mCherry (lentivirus-infected cells, red), Tuj1 (neuron marker, green), and DAPI (nuclei, blue). Insets and white ovals: Tuj1+ cells that are infected with lentivirus (expressing one of the sgRNAs targeting Slc2a4 (GLUT4)). One experiment; representative of 2 independent experiments. h, QuPath image quantification of Tuj1 mean fluorescent intensity in infected (mCherry+) newborn cells (EdU+) in the olfactory bulb of old Cas9 mice (18–21 months old) (mix of males and females), 5 weeks after injection of lentivirus expressing control (unannotated genomic regions), or Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. Mean +/− SEM of the average Tuj1 fluorescence intensity of all enumerated cells in one mouse, sections taken at 100 µm intervals across the olfactory bulb pair for n = 4 old Cas9 mice for each condition, over 2 independent experiments. Each lavender dot represents one mouse. P-values: two tailed Mann-Whitney test. Each grey dot represents a single cell Tuj1 fluorescence intensity, showing all cells across all samples for each respective treatment. i, Immunofluorescence images of olfactory bulb sections from old (20 months old) male Cas9 mouse, 5 weeks after injection of lentivirus expressing Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. One experiment. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. mCherry (lentivirus-infected cells, red), Calretinin (mature neuron marker, green), and DAPI (nuclei, blue). j, Immunofluorescence images of olfactory bulb sections from old (20 months old) male Cas9 mouse, 5 weeks after injection of lentivirus expressing Slc2a4 (GLUT4) sgRNAs directly into the lateral ventricles. Mice were injected with EdU once per week, starting one week after virus injection, for 4 weeks. Left panels: mCherry (lentivirus-infected cells, red), GFAP (astrocytes, blue, top panels), Dcx (immature neuroblast, blue, bottom panels), NeuN (neurons, green). Right panels: mCherry (lentivirus-infected cells, red), DAPI (nuclei, blue), Sox10 (oligodendrocytes, green, top panels), Olig2 (oligodendrocytes, magenta, lower panels). Note that images are the exact same in the top and lower panels to show the different channels, except for the Slc2a4 (GLUT4) sgRNA condition for Dcx (lower panel), as there were no Dcx+ cells (which are very rare) in the same image. One experiment. k, QuPath image quantification of the mean fluorescence intensity with an isotype antibody control (mouse IgG) in sections from the SVZ neural stem cell niche from 4 young (3-4 months old) and 3 old (18–21 months old) male Cas9 mice (control for Fig. 3k). Cell types were identified as follows: qNSC/astrocyte (GFAP+Ki67−), aNSC (GFAP+Ki67+), Neuroblast (GFAP−Ki67+) and other cells (ependymal, microglia; GFAP−Ki67−). Mean +/− SEM of control average fluorescent intensities of each cell type, as identified above, from each mouse, for n = 4 (young qNSCs/astrocytes, young aNSCs, young other cells), n = 3 (young neuroblasts, old qNSCs/astrocytes, old neuroblasts, old other cells), or n = 2 (old aNSCs) Cas9 mice, over 2 experiments. Each dot represents one mouse. P-values determined by two tailed Mann-Whitney test. l, Violin plots comparing the log-normalized expression of Slc2a4 (GLUT4) transcripts between young (blue) and old (red) in single cell RNA-seq of qNSCs/astrocytes, aNSCs, neuroblasts, and all other cells from the neurogenic niche70. P-values determined by two-sided two-sample Welch’s t-test. m, Representative immunofluorescence images of coronal sections of the SVZ neural stem cell niche from young (3-4 months old) and old (18–21 months old) male Cas9 mice. GLUT4 (red), S100a6 (NSC marker, green), GFAP (NSC and astrocyte marker, magenta), and DAPI (nuclei, blue). Dotted squares: regions with NSCs shown in the insets. Insets: zoomed-in images. n, QuPath image quantification of the mean fluorescence intensity of the GLUT4 antibody in S100a6+/GFAP+ cells quantified from sections of the SVZ neural stem cell niche. Mean +/− SEM of the average fluorescent intensities of cells from one mouse, for n = 4 young (3-4 months old) and n = 4 old (18–21 months old) male Cas9 mice, over 2 independent experiments. Each dot represents a mouse. P-values: two tailed Mann-Whitney test.
