Extended Data Fig. 5: Stem cell markers associated with quiescence are differentially expressed between Gli1- and Ascl1-targeted NSCs.
From: Long-term self-renewing stem cells in the adult mouse hippocampus identified by intravital imaging
a, Representative example of a Gli1- tdTom DG section (5 days post tamoxifen injection) stained with multiple antibodies using the 4i protocol. Visualization of 9 different cellular markers in addition to tdTomato. Single channels are shown. b, Representative images of a Gli1 and an Ascl1 DG section stained using 4i protocol, 5 days post tamoxifen injection. Visualization of six different cellular markers plus tdTomato. (Top) Prox1/blue, tdTom/red, SOX2/green, GFAP/light blue, Id4/magenta. (Middle) tdTom/red, Hopx/green, Mt3/magenta. (Bottom) single channels at higher magnification (regions in the white squares). c, Example of a Gli1-targeted R cell stained using the 4i protocol (5 days post tamoxifen injection). Visualization of 5 different R cell markers in addition to tdTomato and DAPI. Displayed are single channels and a merged picture. d, Percentage of cells that express different NSC and proliferation markers in Gli1- and Ascl1-targeted R cells (Gli1 n = 330 cells; Ascl1 n = 316 cells). e, Quantification of Sox2 (Gli1 = 5.24 ± 0.11 n = 330 cells; Ascl1 = 5.22 ± 0.14, n = 316 cells; Mann Whitney test: Mann-Whitney U = 51100; p = 0.661, two-tailed), Id4 (Gli1 = 2.02 ± 0.03, n = 317 cells; Ascl1 = 1.98 ± 0.03, n = 285 cells; Mann Whitney test: Mann-Whitney U = 44567; p = 0.776, two-tailed) and Ki67 (Gli1 = 3.78 ± 0.36, n = 32 cells; Ascl1 = 4.82 ± 0.28, n = 69 cells; Mann Whitney test: Mann-Whitney U = 760; p = *0.011, two-tailed) protein levels (fluorescence intensity of the cell/background) in Gli1- and Ascl1-targeted R cells. f, Quantification of Hopx protein levels (fluorescence intensity of the cell/background) in Gli1- and Ascl1- targeted R cells that are either positive or negative for Mt3 (Gli1 positive: 7.77 ± 0.20, n = 313 cells; Gli1 negative: 5.47 ± 0.5, n = 24 cells; Whitney test: Mann-Whitney U = 2394; **p = 0.003, two-tailed. Ascl1 positive: 6.46 ± 0.18, n = 284 cells; Ascl1 negative: 3.28 ± 0.32, n = 32 cells; Whitney test: Mann-Whitney U = 1651; ****p < 0.0001, two-tailed. Gli1 positive vs Ascl1 positive: Mann Whitney test: Mann-Whitney U = 35578; ****p < 0.0001, two-tailed. Gli1 negative vs Ascl1 negative: Mann Whitney test: Mann-Whitney U = 182; ***p = 0.0006, two-tailed). Values are shown as mean ± s.e.m. Bars in violin plots represent median and quartiles. Scale bars represent 400 μm (a), 100 μm (b) and 20 μm (c, high magnification and b). For detailed statistics, see Supplementary Table 5.4.
