Figure 5

Tlx1-expressing cells function as a HSPC niche for EMH in the spleen. Tlx1^CreER-Venus littermate controls and Tlx1^CreER-Venus; R26^Tlx1 mice (4-week-old) were treated with tamoxifen and stromal cell populations in the spleen were analyzed 24 hours after the final treatment. Data were pooled from 2–4 independent experiments. (a) Representative flow cytometric profiles of CD45⁻ Ter119⁻ CD31⁻ stromal cells from the spleen of tamoxifen-treated Tlx1^CreER-Venus controls (left) and Tlx1^CreER-Venus; R26^Tlx1 mice (middle). Gates used to identify Venus⁺ cell population are outlined, and numbers above the outlined areas indicate percent events in each gate. A histogram (right) represents the intensity of Venus fluorescence of Venus⁺ cells from tamoxifen-treated Tlx1^CreER-Venus controls (black line) and Tlx1^CreER-Venus; R26^Tlx1 mice (red line). (b) Graphs represent the MFI of Venus fluorescence and the numbers of Venus⁺ cells from the spleen of tamoxifen-treated Tlx1^CreER-Venus littermate controls (Ctr) and tamoxifen-treated Tlx1^CreER-Venus; R26^Tlx1 mice (Tg). The relative MFI of Venus⁺ cells in tamoxifen-treated Tlx1^CreER-Venus controls was arbitrarily set to 1. (mean ± SD; n = 7). (c) Total numbers of the indicated stromal cell populations from the spleen of tamoxifen-treated Tlx1^CreER-Venus controls (Ctr) and tamoxifen-treated Tlx1^CreER-Venus; R26^Tlx1 mice (Tg). (mean ± SD; n = 3). (d) Immunohistochemical analysis of the spleen of tamoxifen-treated Tlx1^CreRE-Venus littermate controls and Tlx1^CreER-Venus; R26^Tlx1 mice. Tissue sections were stained with the indicated antibody combinations. Scale bars indicate 100 μm. (n = 3). (e) Clustering analysis of Venus⁺ cells in the spleen upon Tlx1 overexpression. Representative digital images of Venus⁺ cells (white dots) in the spleen sections from the indicated mice. WP, the white pulp area. The right graph represents the Hopkins index of Venus⁺ cells in the spleen of tamoxifen-treated Tlx1^CrER-Venus controls (Ctr) and Tlx1^CreER-Venus; R26^Tlx1 mice (Tg). (n = 6). (f) BrdU incorporation analysis of Venus⁺ cells upon Tlx1 overexpression. Representative flow cytometric histograms of Venus⁺ cells of the indicated mice stained with anti-BrdU antibody (red line) and isotype control antibody (shaded line). The right graph represents the percentage of BrdU⁺ cells among total Venus⁺ cells in the spleen of tamoxifen-treated Tlx1^CreER-Venus controls (Ctr) and Tlx1^CreER-Venus; R26^Tlx1 mice (Tg). (mean ± SD, n = 5). (g) Distance of Venus⁺ cells from CD150⁺CD41⁻Lin⁻ HSPCs. Tissue sections of the spleen from tamoxifen-treated Tlx1^CreER-Venus; R26^Tlx1 mice were stained with the indicated antibody combinations. White arrows indicate HSPCs. The right graph represents the distance of Venus⁺ cells from CD150⁺CD41⁻Lin⁻ HSPCs or randomly generated spots. (n = 93 from 3 mice).