Ptch2 depletion causes an MPN phenotype with leukocytosis, splenomegaly, and mobilization of hematopoietic progenitors. (a) PB samples were analyzed for WBC counts, platelets, and hemoglobin (HGB) at 12 mo of age using the Scil Animal Blood Counter (n ≥ 9 each genotype, unpaired Student’s t test was performed for statistics throughout this figure). (b) Flow cytometry analysis for CD11b⁺ (myeloid cells), B220⁺ (B cells), and CD3⁺CD90⁺ (T cells) in the PB at12 mo of age (n ≥ 5 each genotype). (c) Flow cytometry analysis of PB cells of WT and Ptch2^−/− mice after 3 and 12 mo. CD11b/B220 staining of a representative example is shown for each time point and genotype. B220⁺ cells represents the B cell population, CD11b⁺ cells represents granulocytes and monocytes. (d) Total CD11b⁺ (myeloid cells), B220⁺ (B cells), and CD3⁺CD90⁺ (T cells, subdivided into CD4⁺ and CD8⁺) in the PB (n ≥ 3 each genotype, 12 mo). (e) Ficolized PB was analyzed for LK (Lin⁻cKit⁺Sca1⁻) and LKS (Lin⁻cKit⁺Sca1⁺) cells using flow cytometry (n ≥ 3 each genotype). (f) Total granulocytes (CD11b⁺Ly6G⁺) and monocytes (CD11b⁺Ly6G⁻) within the spleen and femur (n ≥ 5 each genotype, 12 mo). (g) Percentage of CD11b⁺ cells within the BM of WT versus Ptch2^−/− mice. Cells were quantified after IHC staining with a CD11b⁺ antibody using the high content screen scanR (n = 5 each genotype, 3 mo). (h) Representative images from hematoxylin and eosin (H&E)–stained BM slides of WT and Ptch2^−/−mice. Arrowheads indicate megakaryocytes (3 mo). Bars, 100 µm. (i) Megakaryocyte count per 0.5 mm² as assessed on H&E-stained BM slides (n ≥ 4 per genotype). (j) Flow cytometry analysis of megakaryocytic progenitors (MegP, Lin⁻cKit⁺Sca1⁻CD41⁺CD16/32^low, n = 10 each genotype, 3 mo) and erythroid progenitors (EP, Lin⁻cKit⁺Sca1⁻CD41⁻CD16/32⁻). (k) BM and spleen erythropoiesis (n ≥ 4 each genotype, 12 mo). Proerythroblasts (Pro), basophilic eryhtroblasts (Baso), polychromatic erythroblasts (Poly), normoblasts, and reticulocytes (Retic). (l) Flow cytometry analysis of BM and spleen LK (Lin⁻cKit⁺Sca1⁻) cells subdivided into CMPs (Lin⁻cKit⁺Sca1⁻CD34⁺CD16/32^mid), MEPs (Lin⁻cKit⁺Sca1⁻CD34⁻CD16/32⁻), and GMPs (Lin⁻cKit⁺Sca1⁻CD34⁺CD16/32⁺; n ≥ 10 each genotype, 3 mo). MEPs and GMPs are reduced in the BM, but increased in the spleen of Ptch2^−/− mice. (m) Spleen weight shown as single values and as the mean of all samples within WT and Ptch2^−/− mice (n = 5 each genotype, 3 mo). (n) Representative image of WT and Ptch2^−/− spleens. (o) Spleen cell counts (n = 5 each genotype, 3 mo). (p) Colony assay performed with 2 × 10⁴ BM cells, 5 × 10⁵ PB cells, and 2 × 10⁵ spleen cells in MethoCult3434. The numbers of CFU-GM and CFU-E were counted after 7 d (n = 4 each genotype performed in duplicates, 3 mo). *, P < 0.05; **, P < 0.01; ***, P < 0.001.