Enhanced proliferation of HSPCs in the CHT of Pten mutants.C-myb (A-B) and scl expression (C-D) were examined by in situ hybridization at 5 dpf in wild-type and Pten mutant (ptena^−/−ptenb^−/−) embryos as indicated. Representative embryos are depicted with anterior to the left. Brackets indicate the CHT; scale bar (200 µm) in panel D is representative for panels A-D. (E-G) The CHTs of sibling and Pten mutant embryos in the Tg(cd41:eGFP) background were imaged by confocal microscopy with a ×40 objective. Maximum projections of z-planes (step size, 2 µm) are shown (E-F). The number of HSPCs (GFP^low) (indicated by arrowheads) and thrombocytes (GFP^high) were counted in the entire CHT (height of CHT is depicted by a white bar in panels E and F) at 4 dpf using Volocity (siblings, n = 14; ptena^−/−ptenb^−/−, n = 10). (G) Results are expressed as average number of cells per CHT and error bars indicate SEM. Normal distribution of data points was assessed with the Shapiro-Wilk test. Statistical comparisons of groups were performed by the 2-tailed t test, respectively, with the Mann-Whitney U test. *P < .05, **P < .01, ***P < .001 vs control. (H-M) Cell proliferation was assessed in the CHT of 4-dpf-old Pten mutant Tg(cd41:eGFP) embryos and siblings by immunohistochemistry using pHis3-specific antibodies. Representative Tg(cd41:eGFP) (H-I), pHis3 (J-K), and merged images are shown (L-M). GFP and pHis3 double-positive cells are indicated with arrowheads in panels L and M. Scale bar, 50 μm.