Inhibition of PI3K signaling suppressed enhanced proliferation and released differentiation arrest in Pten mutants. Embryos were treated with 4μM LY294002 or DMSO (control) from 82 hpf onward. (A) The CHTs of sibling (sibl) and Pten mutant (mt) embryos in Tg(cd41:eGFP) background were imaged. The number of HSPCs (GFP^low) were counted in the whole CHT at 4 dpf (sibling, n = 14; sibling LY294002 treated, n = 10; ptena^−/−ptenb^−/−, n = 10; ptena^−/−ptenb^−/− LY294002 treated, n = 9). Results are expressed as the 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. (B-U) In situ hybridization markers for blood lineages were used on 5-dpf wild-type and Pten mutant (ptena^−/−ptenb^−/−) embryos: (B-E) c-myb is expressed in HSPCs; (F-I) gata-1, indicative of erythroblasts; (J-M) l-plastin, marking lymphocytes, macrophages, and neutrophils; (N-Q) ikaros, indicating lymphoblasts and (R-U) rag1, expressed in lymphocytes. Representative embryos are depicted; scale bars in panels Q and U (200 µm) are representative for panels B-U. (V) At 5 dpf, SB staining was used to assess the number of neutrophils in various parts of the embryo. SB⁺ cells in control (−, n = 10) and LY294002-treated (+, n = 16) siblings (sibl) and control (−, n = 11) and LY294002-treated (+, n = 17) Pten mutant (mt) embryos were counted in various parts of the embryo as indicated in supplemental Figure 2 and the results are expressed as average number of cells per embryo with the error bars indicating SEM. Normal distribution of data points was assessed with the Shapiro-Wilk test. Statistical comparison of groups was performed by the 2-tailed t test. *P < .05, **P < .01, ***P < .001 as indicated.