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In vitro studies suggest that activation of class I A phosphatidylinositol 3 (PI-3) kinase is necessary for normal erythroid cell development. However, when class I A PI-3 kinase-deficient mice were generated by a targeted deletion of the p85␣ regulatory subunit, fetal erythropoiesis was reportedly unaffected. Given the discrepancies between these studies, we performed a more detailed in vivo analy- IntroductionErythropoiesis is a coordinated process regulated by specific signaling pathways. In vitro studies suggest that activation of class I A phosphatidylinositol 3 (PI-3) kinase following binding of erythropoietin (Epo) and kit-ligand (KitL) to their receptors is required for the proliferation, survival, and differentiation of erythroid progenitors. 1-10 However, the physiologic role of class I A PI-3 kinase in regulating fetal and adult erythropoiesis is not known. A class I A PI-3 kinase knock-out mouse was generated by a targeted deletion of the p85␣ regulatory subunit of this kinase. 11,12 Although p85␣ Ϫ/Ϫ mice die shortly after birth secondary to hepatic necrosis and chylous ascites, 12 we used p85␣ Ϫ/Ϫ embryos to investigate the effect of p85␣ deficiency on fetal liver erythropoiesis in vivo. Study designMice and fetal hematopoietic cell isolation p85␣ ϩ/Ϫ mice (129/SV ϫ C57BL/6) were obtained from Dr Lewis Cantley at Harvard University (Boston, MA). Studies were conducted with a protocol approved by the Indiana University Animal Care and Use Committee. The p85␣ allele was genotyped by polymerase chain reaction (PCR) as previously described. 11,12 p85␣ ϩ/Ϫ mice were mated to produce day-14.5 p85␣ Ϫ/Ϫ and p85␣ ϩ/ϩ embryos. Fetal liver cells were isolated as previously described. 13 Single cell suspensions were prepared by pushing the hepatic tissues through a 23-gauge needle. Peripheral blood analysis and fetal liver erythropoiesisEmbryonic blood was obtained from day-14.5 fetal hearts for peripheral smears, and fetal liver touch preps were performed as previously described 14 and stained with Wright-Giemsa (Dade Behring, Newark, DE). Photomicrographs of peripheral smears and touch preps were taken with an Olympus DP11 microscope (Melville, NY). Colony assaysRecombinant KitL and Epo were obtained from Peprotech (Rocky Hill, NJ) and Amgen (Thousand Oaks, CA), respectively. Erythroid burst-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) assays were performed exactly as previously described. 15 c-kit ؉ cell isolationFetal liver cells were incubated with 1 g phycoerythrin (PE)-conjugated c-kit monoclonal antibody (Pharmingen, San Diego, CA) per 10 6 cells, placed on ice for 20 minutes, pelleted, washed, and resuspended in phosphate-buffered saline (PBS). C-kit ϩ cells were purified by immunomagnetic bead enrichment as previously described. 15Apoptosis and proliferation assays c-kit ϩ fetal liver cells were stained with fluorescein isothiocyanate (FITC)-annexin V (Pharmingen) and propidium iodide (Sigma, St Louis, MO) exactly per manufacturer's protocol followed by flow cytometric analysis as ...

Genetic loss of surface Fas antigen expression leads to reduced apoptosis of myeloid and lymphoid progenitor cells, and a propensity to develop autoimmunity and myeloid leukemia in mouse models. Oncogenic p21 ras decreases surface Fas antigen expression and renders fibroblasts resistant to Fas mediated apoptosis. Neurofibromin, which is encoded by NF1, is a GTPase activating protein that negatively regulates p21 ras activity. NF1 loss leads to deregulation of p21 ras -effector pathways, which control myeloid cell survival. Heterozygous inactivation of Nf1 increases mast cell numbers in Nf1 ؉/؊ mice, and enhances mast cell survival in response to c-kit ligand (kit-L). Here, we show that Nf1-deficient mast cells have reduced surface Fas antigen expression in response to kit-L and are resistant to Fas ligand-mediated apoptosis. Using genetic intercrosses between Nf1 ؉/؊ and class I A -PI-3K-deficient mice, we demonstrate that hyperactivation of the p21 ras -class I A PI-3K pathway is the mechanism for this phenotype. Finally, we demonstrate that mast cells from both Fas antigen-deficient mice and Nf1 ؉/؊ mice are resistant to apoptosis following kit-L withdrawal in vivo. Thus, therapies designed to decrease p21 ras activity and up-regulate Fas antigen expression may limit the pathological accumulation of myeloid cells in disease states where p21 ras is