Early B-cell development is compromised at the CLP stage in tPtch^−/− mice. (A) Lin⁻ BM cells from vehicle- and tamoxifen-treated control animals and of day-19 tPtch^−/− mice were analyzed by flow cytometry using antibodies against c-kit and Sca-1 (left panels). Fraction I (Lin⁻c-kit^highSca-1^−/low) contains mainly precursors of the myeloid lineages, fraction II (Lin⁻c-kit^lowSca-1^−/low) represents the CLP-containing compartment, and fraction III (Lin⁻c-kit^highSca-1^high) contains uncommitted HSCs and MPPs. The IL-7Rα⁺ cells within these fractions are shown in the right panels. Data are representative of 3 independent experiments (each experiment consisted of 1 vehicle-treated Ptch^flox/floxERT2^+/−, 1 tamoxifen-treated Ptch^flox/floxERT2^−/−, and 1 tPtch^−/− mouse). (B) Three hours after intraperitoneal injection of BrdU, Lin⁻ cells of control and day-19 tPtch^−/− mice were isolated and labeled with 7-amino actinomycin D (7-AAD), and cells in fractions I to III (described in A) were analyzed by flow cytometry. Data are representative of 3 independent experiments (each experiment consisted of 1 vehicle-treated Ptch^flox/floxERT2^+/−, 1 tamoxifen-treated Ptch^flox/floxERT2^−/−, and 1 tPtch^−/− mouse). (C) Lin⁻ BM cells of vehicle- and tamoxifen-treated control animals and of day-19 tPtch^−/− mice were isolated, and RT-PCR analysis was performed to monitor expression of the genes indicated on the left. cDNA obtained from day-12.5-old embryos (E12.5) served as positive control. ntc indicates no template control. White vertical lines have been inserted to indicate where a gel lane was cut. All lanes within each individual row are from one and the same experiment.