Intrinsic Hh signaling is dispensable for B-cell development. (A) Diagram of targeted Smo^flox allele. Exons 1 (Ex1) and 2 (Ex2) of Smo and the phosphoglycerokinase-Neo cassette (PGK Neo) are indicated. Arrowheads indicate loxP sites; dotted arrows, primers for amplification of unexcised, floxed allele; solid arrows, primers for detection of recombined allele. (B) Detection of Smo floxed (flox) and recombined (rec) alleles in CD19⁺ splenic B cells from wild-type or Smo^C mice. Amplification was performed on 10-fold serial dilutions of genomic DNA template. (C-D) Quantitative RT-PCR of Smo and Ptch transcripts in CD19⁺ BM (C) or naive splenic B cells (D) from WT or Smo^C mice. (E) Cellularity of BM (left) and spleen (right) in wild-type or Smo^C mice. (F) Examination of B-lymphoid developmental subsets. BM (top and middle panels) and spleen (spln; bottom panels) from 6- to 8-week-old wild-type or Smo^C mice were analyzed by flow cytometry. Early pro-B (B220⁺CD19⁻CD43⁺IgM⁻), pro-B (B220⁺CD19⁺CD43⁺IgM⁻), pre-B (B220⁺CD19⁺CD43⁻IgM⁺), and immature/mature B cell (B220^highCD19⁺CD43⁻IgM⁺) subsets are indicated on representative BM plots. B220⁺IgM^hiIgD^−/lo and B220⁺IgM^loIgD^hi populations, enriched for immature/transitional B cells and mature follicular B cells, respectively, are identified on splenic plots. (G) Percentage representation of B-lymphoid subsets in wild-type or Smo^C mice as defined in panel F (n = 3, mean and SD).