Osteoblastoid-specific ablation of Hh signaling impairs B-cell development. (A) The Smo^fl allele. Exon 1, neo cassette, and τ-lacZ are indicated. Blue arrowheads indicate loxP sites. GT5′ and GT3′, primers for amplification of wild-type and floxed alleles; rec3, reverse primer for detection of recombined allele. (B) Primary OBs examined under light-field (left) or immunostained with an osteocalcin-specific antibody (right). (C) Detection of Smo wild-type (wt), floxed, and recombined (rec.) alleles in spleen, thymus, CD19⁺ BM cells, and OB cells from Cre^+/0, Smo^+/+ and Cre^+/0, Smo^fl/fl mice. Amplification was performed with genomic DNA templates. The Rag1 gene was amplified as a control. (D-E) Examination of B-lymphoid developmental subsets. BM was analyzed from 6- to 10-week-old mice of the indicated genotypes; the number of mice in each group is indicated in the inset (parentheses). Significant differences were determined by the Kruskal-Wallis test. (D) Percentages of pre-/pro-B (B220^loCD19⁻CD43⁺), pro-B (B220^loCD19⁺CD43⁺), and pre-B (B220^loCD19⁺CD43⁻) subsets (mean and standard deviation [SD]). (E) Percentages of Rag1⁺, Rag1⁺ B220⁻ (ELPs/CLPs), and Rag1⁺ B220⁺ subsets (mean and SD). (F) Impaired B lymphopoiesis from LSK progenitors maintained on Smo-depleted OB cells. LSK cells purified from mice of the genotypes indicated at top were maintained on Cre^+/0, Smo^+/+ or Cre^+/0, Smo^fl/fl OB cells under conditions that promote B lymphopoiesis. Lymphoid and myeloid differentiation was assessed by flow cytometry.