HSC, progenitor, and lineage reconstitution by β-/γ-catenin negative BM precursors. (A) Density plots show the abundance of CD45.2⁺ BM cells in the lineage-negative (lin⁻) hematopoietic progenitor compartment of mice reconstituted with a mixture of experimental (CD45.2⁺) and wild-type (CD45.1⁺) BM cells. Shown are the abundances of LSK cells (lin⁻, Sca-1⁺ CD117⁺), multipotent progenitors (lin⁻ Sca-1⁻ CD117⁺), common lymphoid progenitors (lin⁻ CD127⁺ CD117^low), and lymphoid-primed multipotent progenitor (lin⁻ Sca-1⁺ CD117⁺, CD135⁺) among CD45.2⁺ cells. (B) Gated CD45.2⁺ BM cells were analyzed for the presence of myeloid (granulocytes, CD11b⁺, GR1⁺, and monocytes, CD11b⁺ GR1⁻), erythroid (CD71⁺ TER119⁺) and lymphoid (B220⁺) lineage cells. B-cell development was further dissected by the separation into CD43⁺ B220⁺ (fraction A-C′,⁴⁶ ), CD43⁻ B220⁺ IgM⁻ (fraction D), and B220⁺ IgM⁺ (fraction E, F) subsets. Data show a representative analysis (of a total of 5 to 7 performed using 3 independent donor fetuses) after 7 months of reconstitution of secondary recipients. In panels A and B, numbers on graphs are percentage of total cells in the delineated regions. (C) PCR for β- and γ-catenin deletion on genomic DNA isolated from CD45.2⁺ BM cells 7 months after competitive reconstitution with catenin deleted BM. The mutant (indicated as null) and wild-type (wt) γcatenin alleles are detected as 150 bp and 300 bp PCR products, respectively. The floxed (indicated as lox) and deleted (Δ-) β-catenin alleles yield 183 bp and 481 bp bands, respectively. (D) Quantification of β-catenin deletion by Southern analysis of Sac1 restricted genomic DNA isolated from CD45.2⁺ BM cells 7 months after competitive reconstitution with β-catenin–deleted BM. The floxed (indicated as lox), deleted (Δ) and wild-type (wt) β-catenin alleles give rise to 7.4-, 5.2-, and 4.0-kb fragments, respectively. Note the virtually complete deletion of the β-catenin^lox allele on Cre-mediated recombination. (E) Total cellular lysates from flow sorted wild-type (CD45.1⁺) and mutant (CD45.2⁺) BM cells of the indicated genotypes were subjected to immunoblot analysis with mAbs to the COOH terminus of β-catenin (clone 14) and to tubulin (to ensure equal protein loading). Note the virtually complete absence of the full-size β-catenin protein upon Cre–mediated recombination. The smaller β-catenin protein species fail to interact with TCF-1 and do not interfere with TCF/LEF mediated transcription (see “Discussion” and Figure S1 [available on the Blood website; see the Supplemental Materials link at the top of the online article]).