1. Frizzled 9 knock-out mice have abnormal B-cell development
by Erik A. Ranheim, Helen C. K. Kwan, Tannishtha Reya, Yu-Ker Wang, Irving L. Weissman, and Uta Francke
Blood
Volume 105(6):
March 15, 2005
©2005 by American Society of Hematology

2. Knock out of Fzd9 gene.
Knock out of Fzd9 gene. (A) The restriction map of the locus surrounding the single exon of the Fzd9 gene was used to generate the targeting vector for homologous recombination. The Xs indicate sites of recombination in ES cells that are selected by neomycin (positive selection) and G418 (negative selection against TK). In the mutant chromosome, the Fzd9 gene is replaced by the neomycin-resistance (NEO) gene that introduces a new ScaI site. (B) Southern blot of ScaI-digested DNA from a wild-type (+/+), Fzd9 knock-out (-/-), and heterozygous (+/-) animal hybridized with the 3′ probe indicated in panel A. The 6-kb ScaI fragment is derived from the wild-type allele and the 2.5-kb fragment, from the mutant (KO) allele. (C) Northern blot of muscle and testis RNA from wild-type (+/+), Fzd9 knock-out (-/-), and heterozygous (+/-) animals hybridized with an Fzd9 cDNA probe and a β-actin probe as control for RNA loading. The Fzd9-specific 2.4-kb transcript is absent in homozygous mutant animals and reduced in the heterozygote.
Erik A. Ranheim et al. Blood 2005;105:
©2005 by American Society of Hematology

3. Frizzled 9 KO mice show decreased survival and elevated WBC
Frizzled 9 KO mice show decreased survival and elevated WBC. (A) Survival plots of WT129 SvEv mice (• n = 14) versus Fzd9 KO (▴ n = 36).
Frizzled 9 KO mice show decreased survival and elevated WBC. (A) Survival plots of WT129 SvEv mice (• n = 14) versus Fzd9 KO (▴ n = 36). (B) Peripheral blood counts in cells/μL for 17 representative Fzd9-/- mice ranging in age from 6 to 14 months with normal value ranges denoted by rectangles for each cell type. WBC indicates total white blood cell count; Neuts, neutrophils; Monos, monocytes (× 10 for display purposes); Eos, eosinophils (× 10); and RBC, red blood cells (× 10-3).
Erik A. Ranheim et al. Blood 2005;105:
©2005 by American Society of Hematology

4. Histologic analysis of spleen and lymph node.
Histologic analysis of spleen and lymph node. Spleens from WT (A [× 100] and C [× 400]) and KO (B [× 100] and D [× 400]) show expanded red pulp in the KO animals with increased extramedullary hematopoiesis (note megakaryocytes) and hemosiderin deposition. Lymph nodes from WT (E) and KO (F-H) show expansion of pale staining plasma cells expanding the medullary cords (F) or in patches in the interfollicular zones (G) of the nodes at × 100. At higher power (× 1000, H), histologically normal plasma cells are identifiable by their coarse chromatin, perinuclear Hof, and cytoplasmic immunoglobulin inclusions (Russel bodies). Tissue was paraffin embedded and sections were stained with hematoxylin and eosin. Photomicrographs were taken on an Olympus BXS1 microscope (Olympus, Melville, NY) with UPlanFl 10 ×/0.30 and 40 ×/0.75 objectives using an RT Slider camera and SPOT software (Diagnostic Instruments, Sterling Heights, MI).
Erik A. Ranheim et al. Blood 2005;105:
©2005 by American Society of Hematology

5. Decreased bone marrow B cells in Fzd9 KO mice.
Decreased bone marrow B cells in Fzd9 KO mice. (A) Bone marrow from 4-month-old KO (bottom row), WT (top row), and Fzd9 heterozygous littermate (middle row) mice was stained for CD43 and B220 (i), and B220+CD43+ (ii, Hardy fractions A-C) were examined for 6C3 (BP1) and CD24 expression. B220intCD43lo/- cells (iii, predominantly Hardy fraction D-E) and B220hiCD43-/lo (iv, Hardy fraction E-F) were examined for surface IgM and IgD by flow cytometry. Percentages of total BM cells (i) or of gated cells (ii-iv) are shown. Total absolute numbers of bone marrow cells in Fzd9-/- mice varied from equal to 50% of WT levels but were not statistically significantly reduced (data not shown). In this representative experiment, bilateral tibia and femur yielded 2.15 × 107 cells in KO, 1.88 × 107 cells in heterozygotes, and 2.54 × 107 in WT mice. The CD43+B220+ gate contains essentially no sIgM+ or sIgD+ cells (data not shown). Data shown are representative of 14 pairs of KO and WT control mice studied (for CD43, B220, and sIg staining; CD24/6C3 staining was technically more variable with analyzable data in 7 pairs). (B) Individual Fzd9-/- animals are plotted as a function of the relative numbers of each B-cell subset in comparison with WT mice analyzed at the same time, with a value of 1.0 indicating equal numbers and values less than 1 indicating decreased cells in KO animals. Subsets are, from left to right, all B cells (B220+), all Hardy A to C cells (B220+CD43+), Hardy D to E (B220intCD43-), and Hardy F (B220hiCD43-); n = 14; bars denote the mean value for each subset. Individual Hardy subsets are not presented separately due to variability in the antibody combinations used in individual experiments such that data on each subset are unavailable for all data points. B220 and CD43 staining, however, was always performed, and the B220+CD43+, B220intCD43-, and B220hiCD43- subsets consistently contained a vast majority of Hardy A to C, D to E, and F cells, respectively, as determined by staining for CD25, CD24, 6C3, IgM, and IgD (panel A and data not shown).
Erik A. Ranheim et al. Blood 2005;105:
©2005 by American Society of Hematology

6. Bone marrow reconstitution reveals intrinsic and nonintrinsic B-cell abnormalities.
Bone marrow reconstitution reveals intrinsic and nonintrinsic B-cell abnormalities. Equal numbers of flow sorted Lin-Sca1+Kit+ hematopoietic stem cells ( per recipient) from 6- to 10-week-old C57Bl/6-derived, Ly5.2+ WT mice and Fzd9-/- or +/+ mice on the 129SvEv background (Ly5.1+) were used to reconstitute lethally irradiated 6- to 10-week-old Ly recipients (C57Bl/6 derived). Analysis of peripheral blood (A) by gating on Ly5.2+ (WT C57Bl/6, □), Ly5.1+ WT (top row, ) or KO 129 (bottom row, ), or Ly5.1+Ly5.2+ (host, ○) cells and staining for B220 (B cells), CD5 (T cells), or Gr-1+ (myeloid cells). Percent contribution from each donor source is shown for each cell population over time (weeks). (B) Analysis of bone marrow cells 20 weeks after transplantation with percent mature (B220+IgM+) and immature (B220+IgM-) B-cell progenitors derived from WT versus KO 129 donors shown. (C) Analysis of lymph node CD4+ or CD8+ T cells (CD5+B220-) as in panel B. P values by paired or unpaired Student t test are as indicated by brackets. Matching symbols across different columns represent analyses from the same animal.
Erik A. Ranheim et al. Blood 2005;105:
©2005 by American Society of Hematology

7. Expression of Fzd9 mRNA in B-cell progenitor subsets.
Expression of Fzd9 mRNA in B-cell progenitor subsets. (Left) B-cell progenitors subsets were sorted by surface phenotype from WT adult C57/Bl6 and 129SvEv mice, and 1000 to 2000 cell equivalents were subjected to RT-PCR amplification of Fzd9 and beta-actin, as described in “Materials and methods.” Lanes are labeled for the Hardy B-cell subset tested, A to F, from bone marrow. The presence or absence of reverse transcriptase (RT) in the initial cDNA reactions is indicated by + or -, respectively. Mature B cells (CD19+ or B220+) from spleen and peritoneum were flow sorted for CD5 and surface IgD expression prior to RT-PCR (right panel). Splenic plasma cells (PCs) were isolated as CD138+B220lo/negIgD- cells. Data are representative of more than 4 experiments for each cell type.
Erik A. Ranheim et al. Blood 2005;105:
©2005 by American Society of Hematology