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Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects

Xu-Ming Dai, Gregory R. Ryan, Andrew J. Hapel, Melissa G. Dominguez, Robert G. Russell, Sara Kapp, Vonetta Sylvestre and E. Richard Stanley

Article Figures & Data

Figures

  • Fig. 1.

    Targeted disruption of the mouse

    Csf1r gene: decreased growth rate and increased circulating CSF-1 inCsf1r /Csf1r mice. (A) The targeted region of the Csf1r gene, theCsf1r gene-targeting vector, and the correctly targeted allele, showing exons 1 to 7, restriction enzyme sites (As,AseI; H, HindIII; A, ApaI; C,ClaI), PCR primers used (P1-P6, see text), the in-frame humanized green fluorescent protein (hGFP) sequence and the neomycin resistance (PGKneo) and thymidine kinase (PGKtk) cassettes above the flanking intron 7 probe were used to identify the 7-kb wild-type allele and 8-kb targeted allele HindIII fragments depicted below it. (B) Southern blot analyses of the DNA from individual G418 and gancyclovir-resistant ES cell clones using the probe shown in A. Asterisks mark clones possessing the correctly targeted allele. (C) Anti–CSF-1R Western blot analysis of BMM (upper panel). The molecular masses of the mature CSF-1R (165 kDa) and its precursor (130 kDa) are indicated. RT-PCR of BMM RNA with primers P5, P6 (A) specific for the targeted allele (middle panel) and control primers for β-actin (lower panel) are shown. (D) Growth curves of progeny of double heterozygote (Csf1r+/Csf1r ;Csf1+/Csf1op ×Csf1r+/Csf1r ;Csf1+/Csf1op ) crosses (n ≥ 5 for each genotype). (E) Serum CSF-1 concentration determined by a radioimmunoassay that selectively detects biologically active CSF-1 (± SD; n ≥ 5 for each genotype).

  • Fig. 2.

    Comparison of the skeletal development of wild-type,

    Csf1 op /Csf1 op , and Csf1r /Csf1r mice. Radiograms of the heads, bodies, and femurs of mice of the indicated genotypes at different ages (m, months). Each femur is from a different mouse. Arrows indicate regions of increased bone density most easily visualized at this magnification. Also shown is the extent (horizontal line) and fraction (in parenthesis) of the total femur length that is of high radiopacity.

  • Fig. 3.

    Histology of bone marrow of wild type,

    Csf1 op /Csf1 op , and Csf1r /Csf1r mice. (A) Low-power photomicrographs of hematoxylin and eosin–stained midsagittal 5-μm sections of the distal femoral metaphyses of 8-week-old mice (bm, bone marrow; bt, bony trabeculae; ep, epiphyseal plate). Boxes indicate comparable areas of TRAP-stained sections photographed in B. (B) High-power photomicrographs of TRAP staining for osteoclasts in midsagittal 5-μm sections of femurs of 4-week-old mice in areas comparable to those boxed in A. Counterstained with hematoxylin. Arrowheads indicate TRAP+ cells. (C) Percentage of trabecular bone in the entire bone marrow cavity determined from the sections used in A. Original magnification A, × 25; B, × 400.A.

  • Fig. 4.

    FACS analysis of blood leukocytes, peritoneal cavity, and pleural cavity cells.

    (A) Typical FACS analyses of monocytes, granulocytes, and lymphocytes by forward and side light scatter. Separate regions encompassing the monocyte (M), granulocyte (G), and lymphocyte (L) subpopulations are indicated. The means of results of such analyses for 3 mice of each genotype are shown in brackets (± SD). (B) Total pleural cavity and peritoneal cavity cells (n = 3). (C) Typical FACS analyses of CD11b+ peritoneal and pleural cavity cells. Percentage of positive cells for 3 mice of each genotype (± SD) is shown within each FACS distribution. FITC indicates fluorescein isothiocyanate; FSC, forward scatter; SSC, side scatter.

  • Fig. 5.

    F4/80+ cells in liver, kidney, skin, and synovial membrane.

    Tissues from wild-type,Csf1op /Csf1op , andCsf1r/Csf1r mice were immunostained with a monoclonal antibody to F4/80 that selectively stains macrophages and were counterstained with hematoxylin. Sections of (A-C) 2-day-old livers, (D-E) 2-week-old kidneys showing macrophages surrounding the glomeruli (GL) and tubules (T) of wild-type mice (D), (F-I) 2-day-old skin showing immunostaining of both Langerhans cells (LC) and dermal macrophages (DM) from wild- type mice, and (J-L) longitudinal sections of 2-week-old knee joints in the region of the synovial membrane (S) showing immunostaining of cells in the wild-type synovial membranes. Note the more rounded and less dendritic appearance of the F4/80+ cells in the tissues of theCsf1op /Csf1op andCsf1r/Csf1r mice, previously reported forCsf1op /Csf1op mice. Bar, 50 μm. Original magnification A-L, × 400.

  • Fig. 6.

    Hematopoietic progenitor cell concentrations in the spleen and bone marrow of 6-week-old mice.

    (A) Splenic progenitor cell colony numbers per 105 cells (BFU-E, CFU-GM, CFU-GEMM, CFU-C) or per 104 cells (HPP-CFC). (B) Bone marrow progenitor cell colony numbers per 104 cells (BFU-E, CFU-GM, CFU-GEMM, CFU-C) or per 103 cells (HPP-CFC). Means ± SD (3 mice per genotype). *Significantly different from wild type; **Significantly different from wild type andCsf1op/Csf1op (P ≤ .01).

  • Fig. 7.

    Reproductive phenotype of

    Csf1r /Csf1r mice. (A) Duration of estrus cycle in virgin female mice (5 mice per genotype, 8 cycles/mouse, ± SD). (B) Percentage time in proestrus (P), in estrus (E), in metestrus (M), and in diestrus (D) (± SD). (C) Whole-mount alum-carmine staining of the 4th inguinal mammary gland from 18-day pregnant mice. All panels are at the same magnification, approximately only one third of the wild-type gland is shown. LN indicates lymph node. (D) Percentage of successful pregnancies resulting from the consecutive daily mating of wild-type (open) andCsf1r/Csf1r (filled) male mice with superovulated virgin female mice. *Indicates significantly different from wild type; wt, wild type.

Tables

  • Table 1.

    Frequency of genotypes of progeny of single heterozygote crosses of Csf1op andCsf1r mutations

    Age of progenyCrossTotal progenyHomozygous wild typeHeterozygotesHomozygous mutantχ2 test probability*
    1 d Csf1+/Csf1op × Csf1+/Csf1op 4012
    (1.2)
    17
    (1.7)
    11
    (1.1)
    0.62
    Csf1r+/Csf1r × Csf1r+/Csf1r 7221
    (1.17)
    38
    (2.11)
    13
    (0.72)
    0.37
    3 wk Csf1+/Csf1op × Csf1+/Csf1op 21471
    (1.32)
    109
    (2.04)
    34
    (0.64)
    0.002
    Csf1r+/Csf1r × Csf1r+/Csf1r 393107
    (1.09)
    221
    (2.24)
    65
    (0.66)
    0.005
    • * Probability that results do not differ significantly from the 1:2:1 ratio.

    • Ratio compared with expected 1:2:1 ratio of an independently segregating gene.

  • Table 2.

    Genotypic frequencies as a percentage of total 3-week-old progeny from double heterozygote(Csf1r+/Csf1r; Csf1+/Csf1op × Csf1r+/Csf1r; Csf1+/Csf1op) crosses

    Csf1 genotypeCsf1r genotype
    Csf1r+/Csf1r+ Csf1r+/Csf1r Csf1r/Csf1r
    Csf1+/Csf1+ 6.25/7.7* 12.5/17.66.25/3.8
    Csf1+/Csf1op 12.5/15.325.0/31.012.5/4.2
    Csf1op/Csf1op 6.25/4.212.5/12.66.25/3.4
    • * Percentage expected for independently segregating alleles/percentage observed, n = 261.

  • Table 3.

    Bone marrow cellularity of 7-week-old and 8-month-old mice

    AgeTotal femur cellularity/body weight (×10−6/g)
    Wild typeCsf1op/Csf1op Csf1r/Csf1r
    7 wk0.50 ± 0.100.25 ± 0.043-150 0.23 ± 0.103-150
    8 mo1.57 ± 0.281.90 ± 0.141.53 ± 0.82
    • F3-150 Significantly different from wild type (Studentt test P ≤ .01).

  • Table 4.

    Tissue F4/80+ cell densities

    TissueAge of miceWild typeCsf1op/Csf1op Csf1r/Csf1r
    Bone marrow4-150,4-151 2 wk697.7 ± 11.7210.5 ± 14.8173.7 ± 41.0
    Liver4-150,4-151 2 d678.1 ± 108.4413.4 ± 13.0463.7 ± 45.3
    Liver4-150,4-151 4 mo372.9 ± 44.2124.3 ± 7.2150.0 ± 68.2
    Kidney4-150,4-151, 2 wk184.3 ± 83.034.5 ± 1.612.3 ± 0.4
    Thymus4-150,4-151 2 wk376.4 ± 40.1110.6 ± 18.1168.3 ± 15.2
    Langerhans cells4-150,4-151 2 d15.8 ± 1.34-153 3.0 ± 1.44-153 2.0 ± 1.44-153
    Dermis4-150,4-151 2 d253.1 ± 56.015.4 ± 5.132.5 ± 10.0
    Testes4-150,4-151 2 wk178.3 ± 83.246.7 ± 22.234.6 ± 19.4
    • Densities in cells/mm2 average of multiple counts (±SD, n = 4) of tissue sections from at least 2 mice per genotype.

    • F4-150 Significant differences between wild type andCsf1op/Csf1op (P ≤ .05).

    • F4-151 Significant differences between wild type andCsf1r/Csf1r (P≤ .05).

    • Significant differences betweenCsf1op/Csf1op andCsf1r/Csf1r (P≤ .05).

    • F4-153 Densities in cells/mm average of multiple counts (±SD, n = 4) of tissue sections from at least 2 mice per genotype.