|
|
 Previous Article | Table of Contents | Next Article 
Blood, 1 January 2002, Vol. 99, No. 1, pp. 111-120
HEMATOPOIESIS
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
From the Department of Developmental and Molecular
Biology, Albert Einstein College of Medicine, Bronx, NY.
 |
Abstract |
The effects of colony-stimulating factor 1 (CSF-1), the primary
regulator of mononuclear phagocyte production, are thought to be
mediated by the CSF-1 receptor (CSF-1R), encoded by the c-fms proto-oncogene. To investigate the in vivo
specificity of CSF-1 for the CSF-1R, the mouse Csf1r gene
was inactivated. The phenotype of
Csf1 /Csf1r mice closely
resembled the phenotype of CSF-1-nullizygous
(Csf1op/Csf1op) mice,
including the osteopetrotic, hematopoietic, tissue macrophage, and
reproductive phenotypes. Compared with their wild-type littermates, splenic erythroid burst-forming unit and high-proliferative potential colony-forming cell levels in both
Csf1op/Csf1op and
Csf1/Csf1r mice were
significantly elevated, consistent with a negative regulatory role of
CSF-1 in erythropoiesis and the maintenance of primitive hematopoietic
progenitor cells. The circulating CSF-1 concentration in
Csf1r/Csf1r mice
was elevated 20-fold, in agreement with the previously reported clearance of circulating CSF-1 by CSF-1R-mediated endocytosis and
intracellular destruction. Despite their overall similarity, several
phenotypic characteristics of the
Csf1r/Csf1r mice
were more severe than those of the
Csf1op/Csf1op mice. The
results indicate that all of the effects of CSF-1 are mediated via the
CSF-1R, but that subtle effects of the CSF-1R could result from its
CSF-1-independent activation.
(Blood. 2002;99:111-120)
© 2002 by The American Society of Hematology.
| |
Introduction |
Colony-stimulating factor 1 (CSF-1) regulates the
survival, proliferation, and differentiation of mononuclear phagocytic
cells and is the primary regulator of mononuclear phagocyte production in vivo.1,2 However, CSF-1 also regulates cells of the
female reproductive tract and plays an important role in
fertility.3,4 The effects of CSF-1 are mediated by a
high-affinity receptor tyrosine kinase (CSF-1R)5-8 encoded
by the c-fms proto-oncogene.9 The CSF-1R is
expressed on primitive multipotent hematopoietic cells,10,11 mononuclear phagocyte progenitor
cells,12 monoblasts, promonocytes,
monocytes,5,6 tissue macrophages,6,13-15
osteoclasts,16 B cells,17,18 smooth muscle
cells,19 and neurons.20,21 CSF-1R messenger
RNA (mRNA) is expressed in Langerhans cells,22 in the
female reproductive tract, in oocytes and embryonic cells of the inner
cell mass and trophectoderm,23 in decidual
cells,24-26 and in cells of the
trophoblast.24,25 The expression of the CSF-1R on
primitive hematopoietic cells that are unable to proliferate in vitro
in response to CSF-1 alone10,11 but are able to
proliferate and differentiate if stimulated with combinations of CSF-1
and other hematopoietic growth factors10,11,27 suggests
that CSF-1R is involved in the regulation of more primitive
hematopoietic cells than those that form macrophage colonies in vitro
in response to CSF-1 alone.
Mice homozygous for the mutation
osteopetrotic28 possess an inactivating
mutation in the coding region of the CSF-1 gene and are devoid of
detectable CSF-1.29,30 These
Csf1op/Csf1op mice are
osteopetrotic because of an early and marked deficiency of
osteoclasts28 that spontaneously recovers with
age,31,32 probably because of the action of vascular
endothelial growth factor.33 However, the phenotype of
these mice is pleiotropic.3 They are toothless; have low
body weight, low growth rate, and skeletal abnormalities; and are
deficient in tissue macrophages.2,28,30,34,35 They have
defects in both male and female fertility, neural development, the
dermis, and synovial membranes.3 The pleiotropic phenotype of the Csf1op/Csf1op mouse may be
due to a reduction in trophic and/or scavenger functions of the tissue
macrophages regulated by CSF-1, secondary to the reduction of
their concentration in tissues,2 because outside the
female reproductive tract the CSF-1R is primarily expressed in
mononuclear phagocytes.1,3 However, it is possible that some of these effects may also be due to loss of function of other cells such as neuronal cells and muscle precursors, which have also
been reported to express the CSF-1R.20,36
To address the questions of whether CSF-1 activates other receptors
besides the CSF-1R and, conversely, whether the CSF-1R mediates the
response to ligands other than CSF-1, we have carried out the targeted
inactivation of the CSF-1R gene. The present study describes the
phenotype of mice homozygous for a targeted CSF-1R-null mutation and
compares it with the phenotype of
Csf1op/Csf1op mice. The
slightly more severe phenotype of
Csf1r/Csf1r over
Csf1op/Csf1op mice
indicates that the effects of CSF-1 are uniquely mediated through the
CSF-1R and that subtle effects of the CSF-1R could result from its
CSF-1-independent activation.
| |
Materials and methods |
Construction of the Csf1r gene-targeting
vector
Mouse Csf1r 5' promoter region and exon 2 region
primers included cfms-75F, 5'-GGC ACG GGG CTC CCA GCT GCT AGT TCT
GTG-3', and cfms-497R, 5'-AAG GGC AGA TGA GAA AGG TAT GAA GAA TGT-3'; and mouse Csf1r 3' terminal region and exon 22 sequence
primers included cfms-3024F, 5'-CCT CAG CTT GGC CCG ACT CTG ACA ATT
CAG-3', and cfms-3360R, 5'-AGT GAA GGT CAA GAG TGG TGG CCA ATA ATG-3'. These primers were synthesized and submitted to Genome Systems (St
Louis, MO) to screen a 129Sv mouse-derived embryonic stem (ES)
cell genomic P1 library. Three genomic clones containing the
Csf1r gene were obtained. One of these clones was mapped by a combination of restriction enzyme digestion, field inversion gel
electrophoresis, Southern analyses, polymerase chain reaction (PCR),
and sequencing. A 12-kb AseI fragment spanning exons 2 to 6 was subcloned into the SmaI site in the multiple cloning site of pGEM7Z in which the ApaI site had been mutated, and
the subclone was designated pGEM-Ase-Csf1r. The AseI
fragment was subjected to restriction enzyme mapping, PCR, and DNA
sequencing to generate the restriction map shown in Figure
1A. A humanized green fluorescent protein
(hGFP) sequence and the neomycin resistance (PGKneo) cassette was
cloned in-frame into the ApaI site in the exon 3 of
pGEM-Ase-Csf1r, and a PGK-tk (thymidine kinase) cassette was cloned
into the ClaI site in the multiple cloning site of the
pGEM-Ase-Csf1r, yielding the final targeting vector (Figure 1A). A
unique MluI site in the vector was used for
linearization.
View larger version (42K):
[in this window]
[in a new window]
| Figure 1.
Targeted disruption of the mouse
Csf1r gene: decreased growth rate and increased
circulating CSF-1 in
Csf1r/Csf1r
mice. (A) The targeted region of the Csf1r gene, the
Csf1r 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  -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).
|
|
ES cell culture and chimeric mouse production
Mouse ES cells (Go Germline; Genome System) (ESVJ-1182) derived
from 129SvJ strain were cultured on feeder layers of mitomycin C-treated mouse embryonic fibroblasts (MEFs) in ES cell medium (Dulbecco modified Eagle medium containing leukemia inhibitory factor
[LIF]; Gibco BRL, Rockville, MD) at 37°C and 7.5%
CO2. MEF preparation, ES cell propagation, electroporation,
and selection of recombinants with G418 and gancyclovir (Ganc) were
carried out exactly as described in the Genome Systems manual. Briefly, ES cells were expanded on MEF feeder layers; after trypsinization and
preplating to remove MEF, 1 × 107 ES cells were
electroporated with 20 µg of the MluI-linearized targeting
vector DNA. Twenty-four hours later, G418 and Ganc were added into the
LIF-containing ES cell medium for selection over 6 days.
G418r/Gancr ES clones were then picked and
replated onto gelatinized 24-well plates in ES cell medium. After a
further 2 days, the ES cells were trypsinized and split in 2, one half
being used to expand cells for DNA extraction and the other half
cultured for 2 further days before freezing. Genomic DNA was extracted
from the expanded cells, digested with HindIII, and
subjected to Southern blotting with an intron 7 probe to flanking
sequence not included in the targeting vector to identify the targeted
clones. The correctly targeted allele yielded an 8-kb
HindIII band that was clearly resolved from a 7-kb band
derived from the wild-type allele (Figure 1B). In addition, PCR
products of the correctly targeted gene were obtained with forward and
reverse primers corresponding to 5' and 3' sequences flanking the
targeting vector sequence that were used respectively with reverse and
forward primers within the 5' and 3' regions of the targeting vector.
Selected clones bearing the correctly targeted allele were thawed,
expanded, and injected into C57BL/6-derived blastocysts, which were
transplanted into CD1 pseudopregnant females to generate chimeric
founder mice. Subsequent genotyping of mice was carried out by PCR of
tail DNA using the primers P1, P2 (wild-type allele) and P3, P4
(targeted allele) (Figure 1A). Primers P1 (5'-TCT CCT GGG ATG GGA AAC
GAT CCC AAA GGC-3') and P2 (5'-GAT TCA GGG TCC AAG GTC CAG ATG GGA GAG-3') yielded a 536-base pair (bp) product, exclusively from the
wild type allele, whereas P3 (5'-GCC AGC CAC GAT AGC CGC GCT GCC TCG
TC-3') and P4 (5'-CTT CCT GGC CCT CAA CCA CTG TCA C-3') gave rise to a
1.6-kb product exclusively from the targeted allele.
Mice
To obtain germline transmission, chimeras were mated with
C57BL/6J × C3Heb/FeJ-a/a strain mice on which the
Csf1op allele was maintained. All mice were
maintained on this segregating background, behind a barrier at the
Albert Einstein College of Medicine animal facility.
Csf1r/Csf1 and
Csf1op/Csf1op mice were
distinguished from normal siblings at 10 days of age by the absence of
incisor eruption and were fed ad libitum a powdered mixture of mouse
food and infant milk formula (Enfamil) daily to improve their
nutritional status. Control mice received mouse chow ad libitum.
Genotyping of the Csf1op allele was carried out
as described.37
Radiographic analysis of mouse skeletal structure and CSF-1
radioimmunoassay
Radiographs were produced by exposing euthanized or anesthetized
mice in a Faxitron pathology specimen x-ray cabinet (Faxitron X-Ray,
Buffalo Grove, IL). The animals were posed immediately above a
fine-grained Polaroid 665 instant negative film package. Exposure was
set at 50 kV for 1.5 minutes. The negatives were developed and printed
according to the manufacturer's instructions (Polaroid, Cambridge,
MA). CSF-1 in mouse sera was determined by a radioimmunoassay that
selectively detects biologically active CSF-1.38,39
Immunohistochemistry and histochemistry
Rat monoclonal antibody to F4/80 was a gift from Dr David Hume
(Department of Microbiology, University of Queensland). For immunostaining with F4/80 antibodies and histochemical localization of
tartrate-resistant acid phosphatase (TRAP), siblings of the different
genotypes were perfused and tissues were fixed, decalcified (knee joint
only), embedded, sectioned, and immunostained as
described.2,40 TRAP staining was carried out as
described.40 Quantitative histomorphometric analyses of
the hematoxylin and eosin-stained sections were performed using a
digital camera to capture images. Image analysis was performed by using
Image-Pro Plus (Media Cybernetics, Silver Spring, MD). F4/80+ cells in tissue sections of at least 2 mice of a
particular genotype at each age were quantitated as
described.2 Whole-mount preparations of the 4th inguinal
mammary gland were stained with alum carmine as
described.41 For the estrus cycle analyses, daily vaginal smears were stained with hematoxylin and eosin. Mice were assessed as
being in one of the 4 stages: proestrus (100% intact live epithelial cells), estrus (100% cornified epithelial cells), metestrus (50% cornified epithelial cells and 50% leukocytes), or diestrus
(80%-100% leukocytes).42
Hematologic analysis and hematopoietic progenitor cell
assays
Mice (6- to 8-week-old) were euthanized by exposure to a high
concentration of CO2. Blood was collected in heparinized
tubes. Total white blood cells were counted in 6% CH3COOH
by using a hemocytometer. Red cell parameters were analyzed by using an
automatic blood cell counter. Monocytes, granulocytes, and lymphocytes
were resolved by differential flow cytometry analysis as
described.43 Spleen and bone marrow cell suspensions were
assayed for high-proliferative potential colony-forming cells
(HPP-CFCs) and CSF-1-dependent colony-forming units (CFU-Cs) in agar
cultures as previously described.31 Erythroid
burst-forming unit (BFU-E) and granulocyte, erythroid, megakaryocyte,
and macrophage colony-forming unit (CFU-GEMM) assays were performed by
using reagents supplied by Stem Cell Technology (Vancouver, BC, Canada)
in methylcellulose cultures as described by the manufacturers. The
growth factors used for HPP-CFC agar cultures were stem cell factor,
interleukin-6 (IL-6), IL-3, and granulocyte-macrophage CSF (GM-CSF).
CFU-GEMM assays were performed in methylcellulose culture medium
containing stem cell factor, IL-6, IL-3, and EPO.
Reverse transcriptase-PCR and Western blot
To assess Csf1r gene expression, bone marrow-derived
macrophages (BMMs) were prepared from siblings of the different
genotypes as previously described44 but with GM-CSF
replacing CSF-1. BMM cultured in mouse GM-CSF (R & D Systems, MN) was
solubilized in sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) running buffer,45 subjected to
SDS-PAGE, and Western blotted with a 1:1 mixture of 2 affinity purified
goat antimouse CSF-1R cytoplasmic domain peptide
antibodies.46 Total RNA samples were prepared from BMM
using the Trizol reagent (Gibco) and assayed by reverse transcriptase
(RT)-PCR with primers that bind to exon 2 of the Csf1r gene
and 5' coding sequence of the GFP gene (P5 and P6, Figure 1A; P5
[5'-CTAGCAGCTGGGAGCCCCGTGCCCAGCCGACTC-3']; P6
[5'-GGGTAGCGGCTGAAGCACTGCACGCCGTAGGTC-3']). RT-PCR was carried out
with an Advantage one-step RT-PCR kit (Clontech, Palo Alto, CA).
Statistical analyses
The mean, SD, or SEM of all numeric data was calculated. Data
were analyzed by either Student t test or chi-square test,
where appropriate. Comparisons of data sets yielding
P > .05 were considered as not statistically significant.
| |
Results |
Targeted disruption of the mouse CSF-1R gene
A P1 clone containing the entire coding region and flanking DNA of
CSF-1R gene was used to prepare the targeting construct (Figure 1A) as
described in "Materials and methods." This vector was linearized
and electroporated into 129SvJ strain ES cells, and clones of
transfected cells resistant to both G418 and Ganc were screened for
homologous recombinants by Southern blotting and PCR (Figure 1B). Of
the 164 G418/Ganc-resistant clones obtained, 20 were identified as
homologous recombinants and 2 of these (2C5 and 6D3) were injected into
blastocysts. Of the 5 chimeras obtained, 3 (2 derived from 6D3 and one
from 2C5) were transmitted to the germline. These lines were maintained
on the same C57BL/6J × C3Heb/FeJ-a/a background as the
Csf1op/Csf1op mice.
Western blot analysis of whole cell lysates of BMM prepared from
Csf1r/Csf1r and littermate
control mice indicated that the
Csf1r/Csf1r cells were devoid of
CSF-1R (Figure 1C, top panel). Consistent with these results, RT-PCR
analysis using primers P5 and P6 (Figure 1A) indicated that both
Csf1r/Csf1r and
Csf1r+/Csf1r cells expressed mRNA
encoding the CSF-1R-hGFP fusion protein (Figure 1C, lower panels).
However, no significant expression of GFP could be detected in
Csf1r/Csf1r or
Csf1r+/Csf1r cells by fluorescence
microscopy or flow cytometry (data not shown).
Gross phenotype of
Csf1r/Csf1r
mice
Csf1r/Csf1r mice were
identical in appearance to
Csf1op/Csf1op mice. They
were small (see below) and toothless and possessed truncated limbs, a
domed skull, and, occasionally, a kinked tail. Studies with
Csf1op/Csf1op mice
indicate that they have a low body weight and a low growth rate.28,37 Mice homozygous for the
Csf1op or Csf1r
mutations possessed a significantly lower growth rate and lower adult
weight than double-positive control mice or mice heterozygous for the
mutations and were indistinguishable from each other in this respect
(Figure 1D). Furthermore, the growth curves of the double mutants were
indistinguishable from those of mice homozygous for either mutation.
The Csf1r/Csf1r mice, like
Csf1op/Csf1op
mice,47 were also deaf (data not shown).
A comparison of the genotypic frequencies for single heterozygous
crosses (Csf1+/Csf1op × Csf1+/Csf1op and
Csf1r+/Csf1r × Csf1r+/Csf1r) is presented in
Table 1. At birth, the frequency of the
progeny of Csf1op heterozygote and
Csf1r heterozygote crosses was as expected for
a nondeleterious gene inherited in a Mendelian fashion. By weaning,
however, the survival of
Csf1r/Csf1r mice approximated
the survival of
Csf1op/Csf1op mice, and
the survival of both mutant mice was significantly lower than the
survival of wild-type mice, as previously reported for
Csf1op/Csf1op
mice.28 Analysis of the data from double heterozygote
crosses at weaning (Table 2) revealed
that survival of
Csf1op/Csf1op mice was
not significantly different from the survival of
Csf1r/Csf1r mice or of double
mutant
Csf1op/Csf1op;Csf1r/Csf1r,
except for the survival of
Csf1op/Csf1op;Csf1r+/Csf1r
mice, which unexpectedly exhibited normal survival, suggesting that the
Csf1op/Csf1op phenotype
might be slightly less severe than the
Csf1r/Csf1r phenotype.
View this table:
[in this window]
[in a new window]
|
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
|
|
Previous studies from our laboratory indicated that 95% of the
circulating CSF-1 is cleared by CSF-1R-mediated endocytosis and
intracellular destruction by sinusoidally located macrophages and that
circulating CSF-1 has a half-life of only 10 minutes.48 We
therefore determined the CSF-1 concentration in the sera of Csf1r/Csf1r,
Csf1r+/Csf1r+, and
Csf1r+/Csf1r mice (Figure 1E).
Consistent with these previous observations, levels of serum CSF-1 in
Csf1r/Csf1r mice were elevated
approximately 20-fold compared with the levels in
Csf1r+/Csf1r+ littermate control
mice. Interestingly, the levels of circulating CSF-1 in the
Csf1r+/Csf1r mice (19.0 ± 5.4
ng/mL) were within normal range (19.9 ± 6.1 ng/mL).
Comparison of the osteopetrotic phenotypes of
Csf1r/Csf1r and
Csf1op/Csf1op mice
Csf1op/Csf1op mice
exhibit impaired bone resorption associated with a reduction in the
number of osteoclasts.28 Their inability to remodel bone
resulted in general skeletal deformities that, as shown in Figure
2, were shared with the
Csf1r/Csf1r mice. Compared with
wild-type littermates, the long bones of their limbs were short (Figure
2) with increased bone density at the metaphyses (Figure 2),
deformities in the flat bony plates result in a domed skull (Figure 2),
and the increased bone density in the mandible presumably leads to the
failure of tooth eruption (Figure 2). Interestingly, a radiograph
analysis of the temporal changes in the femurs of wild-type and mutant
mice with age (Figure 2) indicates that there is significantly more
radiopacity in the distal metaphysis of the femurs of
Csf1r/Csf1r than of
Csf1op/Csf1op mice.
Similar results were obtained with
Csf1r/Csf1r mice derived from
both 2C5 and 6D3 ES cell lines. All further experiments were carried
out with Csf1r/Csf1r mice
derived from 2C5 ES cells.
View larger version (81K):
[in this window]
[in a new window]
| Figure 2.
Comparison of the skeletal development of wild-type,
Csf1op/Csf1op,
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.
|
|
The metaphyseal radiopacity of the long bones of
Csf1op/Csf1op and
Csf1r/Csf1r mice is the result
of the failure of osteoclast development at this site. Hematoxylin and
eosin staining of longitudinal sections of the distal metaphyseal
regions of the femurs of these mice revealed that both
Csf1r/Csf1r and
Csf1op/Csf1op mice
possessed higher amounts of bony trabeculae than littermate control
mice, consistent with impaired bone remodeling (Figure 3A). In addition, TRAP+ cells
(osteoclasts) were present in low numbers in the bony trabecula regions
of Csf1op/Csf1op femurs
compared with littermate control femurs and even fewer in number in
Csf1r/Csf1r femurs (Figure 3B).
Histomorphometric analyses of the entire bone marrow cavity of the
femurs were consistent with these observations, the percentage of
trabecular bone in Csf1r/Csf1r
and Csf1op/Csf1op femurs
being greater than in wild-type femurs (Figure 3C). In the metaphyseal
region, the retention of trabecular bone was greater for
Csf1r/Csf1r (92%) than for
Csf1op/Csf1op (83%) mice
(compared with wild-type mice [43%]), reflecting the greater
radiopacity of the Csf1r/Csf1r
over Csf1op/Csf1op femurs
in this region (Figure 2).
View larger version (95K):
[in this window]
[in a new window]
| Figure 3.
Histology of bone marrow of
wild type,
Csf1op/Csf1op,
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.
|
|
Consequent to their decreased volume of femoral bone marrow, the total
femoral marrow cellularity of
Csf1op/Csf1op and
Csf1r/Csf1r mice was
significantly lower than the bone marrow cellularity of wild-type mice
(Table 3). As previously reported for
Csf1op/Csf1op
mice,31,32 the bone marrow cellularity
Csf1r/Csf1r mice recovered to
the levels observed in control wild type mice by 8 months of age
(Table 3), despite evidence of some residual osteopetrosis in both
Csf1op/Csf1op and
Csf1r/Csf1r mice (Figure 2).
Reduced mononuclear phagocyte production in
Csf1r/Csf1r
mice
Previous reports34,49 have shown that blood monocyte
and lymphocyte percentages are reduced in
Csf1op/Csf1op mice.
Fluorescence-activated cell sorter (FACS) analysis of these populations
by forward and side-light scatter revealed that there was a decrease in
monocytes and lymphocytes and an increase in granulocytes in the
circulation of Csf1r/Csf1r mice
and that they were not significantly different from
Csf1op/Csf1op mice in
this respect (Figure 4A). Similarly, the
total cellularities of the pleural and peritoneal cavities of
Csf1r/Csf1r mice were reduced to
the same extent as in
Csf1op/Csf1op mice
(Figure 4B) as were the frequencies of Mac1(CD11b)+ cells
in these cavities (Figure 4C). Tissue macrophages expressing macrophage-specific cell-surface protein, F4/80, are significantly decreased in many tissues of
Csf1op/Csf1op
mice.2 The F4/80+ cell densities of several
such tissues were determined in wild-type, Csf1op/Csf1op, and
Csf1r/Csf1r mice at ages in
which the F4/80+ macrophage density had previously been
shown2 to be maximum for the particular wild-type tissue
(Figure 5, Table
4).2,50,51 As previously
shown,2 the F4/80+ cell densities of the
tissues of Csf1op/Csf1op
mice were significantly lower than those of wild-type control mice
(including the Langerhans cells of the epidermis, previously reported
to be normal in
Csf1op/Csf1op
mice2). There was no difference between the lowered
F4/80+ cell densities of these tissues and those of
Csf1r/Csf1r mice, except
that the F4/80+ cell density in the kidney was
significantly lower in
Csf1r/Csf1r mice than in
Csf1op/Csf1op mice (Table
4). Thus, apart from this difference, the mononuclear phagocyte numbers
were equivalently reduced in
Csf1r/Csf1r and
Csf1op/Csf1op
mice.
View larger version (29K):
[in this window]
[in a new window]
| Figure 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.
|
|
View larger version (173K):
[in this window]
[in a new window]
| Figure 5.
F4/80+ cells in liver, kidney, skin, and synovial membrane.
Tissues from wild-type,
Csf1op/Csf1op, and
Csf1r/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 the
Csf1op/Csf1op and
Csf1r/Csf1r mice,
previously reported for
Csf1op/Csf1op mice. Bar,
50 µm. Original magnification A-L, × 400.
|
|
Hematopoietic progenitor cells in
Csf1r/Csf1r
mice
Previous experiments have shown that, consistent with their
reduced space for marrow hematopoiesis (Table 3, Figures 2 and 3),
6-week-old Csf1op/Csf1op
mice have a compensatory splenic hematopoiesis with elevated frequencies of CFU-Cs and HPP-CFCs.31 As in the case of
the Csf1op/Csf1op mice
(3.6 ± 0.2), the splenic weights (milligram per gram of body weight)
of 6-week-old Csf1r/Csf1r mice
(5.1 ± 0.5) were significantly elevated compared with those of
littermate control wild-type mice (3.0 ± 0.3 [± SD], n = 3). The hematopoietic status of the wild-type,
Csf1op/Csf1op, and
Csf1r/Csf1r mice was examined by
determining bone marrow and splenic frequency of hematopoietic
progenitor cells at 6 weeks of age (Figure
6). Splenic BFU-Es and HPP-CFCs were
elevated to a similar extent in both types of mutant mouse. There was
no significant effect of either mutation on CFU-GM or CFU-GEMM. As
expected, in contrast to the elevated frequency of CSF-1-responsive
splenic CFU-C in the
Csf1op/Csf1op mice, there
were no CSF-1-responsive progenitors in spleens of the
Csf1r/Csf1r mice. In the bone
marrow of both
Csf1op/Csf1op and
Csf1r/Csf1r mice, there was no
difference in the frequency of these progenitors compared with their
frequency in wild-type mice, save for the absence of CFU-Cs in the
Csf1r/Csf1r mice. These results
reflect a negative regulatory role of CSF-1 on the in vivo frequencies
of splenic BFU-Es and HPP-CFCs.
View larger version (22K):
[in this window]
[in a new window]
| Figure 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 and
Csf1op/Csf1op
(P  .01).
|
|
Reproductive phenotype of
Csf1r/Csf1r
mice
CSF-1 plays an important role in ovulation, preimplantation,
placental function, regulation of the estrous cycle, and
lactation.3,4,52-55 Estrous cycling times are altered in
mature Csf1op/Csf1op
mice, estrous occurring irregularly and more infrequently compared with
the estrous times of normal mice (~5 days).53 The
duration of estrous in female
Csf1r/Csf1r mice, determined by
the appearance in the vagina of exfoliated anuclear cornified cells and
the absence of macrophages, although lower (8.0 ± 2.0, n = 40;
Figure 7A) than the previously reported cycling time for
Csf1op/Csf1op mice
(~14.5 days), was significantly higher than the duration of estrus in
wild-type mice (5.9 ± 0.5, n = 40; Figure 7 A). As previously
reported for the
Csf1op/Csf1op mice, this
increase in cycling time was primarily due to an increase in the
duration of the diestrus period (Figure 7B). In pregnant Csf1op/Csf1op mice, the
lactating mammary gland fails to develop normally because of a failure
of branching morphogenesis of the ductal epithelium.56 As
shown in the whole-mount stained mammary glands in Figure 7C, a similar
phenotype was observed for mammary glands from pregnant Csf1r/Csf1r mice.
View larger version (55K):
[in this window]
[in a new window]
| Figure 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) and
Csf1r/Csf1r
(filled) male mice with superovulated virgin female mice. *Indicates
significantly different from wild type; wt, wild type.
|
|
Compared with normal males,
Csf1op/Csf1op male mice
had low testosterone levels, low libido, and reduced viable sperm
numbers, as well as mating infrequently and displaying a long latency
between mating when presented serially with female mice in
estrous.42 A similar phenotype was observed for
Csf1r/Csf1r mice, which mated
less frequently with cycling females and produced fewer pregnant
females on successive days following daily exposure to different
superovulated females than wild-type males (Figure 7D).
| |
Discussion |
In this study, we have shown that BMM from mice in which both
Csf1r alleles have been targeted by the insertion of a
PGKneo cassette containing stop codons into exon 3 fail to express
detectable CSF-1R mRNA or protein. In contrast, BMM from wild- type
mice express approximately 50 000 cell surface CSF-1R molecules per cell.7 Consistent with this observation, neither bone
marrow nor splenic cells from the
Csf1r/Csf1r mice are able to
form CSF-1-dependent macrophage colonies in agar cultures.
Furthermore, the circulating concentrations of CSF-1 in the
Csf1r/Csf1r mice were 20-fold
higher than the wild-type CSF-1 concentration, consistent with a
failure of the previously established, CSF-1R-mediated clearance
mechanism for circulating CSF-1.48 These results indicate that the Csf1r/Csf1r mice are
truly CSF-1R nullizygous.
The phenotype of the
Csf1r/Csf1r mice was very
similar to the phenotype of the
Csf1op/Csf1op mice
lacking CSF-1. The growth rates and postnatal lethalities of
Csf1r/Csf1r,
Csf1op/Csf1op, and the
double-mutant
Csf1r/Csf1r;Csf1op/Csf1op
mice are indistinguishable. Of interest was the demonstration that
postnatal lethality of both mutants was not detectable at day 1 but
became apparent by 3 weeks of age (Table 1). Both
Csf1r/Csf1r and
Csf1op/Csf1op mice were
toothless and severely osteopetrotic with the same osteopetrosis-associated skeletal abnormalities and with a reduced bone
marrow cellularity that returns to normal levels by 8 months of age.
There was a similar depletion of circulating monocytes, pleural and
peritoneal cavity cells, peritoneal cavity Mac1+ cells, and
tissue macrophages in both mutant mice. With the exception of
CSF-1-dependent CFU-Cs, which cannot be measured in the
Csf1r/Csf1r mice, there was no
difference in their levels of bone marrow hematopoietic progenitor
cells, and both mutants had splenic BFU-E and HPP-CFC levels that were
significantly higher than in wild-type control mice. Furthermore, both
mutants shared previously reported defects in reproductive function,
including longer estrus cycles, failure of normal lactating mammary
gland development, and a reduced male mating performance. As no
phenotype in the
Csf1op/Csf1op mice was
more severe than in the
Csf1r/Csf1r mice, these results
indicate that all of the effects of CSF-1 are mediated via the CSF-1R
that is encoded by the c-fms gene.
Despite the overall similarity of the
Csf1r/Csf1r and
Csf1op/Csf1op mouse
phenotypes, aspects of the
Csf1r/Csf1r phenotype were more
severe than those of the
Csf1op/Csf1op mouse.
These aspects include a slightly more severe osteopetrosis in the
femurs (Figures 2 and 3) and a more severe depletion of F4/80+ cells in the kidney (Figure 5, Table 4). In
addition, there was a difference in the postnatal survival of mice
homozygous for one allele and heterozygous for the other.
Csf1op/Csf1op;Csf1r+/Csf1r
mouse survival was as expected for 2 independently segregating alleles
with no associated lethality, whereas the survival of Csf1r/Csf1r;Csf1+/Csf1op
mice was one third that rate (Table 2). Because both
Csf1r+/Csf1r+;Csf1op/Csf1op
and
Csf1r/Csf1r;Csf1+/Csf1+
mice also survived at one third of the expected rate, this finding suggests that a single copy of the CSF-1R gene may confer an advantage in the absence of CSF-1. This observation, with significant numbers of
progeny, supports the concept of a CSF-1-independent protective effect
of the CSF-1R, although it is not clear how 2 copies of the CSF-1R gene
fail to protect. It is possible that some of these effects were due to
partial rescue of the
Csf1op/Csf1op mice by
transplacental passage of CSF-1 from the heterozygotic mothers,57 which is not possible in the case of the
Csf1r/Csf1r mice. However,
because no difference was observed between the liver F4/80+
cell densities of 2-day-old
Csf1op/Csf1op and
Csf1r/Csf1r mice and nearly 80%
of the fetal hepatic blood flow is derived directly from the umbilical
vein,58 this seems unlikely. Furthermore, the increased
severity of Csf1r/Csf1r
phenotype has recently been confirmed by using the progeny of mice of a
single strain on which both alleles have been backcrossed for more than
5 generations. On this background, the
Csf1r/Csf1r phenotype is even
more severe and the Csf1r/Csf1r
mice all die by 3 weeks of age, whereas the
Csf1op/Csf1op survival
approximates their survival on the C57BL/6J × C3Heb/FeJ-a/a background. These observations suggest that the CSF-1R can respond to
an additional ligand or can function in a ligand-independent fashion in
some situations.
It has previously been suggested, in part because of the residual
tissue macrophage production seen in certain tissues in Csf1op/Csf1op mice and in
part because of the nature of the mutation, that this mouse is not
completely devoid of a mutated yet active form of CSF-1.59
However, as indicated above, the
Csf1op/Csf1op and
|
Top
Abstract
Introduction