Download Identification of a Novel Point Mutation of Mouse Proto

Copyright © 2005 by the Genetics Society of America
DOI: 10.1534/genetics.104.027177
Identification of a Novel Point Mutation of Mouse Proto-Oncogene c-kit
Through N-Ethyl-N-nitrosourea Mutagenesis
Hai-Bin Ruan,* Nian Zhang*,† and Xiang Gao*,‡,1
*Model Animal Research Center, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing, China 210089, ‡Model Organism Division, E-Institute of Shanghai Unviersity, Shanghai, China
and †Van Andel Research Institute, Grand Rapids, Michigan 49503
Manuscript received February 3, 2004
Accepted for publication August 17, 2004
ABSTRACT
Manipulation of the mouse genome has emerged as an important approach for studying gene function
and establishing human disease models. In this study, the mouse mutants were generated through N-ethylN-nitrosourea (ENU)-induced mutagenesis in C57BL/6J mice. The screening for dominant mutations
yielded several mice with fur color abnormalities. One of them causes a phenotype similar to that shown
by dominant-white spotting (W ) allele mutants. This strain was named Wads because the homozygous mutant
mice are w hite color, a nemic, d eaf, and s terile. The new mutation was mapped to 42 cM on chromosome
five, where proto-oncogene c-kit resides. Sequence analysis of c-kit cDNA from Wads m/m revealed a unique
T-to-C transition mutation that resulted in Phe-to-Ser substitution at amino acid 856 within a highly
conserved tyrosine kinase domain. Compared with other c-kit mutants, Wads may present a novel lossof-function or hypomorphic mutation. In addition to the examination of adult phenotypes in hearing
loss, anemia, and mast cell deficiency, we also detected some early developmental defects during germ
cell differentiation in the testis and ovary of neonatal Wads m/m mice. Therefore, the Wads mutant may
serve as a new disease model of human piebaldism, anemia, deafness, sterility, and mast cell diseases.
N
-ETHYL-N-NITROSOUREA (ENU)-induced mutagenesis has become a powerful tool for the study
of gene functions and generation of human disease
models recently (Nolan et al. 2000; Hrabe de Angelis
et al. 2000; Herron et al. 2002). The growing mouse
mutant archives provide a rich resource for identifying
the novel disease-related genes, deciphering pathogenic
mechanisms, and developing new therapies and new drugs
(Balling 2001; Brown and Hardisty 2003). Recently,
we established ⬎40 lines of mutant mice by screening
the ENU-induced dominant mutants in C57BL/6J mice
(He et al. 2003). Among them, 11 lines displayed an interesting white spotted coat. One of these lines, Wads, displayed similar phenotypes to the mouse W/c-kit strain.
Mutations at the mouse W/c-kit locus on chromosome
five can lead to pleiotropic developmental defects, including sterility, coat color abnormalities, severe macrocytic anemia, loss of interstitial cells of Cajal (ICC), and
mast cell deficiency (Geissler et al. 1981; Chabot et al.
1988; Huizinga et al. 1995; Tsujimura 1996). A total of
76 mutant alleles at this locus have been accumulated in
mouse up to 1997 (see the Mouse Genome Database,
http://www.informatics.jax.org/searches/mlc.cgi?10603).
Human c-kit mutations were also identified in patients
with piebaldism, mastocytosis, gastrointestinal stromal tumors (GISTs), acute myeloid leukemia, and germ cell
tumors (see online Mendelian Inheritance in Man,
OMIM, http://www.ncbi.nlm.nih.gov/entrez/dispomim.
cgi?id⫽164920). Encoded by c-kit, KIT is a type III receptor
tyrosine kinase that binds to stem cell factor (SCF),
which is the product of the mouse Sl locus (Witter
1990). Ligand binding activates KIT through dimerization and autophosphorylation (Heldin 1995). The
phophorylated KIT then further activates downstream
pathways in a variety of cell types (Price et al. 1998;
Timokhina et al. 1998; Linnekin 1999; Hou et al. 2000).
Unfortunately, mechanisms by which the different KIT
mutations cause various diseases are largely unknown.
In this study, we reported the preliminary phenotypic
analysis of the Wads mouse, including hearing ability,
hematopoiesis, mast cell development, and germ cell differentiation. The Wads mutation was also mapped and
cloned. The novel point mutation at nucleotide 2567 of
the c-kit cDNA results in a substitution of Phe to Ser at
KIT amino acid 856 (F856S).
MATERIALS AND METHODS
Sequence data from this article have been deposited with the EMBL/
GenBank Data Libraries under accession nos. AY536430 and AY536431.
1
Corresponding author: Model Animal Research Center, Nanjing University, 308 Xuefu Rd., Pukou District, Nanjing 210089, People’s Republic of China. E-mail: [email protected]
Genetics 169: 819–831 (February 2005)
Animals: C57BL/6J mice were obtained from Shanghai Laboratory Animal Center (Shanghai, China). CAST/Ei mice were
obtained from The Jackson Laboratory (Bar Harbor, ME). Mice
were maintained under specific pathogen-free environment.
Animal welfare and experimental procedures were carried out
820
H.-B. Ruan, N. Zhang and X. Gao
strictly in accordance with the care and use of laboratory
animals (National Research Council, 1996) and the related
ethical regulations of Nanjing University. The G1 generation
dominant mutant mouse (G1M485) of C57BL/6J background
was generated by ENU mutagenesis (He et al. 2003). This male
mouse was mated to C57BL/6J females for confirming the
inheritance test. Homozygous mutants (Wadsm/m) were generated by intercross of the heterozygous mutants (Wadsm/⫹).
Histological analysis: Skin, testes, or ovaries were dissected
and fixed in Bouin’s fixative. They were dehydrated in rising
concentrations of ethanol, embedded in paraffin, and sectioned (7 ␮m). The sections of testis and ovary were stained
with hematoxylin and eosin according to standard procedures.
Toluidine blue in sodium chloride was used to stain mast cells
in skin tissues.
Peripheral blood analysis: Peripheral blood was collected
using a capillary tube from suborbital veins of 5- to 6-weekold mice. Blood cell analysis was performed with a Coulter
(Hialeah, FL) STKS hematology analyzer. The parameters
measured included white blood cell counts (WBC); red blood
cell counts (RBC); hemoglobin concentration (HGB); mean
corpuscular volume (MCV, the average volume of individual
RBC); mean corpuscular hemoglobin (MCH, the average
weight of hemoglobin in a red blood cell); mean corpuscular
hemoglobin concentration (MCHC, the ratio of MCH to MCV);
platelet counts (PLT); and mean platelet volume (MPV, the
average volume of individual platelets). The P-values were
evaluated using an unpaired two-tailed t-test using Prism software (GraphPad Software, San Diego) between the blood parameters of male and female mice and between wild-type mice
and heterozygous or homozygous mice.
Auditory brainstem response test: The auditory brainstem
response (ABR) test was performed with a PowerLab system
(A&D, Castle Hill, Australia). Mice were anesthetized with
Avertin and the stimulating electrode was embedded in the
mastoid of the right ear, the recording electrode subcutaneously in the vertex, and the earth electrode in the mastoid of
the left ear. ABRs were recorded under the sound stimuli of
40, 50, and 70 dB sound pressure levels (Zheng et al. 1999).
Cochlear histology and immunohistochemistry: For cochlear histology, the mice were killed and temporal bones were
removed from the skull and then fixed in 4% paraformaldehyde at 4⬚ overnight. Tissues were then immersed in decalcifying solution (4% EDTA in PBS) for 1 week. The paraffin sections were prepared and stained with hematoxylin and eosin.
For immunofluorescence, the paraffin sections were dehydrated and permeabilized with 20 ␮g/ml proteinase K. Blocking was carried out with 10% goat serum in PBS. Primary rabbit
antibodies to connexin 26 and 30 (Zymed, San Francisco) were
in 2% goat serum/PBS at a dilution of 1:50. A FITC-conjugated
goat anti-rabbit secondary antibody (Sigma, St. Louis) was used
to detect connexins and the fluorescence was observed under
a Leica confocal microscope.
Mutation mapping and cloning: Wads m/⫹ mice of C57BL6/J
were mated to CAST/Ei mice to generate F1. F1 females with
white spots were backcrossed to CAST/Ei male mice to generate N2. The tail DNA samples of N2 mice were extracted and
PCR amplified with D5Mit356 and D5Mit359 microsatellite
markers of the Whitehead/MIT database (http://www-genome.
wi.mit.edu). PCR products were separated by electrophoresis
with 4% agarose II (BioBasic, Markham, ON, Canada) gel.
To examine if the mutation resides in the c-kit gene, total
RNA samples were prepared from the skin of adult C57BL/
6 J and Wads m/m mice using the Trizol kit following the manufacturer’s instructions (Shenergy, Shanghai, China). Fulllength cDNA of c-kit was synthesized with AMV reverse transcriptase (Promega, Madison, WI). Due to the large size of
c-kit cDNA (2925 bp), three overlapping cDNA fragments that
covered the full length were then amplified by PCR with LATaq polymerase (TaKaRa, Shiga, Japan) using the following
primers: kit1-FOR, 5⬘-TCAGAGTCTAGCGCAGCCAC-3⬘; kit1REV, 5⬘-GCCTCGTATTCAACAACCAA-3⬘; kit2-FOR, 5⬘-TGTA
ACCGATGGAGAAAACG-3⬘; kit2-REV, 5⬘-TAAACGAGTCACG
CTTCCTT-3⬘; kit3-FOR, 5⬘-GCCCTAATGTCGGAACTGAA-3⬘;
and kit3-REV, 5⬘-GTTTCTGCTCAGGCATCTTC-3⬘. The three
fragments were then subcloned into TA cloning vector pMD
18-T (TaKaRa) and sequenced (Bioasia, Shanghai, China). All
sequences were confirmed by sequencing from both directions.
Single-strand conformation polymorphism: PCR-single-strand
conformation polymorphism (SSCP) analysis was performed
as described (Strippoli et al. 2001). Briefly, 200 bp of DNA
fragment covering the Wads point mutation was amplified by
PCR using primers of 5⬘-GACTGCCCGTGAAGTGGAT-3⬘ and
5⬘-CTTCCAGAGAGGTGGCAAAT-3⬘. The PCR product was
mixed with 10⫻ alkali denaturating buffer (NaOH, 500 mm;
EDTA, 10 mm) and heated to 94⬚ for 10 min and then quickly
cooled on ice for 2 min. Samples were then mixed with loading
buffer, loaded on an 8% polyacrylamide gel (39:1 acrylamide
to bis-acrylamide; TAE buffer; 0.2% TEMED; 0.05% ammonium persulfate 10%), and run at 90 V for ⵑ1.5 hr. The gel
was stained with 5␮g/ml ethidium bromide.
RESULTS
Pigmentation defects of Wads mutants: The founder
(G1M485) of the Wads strain had a symmetrical cluster
of white coat on the midline of the abdomen and small
white spots on the back (Figure 1, A and B). The coat
of most of the tail as well as the distal part of the legs
also displayed white color. Half of the progenies (112 of
212) from the founder showed a similar pigmentation
abnormality, indicating a single-gene mutation in the
Wads strain.
Interestingly, homozygous mutants (Wads m/m) showed
all white skin and hairs but black eyes (Figure 1C). Because the melanocytes are derived from neural crest
cells (NCCs) whereas retinal pigment epithelium (RPG)
arises from neural tube epithelium, the lack of functional melanocytes suggested that the migration and/or
differentiation of the NCCs were impaired in Wads m/m
mice (Zhao et al. 1997).
The mutation localized at W/c-kit locus: Several signaling pathways, especially the SCF/KIT pathway, have
been reported to participate in proliferation and differentiation during melanoblast development (Bennett
and Lamoreux 2003; Manova and Bachvarova 1991).
Therefore, we examined whether the c-kit gene locus,
42 cM of chromosome 5, links to the Wads mutation.
With the backcrossing protocol for mapping using the
CAST/Ei strain (Reeves and D’Eustachio 1997), we
analyzed the linkage between pigmentation abnormality
of 60 N 2 mice and two microsatellite markers, D5Mit356
of 41 cM and D5Mit359, of 44 cM on chromosome 5. We
found that the ratio of recombinant/nonrecombinant
phase of D5Mit356 was 2/58, while that of D5Mit359
was 5/55. This result strongly suggested that the Wads
mutation was linked to the W/c-kit locus.
Molecular analysis of the mutation: To examine whether
the c-kit gene was mutated in Wads mice, wild-type and
Novel Mutation of the Mouse c-kit Gene
821
Figure 1.—Defects of Wads
mice coat color and mast cells.
(A) A Wads m/⫹ heterozygote with
white spots on the abdomen.
(B) Dorsal view of a Wads m/⫹ heterozygote. (C) Black-eyed white
Wads m/m homozygote. (D) Wildtype, (E)Wads m/⫹, and (F)Wads m/m
skin sections were stained with
Toluidine blue. Black arrows
identify mast cells (purple) and
white arrows identify hair follicles at telogen phase (blue).
Wads m/m c-kit cDNA was cloned and sequenced. The sequence data revealed a T-to-C missense transition at
nucleotide 2567 in Wads m/m coding sequence that resulted in a Phe(F) to Ser(S) change at amino acid 856
(Figure 2A). This mutation position located in the second protein tyrosine kinase (PTK) domain and the Phe
is conserved among all the mammalian KIT proteins.
Combined with the phenotypic profiles, Wads may represent a loss-of-function or hypomorphic c-kit mutation
(Reith et al. 1990).
It is also worth pointing out that our wild-type C57BL/
6J cDNA sequence of c-kit was consistent with that of the
Ensembl Genome Browser (ENSMUSG00000005672).
However, our sequence data differed from the Mus musculus c-kit cDNA sequence listed in GenBank (NM_021099),
with a synonymous mutation and two missense mutations. We believe that our sequence data were more
reliable, according to the protein sequence alignment
with other mammalian KITs (data not shown).
Because the coat abnormality is not available at such
an early stage of animal development, development of
a reliable genotyping method for the Wads mutant is
necessary for studying the embryonic and neonatal defects. For this reason, we developed a PCR-based SSCP
822
H.-B. Ruan, N. Zhang and X. Gao
Figure 2.—Structure and point
mutations of human KIT protein.
(A) Top, the mutation of KitWads
located at position 2567 in exon
18 of the mouse c-kit gene, which
led to exchange from Phe to Ser
at amino acid 856; bottom, lossof-function point mutations in
human KIT. (B) Gain-of-function
point mutations in human KIT.
TM, transmembrane domain; JM,
juxtamembrane domain; KI, kinase insert; K1, kinase domain I;
K2, kinase domain II; Ig, immunoglobulin-like domains.
analysis to genotype Wads m/⫹, Wads m/m, and wild-type mice.
SSCP is the electrophoretic separation of single-strand
nucleic acids on the basis of subtle differences in sequence (often a single base pair) that results in a different secondary structure and a measurable difference in
mobility through a gel (Sunnucks et al. 2000). Primers
were designed to amplify 200 bp of genomic DNA product covering the Wads mutant point. The DNA samples
were denatured and then run on a cold polyacrylamide
gel. Single-strand DNA (ssDNA) samples from heterozygotes had four different gel mobilities, consistent with
two conformations of wild-type ssDNA and two of homozygous mutant ssDNA (Figure 3). The SSCP genotyping
of the newborn mice was used for our germ cell differentiation studies.
Lack of mast cell in Wads mutant skin: Toluidine blue
staining was used to examine the mast cell population on
skin sections. Wild-type and Wads m/⫹ skins contained simi-
Figure 3.—SSCP analysis of KitWads. The arrows indicate wildtype ssDNA, the arrowheads indicate mutant ssDNA. The
bands in the first lane identify 500 and 250 bp of marker.
lar numbers of mast cells (Figure 1, D and E). However,
no mast cells were observed in Wads m/m skin tissues (Figure 1F). This result indicated that Wads-related c-kit mutation affects the development of the mast cell lineage.
Lack of mature germ cell in Wads m/m mutants: The mating test suggested both Wads m/m males and females were
infertile because no offspring were obtained. To evaluate the function of c-kit in spermatogenesis and oogenesis, we performed histological examination on testis
and ovary at different developmental stages.
In testis, the asymmetrical cell division of gonocytes
produces type A1 spermatogonia at postnatal day 6. These
spermatogonia further differentiate into types A2, A3,
A4, and intermediate spermatogonia, which then form
type B spermatogonia, at postnatal day 6. Type B spermatogonia are the precursors of the spermatocytes that
will enter meiosis and finally form mature spermatozoa
at ⵑ1 month (Bellvé et al. 1977). We found no obvious
difference between wild-type testis and Wads m/m testis at
postnatal day 6 (Figure 4, A and B). The seminiferous
tubules of wild-type and Wads m/m testes had similar numbers of germ cells. The spermatogonia cells, intermingled with Sertoli cells, were located near the basement
membrane of the seminiferous epithelium. However,
fewer germ cells were in Wads m/m testis than in wild-type
testis at postnatal day 8. At least two layers of germ cells
along with some type B spermatogonia were present in
the wild-type seminiferous epithelium (Figure 4C), while
the Wads m/m basically had only a single layer of germ cells
and no type B spermatogonia (Figure 4D). At postnatal
day 12, spermatocytes at the zygotene stage became ob-
Novel Mutation of the Mouse c-kit Gene
823
Figure 4.—Spermatogenesis defects in Wads mutants. Sections of wild-type testis of postnatal day 6 (A), day 8 (C), day 12 (E),
and 6 weeks (G) and of Wads m/m testis of day 6 (B), day 8 (D), day 12 (F), 6 weeks (H), and 10 weeks (I) are shown. The type
A and B spermatogonia are indicated by white and black arrows. The Sertoli cells and primary spermatocytes are indicated by
white and black arrowheads. White stars indicate clusters of cells in the center of abnormal seminiferous tubules and the interstitial
space is enlarged by hyperplastic Ledig cells (black stars). Gross morphology of Bouin’s fixative-fixed adult Wads m/⫹ and Wads m/m
testis is shown ( J).
vious in the wild-type seminiferous tubules. In contrast,
no germ cells entered meiosis in the Wads m/m testis (Figure 4, E and F). Overall, the morphology of the seminiferous epithelium of the Wads m/m mice at this stage still
resembled that of the postnatal day 6 mice. At 6 and
10 weeks, only a few germ cells remained at the basal
membrane of the seminiferous tubules of the Wads m/m
testis (Figure 4, H and I). In the adult Wads m/m testis
824
H.-B. Ruan, N. Zhang and X. Gao
Figure 4.—Continued.
we also observed clusters of cells in the center of the
seminiferous tubules. They had germ cell characteristics
and it was possible that the disruption of spermatogenesis in the Wads m/m mice caused shedding of germ cells
into the lumen. Moreover, the interstitial space in adult
Wads m/m testis was filled out with overgrowth of Leydig
cells (Figure 4, H and I). This increase of Leydig cell
numbers was not caused by the reduction of the size of
the seminiferous tubules but by enhanced mitotic activity of Leydig cells (Kissel et al. 2000). And our BrdU assay, which showed that the Leydig cells in Wads m/m testis
were stained positive, was in agreement with this hypothesis (data not shown). Finally, the gross size of the adult
Wads m/m testis is much smaller than that of wild types
(Figure 4J).
In the ovary, Kit expression is observed in oocytes from
the time of birth until ovulation (Horie et al. 1991). Interaction of Kit with KL/MGF expressed by granulosa cells
is essential for follicle development (Kuroda et al. 1988).
In newborn wild-type mouse ovaries, hematoxylin and
eosin staining showed many primordial follicles (Figure
5A). However, no primordial follicles or signal naked
germ cells were present (Figure 5B) in Wads m/m ovaries.
At postnatal day 11, wild-type ovaries showed many developing follicles in the cortex (Figure 5C), compared
with no follicle in Wads m/m ovaries (Figure 5D). Only granulose cells remained in adult Wads m/m ovaries (Figure
5F) and the gross size of the Wads m/m ovary was reduced.
Effect of the Wads mutation on peripheral blood:
Blood cell analysis of peripheral blood was performed
to examine the possible defects in Wads mutant mice
because c-kit is expressed and functions in hemopoietic
progenitor cells (Ogawa et al. 1991). As presented in
Table 1, the total values of each parameter of Wads m/⫹
and Wads m/m were compared to those of wild-type mice.
No differences were seen between wild-type and Wads m/⫹
mice, except Wads m/⫹ mice displayed slight elevation of
MPV (P ⬍ 0.5). Interestingly, the RBC counts of Wads m/m
mice were significant reduced (P ⬍ 0.5), whereas the
MCV of Wads m/m was significantly higher (P ⫽ 0.0001)
compared with that of wild-type and Wads m/⫹ mice. These
results indicated that Wads m/m mice were suffering from
macrocytic anemia. They were also consistent with earlier reports that mutations of c-kit or kitl (encoding KIT
ligand SCF) displayed a similar hemopoietic disorder
(Piao and Bernstein 1996). The platelet counts and
Novel Mutation of the Mouse c-kit Gene
825
Figure 5.—Histological analysis of postnatal ovaries in Wads mice. Sections of ovaries of postnatal day 0 (A and B), day 11 (C
and D), and adult stage (E and F) are shown. (A, C, and E) Wild-type ovaries. (B, D, and F) Wads m/m ovaries. The white arrows
indicate follicles.
mean platelet volume of Wads m/m mice were also significantly elevated compared with that of wild type and
Wads m/⫹ (P ⬍ 0.05 and P ⬍ 0.01, respectively). All blood
parameters showed no significant difference between
males and females within the same genotype.
Hearing loss in Wads m/m mutants: The intermediate
cells that derive from melanoblasts were missing in stria
vascularis of homozygous KitW mutant mice, resulting
in endocochlear degeneration, endocochlear potential
(EP) disappearance, and hearing impairment (Cable
et al. 1994, 1995). To examine the hearing ability of
Wads mice, we carried out an ABR test. Both wild-type
and Wads m/⫹ mice showed normal waves responding to
40, 50, and 70 dB sound pressure levels of stimuli (Figure
3.6
1.1
15.4
1.3
0.4
5.0
268.4
0.3
7.6
7.3
120.8
48.6
16.7
343.0
719.5
5.1
WBC (⫻10 9/L)
RBC (⫻10 9/L)
HGB (g/L)
MCV (fl)
MCH (Pg)
MCHC (g/L)
PLT (⫻10 9/L)
MPV (fl)
WBC, white blood cell counts; RBC, red blood cell counts; HGB, hemoglobin concentration; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin;
MCHC, mean corpuscular hemoglobin concentration; PLT, platelet counts; MPV, mean platelet volume. No difference was seen between males and females within the same
genotype. The average values of the three mice were evaluated using an unpaired two-tailed t-test. *P ⱕ 0.05; **P ⱕ 0.01; ***P ⫽ 0.0001.
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
7.8
5.8
128.2
58.1
18.3
314.6
1039.6
5.6
3.8
1.2
40.4
5.3
1.8
22.0
124.0
0.1
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
8.2
5.2
108.2
60.8
19.9
327.5
1082.3
5.6
3.4
0.6
27.5
4.0
4.5
83.8
283.8
0.6
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
7.4
6.4
148.2
55.4
16.7
301.8
997.0
5.7
2.6
2.0
28.4
1.0
1.7
38.7
410.0
0.5*
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
5.8
7.6
128.1
46.5
17.0
365.6
640.3
5.6
2.6
2.5
39.2
0.8
2.3
52.8
454.5
0.3
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
5.9
6.8
119.2
46.4
17.7
380.8
504.5
5.5
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
6.2
7.3
129.3
46.2
17.2
372.5
755.5
5.0
Female
(n ⫽ 4)
Male
(n ⫽ 4)
1.4
1.1
25.8
2.6
0.3
21.8
420.4
0.2
6.9
7.3
125.0
47.4
16.9
357.8
737.5
5.0
2.6
1.0
20.2
2.3
0.4
21.5
327.1
0.2
5.8
8.4
137.0
46.7
16.4
350.5
776.0
5.8
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
3.0
1.0
11.5
1.2
0.6
10.2
369.4
0.8
Female
(n ⫽ 4)
Male
(n ⫽ 4)
Total
(n ⫽ 8)
Female
(n ⫽ 4)
Total
(n ⫽ 8)
Male
(n ⫽ 4)
Wads m/m
Wads m/⫹
Wild type
Peripheral blood cell analysis
TABLE 1
3.4
1.1*
38.5
5.2***
3.6
58.4
207.8*
0.4**
H.-B. Ruan, N. Zhang and X. Gao
Total
(n ⫽ 8)
826
6A; data of the Wads m/⫹ ABR pattern were not shown).
However, no ABR response waves were detected in
Wads m/m mutant mice 1 month after birth (Figure 6B),
indicating that Wads-related c-kit mutation led to hearing loss. In wild-type cochlear sections, the stria vascularis (SV) was composed of three layers of cells. They
were marginal cells facing the endolymphatic space,
basal cells facing the spiral ligament, and intermediate
cells in the middle (Figure 6, C and D). There were
also abundant blood capillaries in the SV. However, the
SV of the Wads m/m cochlea was much thinner than that
of wild type because of the loss of intermediate cells
and defects of blood capillaries (Figure 6, E–H). Meanwhile, the organ of Corti and hair cells were degenerated in the Wads m/m cochlea (Figure 6, E and G). It was
believed that these phenomena were the later pathological changes of the loss of intermediate cells and EP
(Hoshino et al. 1999). Moreover, the SV became more
disorganized and the blood capillaries were depleted in
the SV in the 1-year-old Wads m/m cochlear sections (Figure 6H).
The gap junction system in the cochlea is critical for
ion cycling, which is the underlying mechanism of the
generation of EP. There are widespread gap junctions
between basal cells and between basal cells and intermediate cells. These cells are coupled together as a syncytium allowing exchange of intracellular contents such
as K⫹. Connexins are gap junction proteins that are
expressed in the cochlea (Lautermann et al. 1998; Figure 6, I and K). In an immunohistological study of
Wads m/m compared with the wild-type mice, the connexin
26 and connexin 30 expression is disorganized and discontinuous in SV of Wads m/m mice, suggesting the gap
junctions in the base of the SV were detrimentally disrupted (Figure 6, J and L).
DISCUSSION
In this report we described the phenotypes and genome alternation of a new mutant strain Wads from
ENU mutagenesis. Genetic mapping indicated that Wads
was a novel allele of the W/c-kit locus, with a missense
point mutation of c-kit proto-oncogene (Phe856Ser) in the
conservative PTK domain. This mutation led to white
spotting in heterozygotes and black-eyed white color,
macrocytic anemia, mast cell deficiency, deafness, and
sterility in homozygotes.
The c-kit gene spans ⬎70 kb of DNA and includes 21
exons (Vandenbark et al. 1992). The longest transcript
is 5230 bp. Notably, c-kit is among those genes with the
highest spontaneous mutations. Various KIT mutations
have been identified in human, mouse, rat, dog, and
pig, etc. (Tsujimura et al. 1991; Pielberg et al. 2002;
Zemke et al. 2002). While the actual molecular mechanism of this high mutation frequency is still not clear,
it may be related to the easy accessibility of the mutagen
to this chromosomal locus as well as the easy recognition
Novel Mutation of the Mouse c-kit Gene
827
Figure 6.—ABR test and cochlear histology of B6 and Wads
mutant mice. (A and B) Normal
ABR waves induced by different
sound pressure level stimuli in a
wild-type (A) and Wads m/m (B)
mouse. (C and E) Cochlear section of a 2-month-old wild-type
(C) and Wads m/m (E) mouse. (G)
Section from a 10-month-old
Wads m/m mouse. (D, F, and H)
Magnification of the stria vascularis regions of C, E, and G. Marginal cells (white arrows), basal
cells (black arrows), intermediate
cells (black arrowheads), and
blood capillaries (white arrowheads) are indicated. (I and H)
Stria vascularis immunostaining
with connexin 26 on wild type and
Wads m/m. (K and L) Stria vascularis
immunostaining with connexin 30
on wild type and Wads m/m. The
arrows indicate the margin of the
SV. The arrowheads indicate the
gap junction between basal cells
and intermediate cells. The stars
point out the disruptions of gap
junction in Wads m/m SV.
of dominant coat abnormality. It is possible that the c-kit
gene is transcriptionally active in dividing germ cells;
therefore the locus is in a constant “open” status. However, by examining the expression file of testis, c-kit is
certainly not among the highest-expressing genes, although the testis contains mixed types of cells (Su et al.
2002).
Two types of c-kit mutations have been reported in
humans. Loss-of-function mutations, which often take
place at the tyrosine kinase domains and immunoglobulin-like loops, may cause a deficiency/defect of
melanocyte, mast cell, germ cell, and hematogenic cells
(Figure 2A; Spritz 1994; Fleischman et al. 1996; OMIM
{*164920}). In contrast, gain-of-function mutations, which
828
H.-B. Ruan, N. Zhang and X. Gao
Figure 6.—Continued.
are located in the cytoplasmic juxtamembrane domain
or catalytic domain, were identified as the cause of mastocytosis, acute myeloid leukemia, gastrointestinal stromal tumors, and germ cell tumors (Figure 2B; Kitayama et al. 1996; Tsujimura 1996; Hirota et al. 1998;
Tian et al. 1999; OMIM {*164920}). The phenotypes of
Wad m/m suggested that the F856S substitution (equal to
F858S in human KIT) is a loss-of-function or hypomorphic mutation (Figure 2A).
Previous studies showed that the level of KIT kinase
activity and the severity of the phenotypic expression
for each W/c-kit allele correlated with the type of mutation. Mutations that abolish activity by deletion (e.g., KitW,
KitW-19H) or point mutation (e.g., KitW-37J, KitW-42J) are homozygous lethal, while mutations with residual kinase activity (e.g., KitW-V, KitW-57J) are homozygous viable (Bernstein et al. 1991). From these studies we speculated
that KIT of Wads m/m maintains residual kinase activity
although further proof is needed. Interestingly, the
Wads m/m mice displayed noticeable reduced prenatal or
neonatal viability. Among a total of 207 mice from a
heterozygous intercrossing breed, only 12 of them were
homozygotes, 4 of which died within 10 weeks after
birth. The proportion of viable homozygous mutant
mice was 4.35% and significantly below the expected
25%. The cause of this lethality is still unknown.
Novel Mutation of the Mouse c-kit Gene
829
Figure 6.—Continued.
Among all the known c-kit mutants, only KitW-V and
our KitWads show all the defects that have been reported
in c-kit mutations, including white color, mast cell loss,
anemia, hearing loss, and sterility. Nevertheless, KitW-V
is a missense Thr-to-Met point mutation in position 660
(T660M) of the kinase domain I of the KIT protein,
whereas the KitWads mutation localized in the kinase domain II. There are also some phenotypic differences
between these two strains. For instance, The KitW-V heterozygotes had a slight macrocytic anemia but the KitWads
heterozygotes did not suffer from anemia (Russell
1949). It may reflect that residual kinase activity in KitWads
830
H.-B. Ruan, N. Zhang and X. Gao
is more than that in KitW-V mice. Alternatively, the phenotype variation may result from different mutant sites
in the kinase domain, which might subsequently affect
different downstream signaling pathways. Similar scenarios have been reported, where blockage of phosphatidylinositol 3⬘-kinase (PI3K) signaling from KIT did not
affect other KIT downstream responses (Blume-Jensen
et al. 2000; Kissel et al. 2000).
The partial viability of the KitWads homozygote really
facilitated the functional analysis of the c-kit gene on late
developmental events such as gametogenesis. Previous
studies suggested that the migration and/or proliferation of primordial germ cells (PGCs) were impaired in
the KitW-V embryos. However, the testes of newborn KitWads
homozygous mice showed no differences from those of
wild-type mice up to postnatal day 6. It is possible that
the KitWads mutation affects only postnatal differentiation
and maturation of spermatogenesis but not embryonic
PGC development. Alternatively, the KitWads mutation did
reduce the number of PGCs by affecting their proliferation and/or survival, but the remaining PGCs were enough
to form a relatively normal gonad structure at birth.
In contrast with testis, the ovary lacks primordial follicles in newborn KitWads homozygous females, and this
directly caused the female to be infertile. This suggested
that the c-kit mutation affected the female reproductive
system before birth. When and how the mutation reacts
with female primordial germ cell development remain
unclear.
It is interesting to dissect out the differential response
of the KitWads mutation in the male and the female reproductive systems. Transgenic study indicated that the melanocyte stem cell (MSC) could survive independently
of c-kit signaling transduction. However, MSCs migrated
outside of the hair follicles in a c-kit-dependent manner
(Kunisada et al. 1998). Hematopoietic stem cells expressed c-kit but did not depend on it (Ikuta and Weissman 1992). Similarly, spermatogonia stem cells (SSCs)
did not express or depend on c-kit (Schrans-Stassen
et al. 1999; Ohta et al. 2003). We suspect only the further
differentiation of the SSCs requires c-kit gene function.
In male mice, the differentiation of SSCs and spermatogenesis start ⵑ6 days after birth. The meiosis happens
much later. In contrast, the oogonia start meiosis at
around embryonic day 13.5 and arrest at the diplotene
stage before birth. We hypothesize that, therefore, KIT
function is crucial at the time of stem cell differentiation
and/or meiosis in the reproductive system. Because of
the different time courses for male and female germ cell
differentiation, the KitWads mutation displayed distinct
patterns of defects in the male and the female reproductive systems.
In this study, we generated and identified a new strain
for the mouse c-kit mutant archive. This KitWads mouse
showed almost all the phenotypes associated with protooncogene c-kit mutation. There were detailed differences between KitWads and other c-kit alleles. It provided
us a new powerful animal model to comprehensively
reveal the function of c-kit and most importantly to figure out the complex downstream signaling pathways
and targets of c-kit in related different systems.
We thank Xingxing Gu, Fang He, Zixin Wang, and Haibo Sha for
technical help. This work is supported by the National Natural Science
Fund of China (30300425), the National Gongguan Project of China
(2001BA710B), the Joint Research Fund for Overseas Chinese Young
Scholars (30228008), and EISU(E03003).
LITERATURE CITED
Balling, R., 2001 ENU mutagenesis: analyzing gene function in
mice. Annu. Rev. Genomics Hum. Genet. 2: 463–492.
Bellvé, A. R., J. C. Cavicchia, C. F. Millette, D. A. O’Brien, Y. M.
Bhatnagar et al., 1977 Spermatogenic cells of the prepuberal
mouse. Isolation and morphological characterization. J. Cell Biol.
74: 68–85.
Bennett, D. C., and M. L. Lamoreux, 2003 The color loci of
mice—a genetic century. Pigment. Cell. Res. 16: 333–344.
Bernstein, A., L. Forrester, A. D. Reith, P. Dubreuil and R. Rottapel, 1991 The murine W/c-kit and Steel loci and the control of
hematopoiesis. Semin. Hematol. 28: 138–142.
Blume-Jensen, P., G. Jiang, R. Hyman, K. F. Lee, S. O’Gorman
et al., 2000 Kit/stem cell factor receptor-induced activation of
phosphatidylinositol 3⬘-kinase is essential for male fertility. Nat.
Genet. 24: 157–162.
Brown, S. D., and R. E. Hardisty, 2003 Mutagenesis strategies for
identifying novel loci associated with disease phenotypes. Semin.
Cell. Dev. Biol. 14: 19–24.
Cable, J., D. Huszar, R. Jaenisch and K. P. Steel, 1994 Effects of
mutations at the W locus (c-kit) on inner ear pigmentation and
function in the mouse. Pigment. Cell. Res. 7: 17–32.
Cable, J., I. J. Jackson and K. P. Steel, 1995 Mutations at the W
locus affect survival of neural crest-derived melanocytes in the
mouse. Mech. Dev. 50: 139–150.
Chabot, B., D. A. Stephenson, V. M. Chapman, P. Besmer and A.
Bernstein, 1988 The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus.
Nature. 335: 88–89.
Fleischman, R. A., T. Gallardo and X. Mi, 1996 Mutations in the
ligand-binding domain of the kit receptor: an uncommon site
in human piebaldism. J. Invest. Dermatol. 107: 703–706.
Geissler, E. N., E. C. McFarland and E. S. Russell, 1981 Analysis
of pleiotropism at the dominant white-spotting (W) locus of the
house mouse: a description of ten new W alleles. Genetics 97:
337–361.
He, F., Z. Wang, J. Zhao, J. Bao, J. Ding et al., 2003 Large scale
screening of disease model through ENU mutagenesis in mice.
Chin. Sci. Bull. 48: 2665–2671.
Heldin, C. H., 1995 Dimerization of cell surface receptors in signal
transduction. Cell 80: 213–223.
Herron, B. J., W. Lu, C. Rao, S. Liu, H. Peters et al., 2002 Efficient
generation and mapping of recessive developmental mutations
using ENU mutagenesis. Nat. Genet. 30: 185–189.
Hirota, S., K. Isozaki, Y. Moriyama, K. Hashimoto, T. Nishida
et al., 1998 Gain-of function mutations of c-kit in human gastrointestinal stromal tumors. Science 279: 577–580.
Horie, K., K. Takakura, S. Taii, K. Narimoto, Y. Nada et al., 1991
The expression of c-kit protein during oogenesis and early embryonic development. Biol. Reprod. 45: 547–552.
Hoshino, T., K. Mizuta, J. Gao, S. Araki, K. Araki et al., 1999 Cochlear findings in the white spotting (Ws) rat. Hear. Res. 140: 145–156.
Hou, L., J. J. Panthier and H. Arnheiter, 2000 Signaling and
transcriptional regulation in the neural crest-derived melanocyte
lineage: interactions between KIT and MITF. Development 127:
5379–5389.
Hrabe de Angelis, M., H. Flaswinkel, H. Fuchs, B. Rathkolb, D.
Soewartom et al., 2000 Genome-wide, large-scale production
of mutant mice by ENU. Nat. Genet. 25: 444–447.
Huizinga, J. D., L. Thuneberg, M. Kluppel, J. Malysz, H. B. Mikkel-
Novel Mutation of the Mouse c-kit Gene
esen et al., 1995 W/kit gene required for interstitial cells of Cajal
and for intestinal pacemaker activity. Nature 373: 347–349.
Ikuta, K., and I. L. Weissman, 1992 Evidence that hematopoietic
stem cells express mouse c-kit but do not depend on steel factor
for their generation. Proc. Natl. Acad. Sci. USA 89: 1502–1506.
Lautermann, J., W.-J.F. ten Cate, P. Altenhoff, R. Grummer, O.
Traub et al., 1998 Expression of the gap-junction connexin 26
and 30 in the rat cochlea. Cell Tissue Res. 294: 415–420.
Linnekin, D., 1999 Early signaling pathways activated by c-Kit in
hematopoietic cells. Int. J. Biochem. Cell Biol. 31: 1053–1074.
Kissel, H., I. Timakhina, M. P. Hardy, G. Rothschild, Y. Tajima
et al., 2000 Point mutation in Kit receptor tyrosine kinase reveals
essential roles for Kit signaling in spermatogenesis and oogenesis
without affecting other Kit responses. EMBO. J. 19: 1312–1326.
Kitayama, H., T. Tsujimura, I. Matsumura, K. Oritani, H. Ikeda
et al., 1996 Neoplastic transformation of normal hematopoietic
cells by constitutively activating mutations of c-kit receptor tyrosine kinase. Blood 88: 995–1004.
Kunisada, T., H. Yoshida, H. Yamazaki, A. Miyamoto, H. Hemmi
et al., 1998 Transgene expression of steel factor in the basal layer
of epidermis promotes survival, proliferation, differentiation and
migration of melanocyte precursors. Development 125: 2915–2923.
Kuroda, H., N. Terada, H. Nakayama, K. Matsumoto and Y. Kitamura, 1988 Infertility due to growth arrest of ovarian follicles
in Sl/Slt mice. Dev. Biol. 126: 71–79.
Manova, K., and R. F. Bachvarova, 1991 Expression of c-kit encoded at the W locus of mice in developing embryonic germ
cells and presumptive melanoblasts. Dev. Biol. 146: 312–324.
Nolan, P. M., J. Peters, M. Strivens, D. Rogers, J. Hagan et al.,
2000 A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat.
Genet. 25: 440–443.
Ogawa, M., Y. Matsuzaki, S. Nishikawa, S. Hayashi, T. Kunisada
et al., 1991 Expression and function of c-kit in hemopoietic
progenitor cells. J. Exp. Med. 174: 63–71.
Ohta, H., A. Tohda and Y. Nishimune, 2003 Proliferation and
differentiation of spermatogonial stem cells in the W/Wv mutant
mouse testis. Biol. Reprod. 69: 1815–1821.
Piao, X., and A. Bernstein, 1996 A point mutation in the catalytic
domain of c-kit induces growth factor independence, tumorigenicity, and differentiation of mast cells. Blood 87: 3117–3123.
Pielberg, G., C. Olsson, A.-C. Syvanen and L. Andersson, 2002
Unexpectedly high allelic diversity at the KIT locus causing dominant white color in the domestic pig. Genetics 160: 305–311.
Price, E. R., H. F. Ding, T. Badalian, S. Bhattacharya, C. Takemoto et al., 1998 Lineage-specific signaling in melanocytes: c-Kit
stimulation recruits p300/CBP to microphthalmia. J. Biol. Chem.
273: 17983–17986.
Reeves, R. H., and P. D’Eustachio, 1997 Genetic and comparative
mapping in mice, pp.71–131 in Genome Analysis: A Laboratory Manual,
edited by E. D. Green, B. Birren, S. Klapholz, R. M. Myers and
P. Hieter. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
831
Reith, A. D., R. Rottapel, E. Giddens, C. Brady, L. Forrester
et al., 1990 W mutant mice with mild or severe developmental
defects contain distinct point mutations in the kinase domain of
the c-kit receptor. Genes Dev. 4: 390–400.
Russell, E. S., 1949 Analysis of pleiotropism at the W-locus in the
mouse: relationship between the effects of W and W-v substitution
on hair pigmentation and on erythrocytes. Genetics 34: 708–723.
Schrans-Stassen, B., H. Van De Kant, D. De Rooij and A. Van Pelt,
1999 Differential expression of c-kit in mouse undifferentiated
and differentiating type A spermatogonia. Endocrinology 140:
5894–5900.
Spritz, R. A., 1994 Molecular basis of human piebaldism. J. Invest.
Dermatol. 103: 137S–140S.
Strippoli, P., S. Sarchielli, R. Santucci, G. P. Bagnara, G. Brandi
et al., 2001 Cold single-strand conformation polymorphism analysis: optimization for detection of APC gene mutations in patients
with familial adenomatous polyposis. Int. J. Mol. Med. 8: 567–572.
Su, A. I., P. M. Cooke, K. A. Ching, Y. Hakak, J. R. Walker et al.,
2002 Large-scale analysis of the human and mouse transcriptomes. Proc. Natl. Acad. Sci. USA 99: 4465–4470.
Sunnucks, P., A. C. Wilson, L. B. Beheregaray, K. Zenger,
J. French et al., 2000 SSCP is not so difficult: the application
and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Mol. Ecol. 9: 1699–1710.
Tian, Q., H. F. Frierson Jr., G. W. Krystal and C. A. Moskaluk,
1999 Activating c-kit gene mutations in human germ cell tumors.
Am. J. Pathol. 154: 1643–1647.
Timokhina, I., H. Kissel, G. Stella and P. Besmer, 1998 Kit signaling through PI3-kinase and Src kinase pathways: an essential role
for Rac1 and JNK activation in mast cell proliferation. EMBO J.
17: 6250–6262.
Tsujimura, T., 1996 Role of c-kit receptor tyrosine kinase in the
development, survival and neoplastic transformation of mast cells.
Pathol. Int. 46: 933–938.
Tsujimura, T., S. Hirota, S. Nomura, Y. Niwa, M. Yamazaki et al.,
1991 Characterization of Ws mutant allele of rats: a 12-base deletion in tyrosine kinase domain of c-kit gene. Blood 78: 1942–1946.
Vandenbark, G. R., C. M. deCastro, H. Taylor, S. Dew-Knight
and R. E. Kaufman, 1992 Cloning and structural analysis of the
human c-kit gene. Oncogene 7: 1259–1266.
Witter, O. N., 1990 Steel locus defines new multipotent growth
factor. Cell 63: 5–6.
Zemke, D., B. Yamini and V. Yuzbasiyan-Gurkan, 2002 Mutations
in the juxtamembrane domain of c-KIT are associated with higher
grade mast cell tumors in dogs. Vet. Pathol. 39: 529–535.
Zhao, S., L. J. Rizzolo and C. J. Barnstable, 1997 Differentiation
and transdifferentiation of the retinal pigment epithelium. Int.
Rev. Cytol. 171: 225–266.
Zheng, Q. Y., K. R. Johnson and L. C. Erway, 1999 Assessment of
hearing in 80 inbred strains of mice by ABR threshold analyses.
Hear. Res. 130: 94–107.
Communicating editor: C. Kozak