Identification of a Novel Point Mutation of Mouse ... - Semantic Scholar

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

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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

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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

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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 (Bellve´ 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-


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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

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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


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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**


Total (n ⫽ 8)

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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


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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

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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.


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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

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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. Bellve´, 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.

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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