ABSTRACT
The receptor tyrosine kinase c-kit and its cognate ligand KL are encoded at the white spotting (W) and steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl locus cause deficiencies in gametogenesis, melanogenesis and hematopoiesis (erythrocytes and mast cells). The W-sash mutation differs from most W mutations in that it affects primarily mast cells and melanogenesis but not other cellular targets of W and Sl mutations. Thus, Wsh/Wsh mice are fertile and not anemic, but they lack mast cells in their skin and intestine and are devoid of coat pigment. Heterozygotes are black with a broad white sash/belt in the lumbar region. In order to determine the basis for the phenotypes of W-sash mice, we investigated c-kit RNA and protein expression patterns in adult Wsh/Wsh mice and during embryonic development. We show that c-kit expression is absent in bone-marrow-derived Wsh/Wsh mast cells, the fetal and the adult lung, and the digestive tract at embryonic day , tissues that normally express c-kit. Unexpectedly, in and Wsh/Wsh embryos, we found c-kit expression in the dermatome of the somites, the mesenchyme around the otic vesicle and the floorplate of the neural tube, structures known to express the c-kit ligand in wild-type embryos. The ectopic c-kit expression in Wsh homozygous embryos does not affect c-kit ligand expression. The presumed Wsh/Wsh melanoblasts appeared to be normal and, at similar numbers were found in normal and homozygous mutant embryos. At embryos had a graded distribution of melanoblasts from cranial to caudal with a minimum in the lumbar region. Whereas homozygous Wsh/Wsh embryos essentially lacked c-kit-positive cells in the skin, heterozygous Wsh/+ embryos had reduced numbers of melanoblasts compared to +/+ with few or none in the lumbar region (future sash). It is proposed that ectopic c-kit expression in the somitic dermatome affects early melanogensis in a dominant fashion. Molecular analysis of Wsh chromosomal DNA revealed a deletion or rearrangement in the vicinity of the c-kit gene. These results provide an explanation for the Wsh phenotype and have implications for the control of c-kit expression.
INTRODUCTION
Mutations at the white spotting (W) and steel (Sl) loci in mice result in deficiencies in the production of three classes of cells: melanocytes, germ cells and hematopoietic cells (specifically erythrocytes and mast cells) (for reviews, see Russell, 1979; Silvers, 1979). The W and the Sl loci of mice, respectively, encode the proto-oncogene c-kit, a receptor tyrosine kinase of the PDGF receptor subfamily and the kitligand, KL, also designated steel factor (Besmer et al., 1986;Yarden et al., 1987; Majumder et al., 1988; Qiu at al., 1988; Chabot et al., 1988; Geissler et al., 1988; Nocka et al., 1989, 1990b,c; Zsebo et al., 1990a,b; Williams et al., 1990; Copeland et al., 1990).
Many independent mutations at the W locus have been described. The different alleles vary in their degree of severity in the heterozygous and the homozygous state, but most of them affect the different cellular targets of the mutations to comparable degrees (Silvers, 1979; Geissler et al., 1981; Lyon and Searle, 1989). The W-sash mutation differs from most other W mutations in that only pigmenta-tion and tissue mast cells are affected in homozygous mutant mice (Lyon and Glenister, 1982; Stevens and Loutit, 1982). Thus, homozygous Wsh mutant mice are fertile and not anemic, but they are black-eyed whites with small black patches around the ears and eyes and lack mast cells in their skin and intestine. Heterozygotes, from which the name is derived, have a broad white sash or band in the lumbar region.
The white sash of heterozygous Wsh mice differs from the pigmentation pattern of most mice with W mutations and reflects a dominant property of the mutation (Stephenson et al., 1985). Heterozygous W mutant mice with null mutations (W and W19H) have a white belly spot of varying size, while heterozygous mutant animals with dominant W mutations are either black-eyed whites (W42), or have a flecked appear-ance (W37).
Melanocytes are derived from neural crest cells that migrate along a dorsolateral pathway away from the neural tube over the dermatomes of the somites (for a review see LeDouarin, 1982). They then spread through the dermis down to the ventral region of the body. Neural crest cell migration is first seen at rostral levels around and then proceeds in an anterior-to-posterior fashion (Rawles, 1947). By melanoblasts are found in the dorsal dermis in the trunk region (Rawles, 1947; Mayer, 1965; Serbedzija et al., 1990) and by the precursor cells have reached the ventral midline (Rawles, 1947). At some melanoblasts have moved from the dermis into the epidermis (Rawles, 1947; Mayer, 1973). The white skin of Wv/Wv mice as well as the white spots of W/+ and Wv/+ mice lack melanocytes (Silvers, 1956, 1979). The absence of melanocytes can be interpreted as a failure of the precursor cells to migrate, or as a failure to proliferate, or to survive. However, a defect in migration or failure to proliferate are not sufficient to explain the white belt in Wsh/+ mice.
During melanogenesis, c-kit function is necessary at around , when melanocyte precursors migrate from the dermis into the epidermis (Mayers, 1973; Nishikawa et al., 1991). An earlier function for c-kit is suggested by the finding of synchronous c-kit and KL expression in melanoblasts found dorsally and laterally of the somites and in the somitic dermatome, respectively, at around embryonic day (Manova and Bachvarova, 1991; Matsui et al., 1990).
The molecular basis of the Wsh mutation is not known. In mutant mice in which all three targets are affected similarly, an intrinsic defect of c-kit receptor function is the probable cause of the mutation. Molecular analysis of several W alleles revealed missense mutations in c-kit which affect the c-kit kinase (Nocka et al., 1989, 1990a; Tan et al., 1990; Reith et al., 1990; for a review see, Besmer, 1991). The nonparallel display of mutant characteristics in Wsh mice might indicate that the mutation affects c-kit function in some cell types and not in others, possibly as a result of differential effects on c-kit transcription or post-transcriptional process-ing.
In an attempt to determine the basis of the phenotype of Wsh mice, we investigated c-kit expression in various tissues and cell types of homozygous mutant mice at various stages of development. We show here that some tissues and cell types that normally express c-kit lack expression in Wsh/Wsh mice. Interestingly, inappropriate c-kit expression was found in some tissues known to express the kit-ligand in normal mice during embryonic development. Analysis of chromosomal DNA from Wsh/Wsh mice by conventional and pulsedfield gel electrophoresis (PFGE) DNA blots indicated no alterations of c-kit exon and intron sequences but a deletion or rearrangement in the vicinity of the c-kit gene. Our results provide possible explanations for the pigmentation pattern of heterozygous and homozygous Wsh mice and define time points of c-kit function during melanogenesis. Also, our results have implications for transcriptional control of the c-kit proto-oncogene.
MATERIALS AND METHODS
Mice and embryos
C57BL/6J and C3H/HeJ mice were purchased from the Jackson Laboratory. C57BL/6 Wsh/Wsh mice were kindly provided by Dr Rudolf Jaenisch, MIT and 101 mice were provided by Dr L. B. Russell, Oak Ridge National Laboratories. Embryos were derived from appropriate matings of C57BL/6 Wsh/Wsh and C57BL/6J. The morning of vaginal plug was considered as day 0.5 p.c.
Mast cell cultures
Mast cells were grown from bone marrow of adult mice in RPMI-1640 supplemented with 10% fetal calf serum, 6% conditioned medium from WEHI-3B cells, non-essential amino acids, sodium pyruvate and 2-mercaptoethanol (5×10−5 M). Non-adherent cells were harvested and fed weekly and maintained at a cell density of 5×105 cells/ml.
RNA and DNA isolation and blot analysis
Mast cells and mouse tissues were homogenized in guanidinium isothiocyanate and RNA isolated according to the method of Chirgwin et al. (1979). Total cellular RNA (mast cells: 1 μg, 2 μg and 5 μg; lung, brain and testis: 15 μg) was fractionated in 1% agarose-formaldehyde gels, transferred to nylon membranes (Nytran, Schleicher and Schuell) and prehybridization and hybridization performed as previously described (Lehrach et al., 1977; Nocka et al., 1989). The murine 1.4 kb c-kit cDNA labeled with 32P-dCTP, prepared by the random primer method, was used as a probe for hybridization (Feinberg and Vogelstein, 1983).
DNA was extracted by standard techniques (Sambrook et al., 1989) and digested with restriction endonucleases (Boehringer Mannheim) in restriction buffer provided by the manufacturer. The DNA was fractionated in 0.8% agarose gels. The DNA was depurinated with 0.25 M hydrochloric acid for 5 minutes, denatured with 0.5 N sodium hydroxide for 5 minutes, neutralized with 1 M Tris, pH 5.0, 2 M sodium chloride for 5 minutes and vacuum-transferred (LKB, Pharmacia) to nylon membranes (Zetabind; AMF Cuno, Meridian, CT) in 1× SSC (0.15 M sodium chloride, 0.015 M sodium citrate) for 3 hours. DNA was crosslinked to filters with ultraviolet irradiation in a Stratalinker (Stratagene, La Jolla, CA). Filters were prehybridized in 50% formamide, 6× SSPE (1× SSPE: 0.15 M sodium chloride, 0.01 M sodium phosphate, pH 7.5, 1 mM EDTA), 0.5% sodium dodecyl sulfate (SDS), 100 μg denatured salmon sperm DNA, 10× Denhardt’s (1× Denhardt’s: 0.01% Ficoll 400, 0.01% polyvinylpyrrolidone, 0.01% BSA) at 42°C for 2 to 6 hours. Filters were hybridized in 50% formamide, 6× SSPE, 0.5% SDS, 1× Denhardt’s, 100 μg denatured salmon sperm DNA, 10% dextran sulfate with 106 cts/minute/ml at 42°C for 16 hours. The 3.7 kb murine c-kit cDNA labeled with 32P-dCTP, prepared by the random primer method, was used as a probe. Filters were washed 4× 15 minutes at room temperature in 2× SSC, 0.5% SDS and once for 15 minutes at room temperature in 0.1× SSC, 0.5% SDS. The final wash was in 0.1× SSC, 0.5% SDS at 42°C for 30 minutes.
Pulse field gel electrophoresis (PFGE)
Samples for PFGE were prepared as described (Zelenetz et al., 1991). Briefly, spleen cells were purified on a Ficoll-Hypaque density gradient (Pharmacia). Cells were resuspended in T10E100 (10 mM Tris, pH, 100 mM EDTA) at 3.36×107 cells/ml and mixed with an equal volume of molten agarose solution (2% InCert agarose (FMC, Rockland, ME) in T10E100) cooled to 42°C. Aliquots (250 μl) of the molten agarose-cell suspension were placed in plug molds (BioRad, Richmond, CA) and allowed to solidify at 4°C. Plugs were then suspended in 10 ml of T10E containing 1% N-lauryl sarcosine (Sigma, St Louis, MO) and 200 μg/ml Proteinase K (Boehringer-Mannheim, Indianapolis, IN) and incubated at 55°C for 16 hours. Plugs were then equilibrated with a large volume of TE to remove the detergent. Next, the plugs were equilibrated in TE containing 1 mM phenylmethylsulfonyl fluoride (Sigma) to inactivate the Proteinase K. Finally the plugs were extensively washed in TE and stored in T10E100 at 4°C. For analysis, the plugs were divided into 6 parts of approximately 40 μl, with each 40 μl containing 4-5 μg of DNA. Before restriction with restriction endonucleases the 40 μl plugs were equilibrated for 30 minutes at room temperature (repeated twice) in the restriction buffer provided by the manufacturer. The buffer was replaced by fresh buffer containing 50 U of restriction endonuclease and the enzyme was allowed to diffuse into the plug for 4 hours on ice before incubation at 37°C for 16 hours. The plug was transferred into a well in the pulsed-field gel and sealed with agarose.
PFGE was performed in a contour-clamped homogenous electric field (CHEF) apparatus. Electrophoresis was performed at 14°C in 0.5× TBE with a pulse time (Tp) of 1 minute to 2 minutes in an electric field of 6 V/cm for 21 hours. Saccharomyces cerevisiae chromosomes (BioRad) were used for size marker.
DNA transfer was the same as for the conventional gels but the times were as follows: depurination: 5 minutes, denaturation: 30 minutes, neutralization: 30 minutes, transfer: 4 hours. Conditions for prehybridization, hybridization and washing of the filters were the same as for the Southern blots. A PvuII-SacI c-kit fragment (−617 to +37, 5′ exon probe) was used as a probe.
In situ hybridization
In situ hybridization protocols were essentially as described (Manova et al., 1990). Briefly, dissected embryos were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7 overnight, washed, dehydrated and embedded in paraffin (Tissue Prep). Sections 5 μm thick were mounted on poly-L-lysine-coated slides and dried. 35S-labeled antisense and sense riboprobes were synthesized with SP6 and T7 polymerase, respectively, and hydrolysed in mild alkali to a length of 100-200 nucleotides. The c-kit probe was transcribed from a 3.6 kb insert including the entire coding region. The KL probe was synthesized from the pcDNA1-KL plasmid which contains the entire KL coding sequence after cleavage with BglII. The slides were hydrated, postfixed in paraformaldehyde followed by treatment with proteinase K and acetic anhydride and prehybridized. Slides were hybridized overnight at 65°C in a moist chamber using 0.3 M NaCl and 60% formamide in the mix and a probe concentration of 0.25 ng/μl. Slides were washed as described except that the RNase conditions were 50 μg/ml in 0.3 M NaCl, 0.01 M Tris pH 8, 1 mM EDTA, for 30 minutes at 37°C. Slides were dipped in NTB2 emulsion, exposed for 1-2 weeks, developed and stained with eosin and hematoxylin according to standard techniques.
Immunohistochemistry
Antibody to the c-kit receptor was raised in rabbits by immu-nization with recombinant vaccinia modified to express the entire c-kit protein (Nocka et al., 1990a). Antisera and control rabbit serum were partially purified by DEAE Affi-gel Blue (BioRad) chromatography to enrich for immunoglobulins. Column fractions of the c-kit anti-sera were used at dilutions of 1:50. Dissected embryos were washed twice in 0.1 M sodium phosphate buffer (pH 7), some were fixed in 2-4% buffered paraformaldehyde and embedded in O.C.T. (Tissue Tek). Cryostat sections were cut at 10 μm and stained with the Vectastain Elite ABC kit (Vector Laboratories). The manufacturer’s instructions for the procedure were followed except that the quenching of endogenous peroxidase was carried out in 0.1% H2O2 in phosphate-buffered saline (PBS) for 15 minutes, and incubation in 10% normal goat serum in PBS with 2% BSA for 30 minutes was included as a blocking agent for the secondary antibody, as well as an extra step to block nonspecific binding using the avidin/biotin blocking kit from Vector Laboratories. Some sections were counter-stained with Harris’ hematoxylin, before dehydration and mounting in Permount.
RESULTS
Determination of c-kit RNA expression in tissues of adult Wsh/Wsh mice: lack of c-kit expression in mast cells and lung
In order to determine if Wsh is a c-kit expression mutation, we first investigated c-kit mRNA levels in tissues of adult Wsh/Wsh mice by RNA blot analysis. Wsh homozygous mice lack mast cells in their skin and intestine. In W mutant mice, mast cell progenitors are present in the bone marrow and, from these, mature mast cells can be grown in vitro in the presence of interleukin-3 (IL-3) (Yung and Moore, 1982; Nocka et al., 1990c). Wsh/Wsh mast cells were derived from bone marrow of homozygous mutant animals. After 4 weeks in culture, a pure population of bone-marrowderived mast cells containing toluidine-blue-stained granules and indistinguishable from normal bone-marrowderived mast cells were obtained (Fig. 1B) and RNA was extracted for RNA blot analysis. Wsh/Wsh mast cells lack c-kit RNA transcripts in contrast to the high levels seen in +/+ mast cells (Fig. 1A). Thus the absence of mast cells in homozygous mutant mice probably results from the lack of c-kit expression in mast cells. Consistent with the lack of effect of the Wsh mutation on fertility, c-kit RNA expression in Wsh/Wsh testis was normal (Fig. 1A). Moreover, in brain, a tissue not known to be affected by W mutations, c-kit RNA expression was normal, while Wsh/Wsh lung lacked c-kit transcripts (Fig. 1A). These results indicate that the Wsh mutation affects c-kit RNA expression in a tissue-specific manner.
Characterization of c-kit RNA and protein expression during embryonic development of Wsh/Wsh mice
In order to investigate tissue-specific effects of the Wsh mutation on c-kit expression during embryogenesis with an emphasis on melanogenesis, we characterized c-kit RNA and protein expression at various stages of development in mutant mice by in situ hybridization and immunohisto-chemistry.
Embryonic day Absence of presumptive melanoblasts and lack of c-kit expression in digestive tract and lung
At melanoblasts have reached the ventral part of the body at all levels along the anterior-posterior axis and some have migrated into the epidermis. In normal embryos, melanoblasts of this stage express the c-kit proto-oncogene (Fig. 2A,E: cross section at the level of the lung, panel C and G: slightly more caudal cross section at level of testis). In Wsh/Wsh embryos, however, no c-kit-positive cells were detected in the skin (Fig. 2B,F and D,H: cross sections at the same level as sections in Fig. 2A,E and C,G, respectively). Thus, in Wsh/Wsh embryos, the melanoblasts are either absent or they are present but do not express c-kit (see later). At the fetal liver is the major site of fetal hematopoiesis. We found that, in Wsh/Wsh embryos, the fetal liver shows normal c-kit expression (data not shown). Since homozygous Wsh mice are not anemic, this result is in accordance with the phenotype. Also in agreement with the Wsh phenotype, we found that the testis of Wsh/Wsh embryos express c-kit (Fig. 2I,J). In normal embryos, mesenchymal cells around the bronchi of the developing lung express c-kit at (Fig. 2A,E). Consistent with the northern blot results of RNA from adult tissues, these cells do not express c-kit in Wsh/Wsh embryos (Fig. 2F). In addition, there is no c-kit expression in the mesenchymal layer of the digestive tract in Wsh/Wsh embryos (Fig. 2, compare panels I and J: Wsh/Wsh with panels K and L: +/+). c-kit expression in Wsh/Wsh embryos is normal in most other tissues, including the brain (tel-, met- and myelencephalon), trigeminal ganglion, endolymphatic sac and duct, thymus, pancreas, kidney, neural tube, dorsal root and sympathetic ganglia (data not shown). Most of the results on c-kit expression have been confirmed at the protein level using c-kit antibody (data not shown).
Embryonic day : inappropriate c-kit RNA and protein expression in the dermatome of somites, the mesenchyme around the otic vesicle and the floor plate of the neural tube
In situ hybridization of sections of E10 Wsh/Wsh embryos gave the most surprising result. We found that in homozygous Wsh embryos, c-kit RNA is expressed ectopically in the floorplate of the neural tube extending anteriorly to the floorplate of the myelencephalon (Fig. 3B,E,H,K), in the mes-enchyme around the otic vesicle (Fig. 3B,E) and in the dermatome of the somites (Fig. 3H,K). Expression in the dermatome was found in all somites along the anteriorposterior axis, except the most recently formed caudal somites. Sections of +/+ embryos hybridized with a c-kit probe show that c-kit is not expressed in these structures normally (Fig. 3A,D,G,J). In contrast, normal embryos of this stage express the kit-ligand in the floorplate, the mesenchyme around the otic vesicle and the dermatome (Fig. 3C,F,I,L).
Ectopic c-kit expression in Wsh/Wsh embryos did not affect kit-ligand expression. In Fig. 4A,B, similar kit-ligand expression was observed in the dermatome and the floorplate of Wsh/Wsh embryos as in +/+ embryos. Importantly, c-kit and kit-ligand are expressed in essentially every cell in these structures (Fig. 4C-F) indicating that kit-ligand and c-kit are coexpressed in floorplate and dermatomal cells in Wsh/Wsh embryos. The ectopic c-kit expression in Wsh/Wsh embryos may provide an explanation for the defect in melanogenesis in Wsh mutant mice (see discussion).
Next, we performed immunohistochemical staining in order to determine whether c-kit protein is also present in these structures. The results show that c-kit protein is present in the floorplate of Wsh/Wsh embryos (Fig. 5B), while absent in the wild-type embryo (Fig. 5A). No staining was seen in Wsh/Wsh control sections treated with preimmune serum (Fig. 5C). In Wsh/Wsh embryos c-kit protein was found in the mesenchyme around the otic vesicle (Fig. 6A) and in the dermatome (Fig. 6E-G). As expected, in the wild-type embryo, these structures are negative (Fig. 6B,D). Interestingly, in Wsh/Wsh embryos, the floorplate no longer expresses c-kit protein (Fig. 6A,E); thus this ectopic c-kit expression in Wsh/Wsh embryos is transient.
No c-kit-positive cells were seen in the skin of Wsh homozygous embryos (see above). Therefore, we next determined whether melanoblasts are present in Wsh/Wsh embryos at and , a time when c-kit-expressing melanoblasts are normally found at the level of the somites in the trunk region (Fig. 7A,B). In Wsh/Wsh embryos, c-kit-positive melanoblasts were detected in low numbers, but comparable to those in +/+ embryos (Fig. 7C,F, sections at the level of the hindlimb). In Wsh/Wsh embryos, c-kit-positive melanoblasts were still seen but their numbers were already reduced compared to those in +/+ embryos. In Fig. 7D,F, c-kit-positive cells are seen migrating over the dermatome and, in Fig. 7E, c-kit protein expression in the dermatome is obvious. Therefore, in Wsh/Wsh embryos, melanoblasts express c-kit gene products and the absence of melanoblasts at later stages may be the result of ectopic c-kit expression in the dermatome.
Melanocyte progenitors in +/+ and Wsh/+ embryos: graded distribution is reduced in Wsh/+ embryos along the body axis
To gain further insight into the timing and mechanism of disappearance of melanoblasts in Wsh mutant embryos, a detailed analysis of the distribution of melanoblasts in +/+ and Wsh/+ embryos was undertaken. Since wild-type melanoblasts express c-kit from onwards (Manova and Bachvarova, 1991), in Wsh/+ embryos unlike in homozygous mutant embryos, an absence of c-kit-positive cells indicates an absence of melanoblasts. wild-type and heterozygous embryos were serially sectioned in sagittal and cross section orientation and regularly spaced sections processed for immunohisto-chemistry using the anti-c-kit serum. At melanoblasts are located in the dermis and the epidermis, but some c-kit-positive mast cell progenitors may also be present in the dermis (Hayashi et al., 1985). Therefore, the distribution of melanoblasts was assessed by counting only c-kit-positive cells in the epidermis. Examples of stained melanoblasts in different regions of these embryos are shown in Fig. 8. Little staining was seen in control sections stained with preimmune serum (not shown). Note continued ectopic expression of c-kit in the dermis of Wsh/+ embryos (also in Wsh/Wsh embryos, not shown), which extends to the ventral midline at this stage.
A quantitative representation of the results was obtained by plotting epidermal melanoblasts in sagittal sections (Fig. 9A) or by plotting the density of cells in the dorsal half of cross sections (Fig. 9B). The results indicate that, in wild-type embryos, melanoblasts are distributed in a graded fashion with maximal numbers in the rostral and caudal regions and a minimum in the lumbar region. In Wsh/+ embryos, the density of the melanoblasts is reduced throughout resulting in a virtual absence in the trunk region.
At E11 melanoblasts are already distributed at varying densities along the body axis with a minimum in the lumbar region and their number is reduced in Wsh/+ embryos (Fig. 10). Mast cells essentially are absent from the skin at this age (Hayashi et al., 1985). In Wsh/+ embryos, as in Wsh/Wsh embryos, regions of head mesenchyme, particularly around the otic vesicle, and all dermatomes along the axis of the embryo expressed c-kit at similar levels. At this stage, dermis expressing c-kit has almost reached the ventral midline in the thoracic region. Since, melanoblasts are essentially normal in embryos (see above), these results establish that the melanocyte defect in Wsh/+ embryos, the”sash”, occurs between and .
Analysis of Wsh chromosomal DNA
In order to determine whether the Wsh mutation arose as a result of a DNA rearrangement or deletion, we investigated the c-kit gene in Wsh chromosomal DNA using conventional and pulsed-field gel electrophoresis (PFGE) DNA blots. Southern blot analysis of DNA from normal and Wsh/Wsh mice shown in Fig. 11A using either the c-kit cDNA or sequences including the first exon of the c-kit gene (not shown) as probes revealed no differences between the mutant and the normal DNA suggesting that the c-kit gene in the Wsh DNA is unaltered. However, analysis of blots obtained after resolution of megabase DNA by PFGE, digested with the restriction enzymes NotI, NruI and NarI and hybridization with a c-kit probe revealed Wsh DNA fragments of a reduced size with all three enzymes (Fig. 11B). Since the Wsh mutation arose in F1 C3H/HeH × 101/H mice, we also analyzed DNA from C3H/HeJ and 101.
Digestion of DNA from C3H/HeJ, 101 and C57BL/6J mice with NotI (Fig. 11C lanes 1,2 and 3) and NruI (Fig. 11C lanes 5,6 and 7) resulted in indistinguishable restriction fragments. Taken together these results suggest that the Wsh allele arose as a result of a deletion or a rearrangement in the vicinity of the c-kit gene.
DISCUSSION
The non-parallel display of mutant characteristics in mice containing the Wsh allele suggested that the phenotypes of this mutation could result from tissue-specific effects on c-kit transcription or post-transcriptional processing. We therefore characterized c-kit expression patterns in various tissues and cell types of homozygous mutant mice at various stages of development. Our experiments show that several tissues that normally express c-kit do not express c-kit in Wsh/Wsh mice; they include bone-marrow-derived mast cells of adult Wsh/Wsh mice, adult and fetal lung and the digestive tract at embryonic day . c-kit expression is not affected in most other adult and fetal tissues in Wsh/Wsh mice. Wsh/Wsh embryos have normal numbers of c-kit-expressing melanoblasts but at the homozygous and heterozygous mutant embryos lack c-kit-positive cells in the skin or have proportionally reduced numbers, respectively. A surprising but major finding was the expression of c-kit in mutant day embryos in some tissues and cell types that do not normally express c-kit but do express the kit-ligand, i.e. the dermatome of the somites, the mesenchyme around the otic vesicle and the floorplate of the neural tube. The inappropriate c-kit expression does not affect kit-ligand expression in homozygous mutant embryos. Furthermore, molecular analysis of Wsh chromosomal DNA revealed a deletion or rearrangement in the vicinity of the c-kit gene. These results provide possible explanations for the phenotypes of homozygous and heterozygous Wsh mice, and they have implications for the transcriptional control of the c-kit protooncogene.
The Wsh mutation differs from other W mutations in that only mast cells and melanocytes are affected, whereas germ cells and erythrocytes areaffected only in minor ways (Lyon and Glenister, 1982; Stevens and Loutit, 1982). These characteristics can be explained by the normal expression of c-kit in fetal liver and testis of Wsh mice and the lack of c-kit expression in mast cells. Recently, it has also been shown that CFU-S from bone marrow of Wsh/Wsh animals are severely reduced in number (Tono et al., 1992), possibly due to mis-regulated c-kit expression in the hematopoietic stem cell hierarchy.
The inappropriate c-kit expression in the dermatome of and E11 mutant embryos is an important feature of the Wsh mutation, which may provide an explanation for the pigmentation defect in mutant mice. We propose that inappropriate expression of c-kit receptor in the dermatome of Wsh/Wsh mice may sequester (bind and neutralize) kitligand. As a result the amount available for c-kit-expressing melanoblasts migrating over the dermatome is reduced, thus affecting their survival and/or proliferation. It is also conceivable that the co-expression of c-kit and its ligand results in the activation of dermatomal cells by an autocrine or paracrine mechanism and this in turn may lead to changes in the extracellular matrix. In either model, the Wsh/Wsh melanoblasts would die between day and day . In support of our model is the fact that, in the trunk region, at , melanoblasts migrate over the somites and continue to migrate as dermatome cells spread under the ectoderm from , a period that coincides with the time of inappropriate c-kit expression in the dermatome. Also in agreement with this paradigm, we found normal numbers of c-kit-expressing melanoblasts in Wsh/Wsh embryos apparently on a dorsolateral pathway, whereas in day embryos no c-kit-positive cells were detected in the skin. Although melanoblasts appear to express c-kit normally in day Wsh/Wsh embryos, our results do not preclude the possibility that c-kit expression in more mature melanoblasts/melanocytes is affected by the Wsh mutation. But the presence of small patches of pigmentation around the ears and eyes in homozygous Wsh mice suggests that melanocytes are normal in these mice. In agreement with this notion, melanocytes have been grown from neural crest cells of Wsh/Wsh embryos (Huszar et al., 1991).
c-kit is expressed in all somites and the mesenchyme near the otic vesicle of Wsh/Wsh and Wsh/+ embryos apparently in the same pattern as KL. Hence, ectopic c-kit expression in the dermatome may not be a sufficient explanation for a differential effect of the mutation on melanocyte development in the anterior, mid and posterior regions in Wsh/+ mice. However, in the epidermis of +/+ embryos, the melanoblasts are present in a graded distribution with high densities in the face, ear and tail, moderate densities in the shoulder and rump, and a minimal density in the lumbar region. As expected from our model, melanoblasts in Wsh/+ embryos are reduced at all levels, with very few present in the lumbar region. Therefore, the formation of the SASH in heterozygous mutant animals may stem from a reduction of cells in the anterior, mid level and posterior regions during embryogenesis and reflects the uneven distribution of melanoblasts in normal embryos. Several patterns of mice with coat color mutations can be explained on the basis of general effects on melanocyte precursors and the graded melanoblast distribution that we have observed at (Charles, 1938; Silvers, 1979). Taken together these results suggest that c-kit is required in melanogenesis between and . This is a period of substantial proliferation of melanoblasts from a sparse and quite even distribution at to a more numerous population distributed differentially over the surface at .
Aggregation chimeras produced from W/W↔ +/+ embryos have a ventral spot and blaze on the forehead similar to heterozygous W/+ animals (Mintz, 1974). Interestingly, Wsh/Ws↔ +/+ aggregation chimeras display a pigmentation pattern similar to or more pronounced than that of heterozygous Wsh/+ animals (Stephenson et al., 1985), in agreement with the non-cell autonomous dominant mode of action of the Wsh mutation that we have proposed. By contrast, transplantation of normal neural crest cells into the amniotic cavity of E8.75-9.0 Wsh/Wsh embryos results in the formation of pigment patches in apparent contradiction to our findings (Huszar et al., 1991). However, several considerations suggest that events after transplantation may not necessarily faithfully reflect normal developmental processes: first, the neural crest cells used for transplantation were cultured prior to use and may differ from the normal melanoblasts; second, the neural crest cells can be transplanted only into E8.75-9.0 embryos, and the timing of migration of the transplanted cells therefore may differ from the normal developmental program; third, the number of transplanted cells may exceed those found in normal embryogenesis and this might affect the outcome of the experiment; and fourth, the path of migration of the transplanted cells from the site of injection in the amniotic cavity is not known and may differ from the normal situation.
The Wsh mutation blocks c-kit expression in mast cells and mesenchymal cells in the lung and the digestive tract at , while expression in other tissues is normal. This suggests that c-kit expression in different tissues is regulated by different cis-acting elements and that positive regulatory elements specific for mast cells and mesenchymal cells in the lung and the digestive tract are affected by the Wsh mutation. Furthermore, our observation of c-kit expression in tissues that normally express the c-kit ligand implies that the regulatory region of the c-kit gene includes negative control elements which are also affected by this mutation. Our Southern blot analysis indicates no alteration of c-kit exon and intron sequences in the Wsh allele in agreement with the recent demonstration that the c-kitWSH coding sequence is unaltered (Tono et al., 1992). However, the results from the pulsed-field gel electrophoresis indicate that the Wsh mutation involves a large deletion or rearrangement and this is in agreement with our observations that several control elements are affected by this mutation. We speculate that the deletion is upstream of the c-kit locus based on the following observation. The Patch locus encodes the PDGFRA receptor and is closely linked to the c-kit/W locus (Smith et al., 1991; Stephenson et al., 1991; Duttlinger, unpublished). Interestingly, mice heterozygous for the Patch mutation display the same pigmentation pattern as do Wsh/+ mice, while homozygous Ph/Ph mice die during embryonic development. The original Patch mutation arose as a result of a deletion that includes the entire PDGFRA receptor. The 3′-deletion endpoint, which is in juxtaposition with the 5′ end of the c-kit gene, is not known precisely, but it does not include c-kit coding sequences. Because of the similar het-erozygous pigmentation phenotypes of the Wsh and the Patch mutations, it is reasonable to speculate that the pigmentation defect in Ph/+ mice is the result of inappropriate c-kit expression similar to that in Wsh/+ mice and that the two mutations, therefore, affect some of the same elements in the 5′ control region of the c-kit proto-oncogene. In agreement with these predictions, preliminary results have placed the Wsh alteration upstream of the c-kit (R. Dut-tlinger, unpublished). We are currently investigating the precise nature of the Wsh mutation, its relationship to PDGFRA and its effects on PDGFRA.
Our observation of co-expression of c-kit and KL in Wsh/Wsh embryos in head mesenchyme, dermatomal cells and cells of the floorplate raises the interesting possibility that mechanisms regulating their expression may be related. Thus, negative control elements might be necessary to insure that c-kit is not expressed in tissues normally expressing KL. Such elements may be affected by the Wsh mutation uncovering positive regulatory elements common to c-kit and KL.
ACKNOWLEDGEMENTS
We would like to thank Sandra Sanchez and Michael Angeles for help with histochemistry and in situ hybridizations, Dr Hai Nguyen for histopathology and Drs Karl Nocka, Tang Yang Chu and Ellen Pritzer for advice and stimulating discussions. This work was supported by grants from the National Institutes of Health to P. B. (R37 CA 32926) and R. F. B. (HD 06910).