Mutations in either the dominant white-spotting (W) or Steel (Sl) loci of the mouse lead to coat color, primordial germ cell and hematopoietic defects. Consistent with the cell autonomous and microenvironmental nature of W and Sl mutations, respectively, it has recently been shown that W encodes the c-kit receptor tyrosine kinase while Sl encodes a ligand for this receptor. Previous in situ hybridization analysis has shown that both c-kit and steel are expressed in the embryo in anatomical sites known to be affected by W and Sl mutations and in various tissues in which no corresponding phenotype has been described. To investigate the possible involvement of the Kit transduction pathway in developmental processes, we compared the patterns of expression of c-kit and steel in wild-type embryos and in embryos homozygous for severe (lethal) and mild (viable) alleles at the W and Sl loci. In addition, we analyzed the patterns of expression of both genes in adult wild-type and mutant gonads and brain. Both c-kit and steel are contiguously expressed in a wide variety of anatomical locations in both the developing embryo and in the adult. In adult gonads, steel is expressed in the follicular cells of the ovary and in Sertoli cells of the testis, the layers that immediately surround the c-kit expressing germ cells. In adult brain, the complementary patterns are particularly striking in the olfactory bulb, cerebral cortex, hippocampus region and cerebellum, steel expression in brain is probably restricted to neurons in certain areas, while c-kit is expressed in neurons and in some glial cells. Severe mutations in the W or Sl loci result in dramatic reduction or absence of c-kit positive cells in lineages known to be affected by these mutations. In contrast, these mutations do not affect the number or histological organization of c-kit positive cells in the embryonic peripheral or central nervous systems, nor is the number or organization of c-kit positive cells detectably altered in Wv/Wv or Sf’/S adult brain. Taken together, these results suggest that the Kit signaling pathway is not obligatory for the viability and/or migration of most c-kit expressing cells either because of functional redundancy with another signaling pathway or because the Kit pathway is involved in post-developmental processes of mature cells.

Members of the receptor tyrosine kinase (RTK) family appear to play central roles in the regulation of normal and abnormal cell proliferation and differentiation (Hunter and Cooper, 1985; Yarden and Ullrich, 1988). These receptors, and their ligands, are an integral part of the mechanisms by which cells receive and transmit signals to ensure orderly patterning and tissue formation during embryogenesis (Hunter, 1987; Pawson and Bernstein, 1990; Mercóla and Stiles, 1988). Unequivocal demonstration of the importance of RTKs in mammalian development emerged three years ago with the identification of the c-kit RTK as the product of the murine dominant white-spotting (W) locus (Chabot et al. 1988; Geissler et al. 1988). Recently a ligand for c-kit was cloned and was designated mast cell growth factor or MGF (Williams et al. 1990; Anderson et al. 1990), stem cell factor or SCF, (Zsebo et al. 1990b; Martin et al. 1990), and c-kit ligand (KL) (Nocka et al. 1990a; Flanagan and Leder, 1990) and mapped to the Steel (Sl) locus (Copeland et al. 1990; Zsebo et al. 1990a ; Huang et al. 1990) and therefore called Steel factor here. Earlier chimera analysis indicated that W mutants have an intrinsic defect in the affected cells (Russell et al. 1956; Kitamura et al. 1978; Mayer and Green, 1968), while in Steel mutant mice the defect is in the micro-environment in which these cells develop (McCulloch et al. 1965; Kitamura and Go, 1979; Mayer and Green, 1968), consistent with the receptor-ligand relationship of their products.

W and Sl mutations are known to have very similar pleiotropic effects on diverse developmental pathways. Mice homozygous for severe W or Sl mutations are black-eyed white due to the failure of neural crest-derived melanoblasts to migrate and incorporate into the developing hair follicles (Mayer, 1970). These mutants are also deficient in germ cells as a result of impairment in proliferation and/or migration of the primordial cells to the gonads (Mintz and Russell, 1957; McCoshen and McCallion, 1975). Finally, this receptor-ligand system also affects hematopoiesis and the growth of mast cells (for review, see Russell, 1979; Bernstein et al. 1991). In embryos homozygous for null alleles at the W and Sl loci (e.g. W, W37, Sl), the resulting severe anemia leads to lethality late in gestation.

The cell-autonomous, intrinsic nature of W mutants predicts that c-kit would be expressed in germ cells, melanoblasts and hematopoietic cells while, in contrast, steel transcripts would be found in the cells surrounding Kit-positive cells. Surprisingly, however, northern (Nocka et al. 1989) and RNA in situ analysis (Orr-Urtreger et al. 1990) has shown that, in addition to the expected tissues, c-kit is expressed in other organs, most notably the embryonic lung, kidney, gut and brain. Similar observations have recently been reported for steel expression. Compatible with the phenotype of Sl mutant mice, steel is expressed in hematopoietic tissues, along the migratory pathways of melanoblasts and germ cells and in their final sites of destination (Matsui et al. 1990). However, steel expression has also been detected in the mesoderm of the kidney, lung, heart and stomach, in the spinal cord and in brain (Matsui et al. 1990).

The expression of both the receptor and its ligand in a variety of different anatomical sites raises the possibility that the c-kit pathway may play a role at these locations despite the absence of any obvious phenotype associated with these sites of expression in W or Sl mutants. The availability of c-kit and steel probes allows a more detailed examination of this question, using the genes as molecular markers for following these lineages in W or Sl mutant animals. Therefore, we have analyzed the patterns of c-kit and steel expression by in situ hybridization RNA analysis to adjacent sections of wild-type embryos and adult tissues, as well as in embryos homozygous for null mutations at either the W or Sl loci and adults carrying mild W or Sl mutations. We show here that c-kit and steel are expressed contiguously in a wide variety of distinct anatomical sites in wild-type embryos. The numbers of cells expressing c-kit RNA was profoundly reduced in phenotypically affected lineages in the mutant embryos. In contrast, the morphology and number of c-kú-expressing cells was unaffected in embryonic and adult tissues that are not known to be affected by W or Sl mutations. The absence of c-kit expressing cells in the affected lineages of mutant embryos confirms a role for the c-kit pathway in the proliferation, migration and/or survival of cells in the lineages affected by W or Sl mutations. However, the continued presence of c-kit expressing cells in lineages not known to be affected by W or Sl mutations argues either for functional redundancy in these cell types or that any role for Kit in these lineages must involve not the development and proliferation of these cells but their later differentiative properties.

Mice, embryos and tissues

C57BL/6J, C57BL/6 W37/+, C57BL/6 W/+, WB/Re Sl/+ and C57BL/6 . Sld/+ mice were obtained from the Jackson Laboratories (Bar Harbor, ME) and maintained at the Mt. Sinai animal colony. For embryonic staging, the noon after vaginal plug observation was considered as 0.5 day post-coitum (dpc). Embryos homozygous for the Sl allele (at 13.5 dpc) and for W, W37 and Sld alleles (at 16 dpc) were recognized by their pale appearance relative to heterozygous and wild-type littermates. Mice were killed by cervical dislocation and embryos or adult tissues were transferred immediately to ice-cold 4% paraformaldehyde in PBS and fixed overnight (O/N) at 4°C. Samples were incubated in 0.5 M sucrose in PBS at 4°C O/N before embedding in Tissue-Tek O.C.T. (Miles).

In situ hybridization

In situ hybridization was performed essentially as described by Hogan et al. (1986). Briefly, tissues were cryostat-sectioned at 10 am, mounted on glass slides treated either with poly-L-lysine or 2% 3-aminopropyltriethoxysilane (Sigma), and refixed in 4% paraformaldehyde. Prehybridization treatments were performed as described (Hogan et al. 1986). 35S-UTP-labeled single-stranded probes were prepared using the Stratagene RNA transcription kit. Adjacent sections were hybridized with c-kit antisense, steel antisense and steel sense probes. For c-kit antisense probe, a Socl-Wzndlll fragment derived from a previously described c-kit cDNA clone (Reith et al. 1990) was subcloned into the Bluescript KS vector (Stratagene). This fragment, spanning nucleotides 2607 to 3710 of murine c-kit gene (Qiu et al. 1988) was linearized with BglII at nucleotide 2802 and the RNA was produced using T3 RNA polymerase. For steel antisense and sense probes, the MGF-10 clone (Anderson et al. 1990) was linearized with BamHl or Xhol, respectively, and the RNAs were produced using T7 or T3 RNA polymerases, respectively. The probes were degraded to 100–150 bp average length by alkaline hydrolysis (Cox et al. 1984). Hybridization was carried out at 50°C at a probe concentration of 108 cts min-1ml-1. Posthybridization washings included treatment with 50μgml-1 RNAase A (Sigma) at 37 °C for 30 min and either an incubation at 37°C O/N in 50% formamide/0.3 M NaCl or incubation at 50°C for 2 × 20 min in 0.l × SSC. Both conditions resulted in qualitatively similar results but the former resulted in a slightly higher background and faint labeling with steel antisense and sense (control) probes over the pyramidal layer of the hippocampus. Following dehydration, the slides were dipped into NTB-2 emulsion (Kodak), exposed at 4°C for 4-6 days, developed, and stained with toluidine blue. All experiments were done on multiple sections from different animals, and embryos from at least two different litters for the mutants.

To investigate the relationship between the patterns of c-kit and steel expression, adjacent sections of embryos and adult tissues were hybridized in situ to c-kit and steel antisense probes, and to a steel sense probe as a control. The single-stranded 35S-labeled c-kit probe was synthesized from a 908 bp fragment spanning 152 bp of coding sequences and 756 bp of 3’ untranslated region. The coding region is 3’ to the conserved tyrosine kinase domains of the RTK family (Hanks et al. 1988). To detect steel expression, an antisense riboprobe from the 2060 bp steel cDNA (MGF-10 in Anderson et al. 1990) was used. This clone contains the entire coding sequence of steel as well as 3’ and 5’ untranslated regions. The same fragment was used for the steel sense probe. The sense probe did not give a specific signal above background in any of the tissues examined. The fragments that were used to make the c-kit and steel riboprobes were also labeled with [32P]dCTP by random priming and hybridized in northern blot analysis to RNA derived from embryonic and adult tissues; only the expected RNA species were observed (data not shown).

In addition to wild-type embryos, embryos homozygous for two severe (W37,Sl) and two mild (Wv,Sld) mutations were examined. W37/W37 and Sl/Sl embryos die late in gestation, probably from severe anemia (Geissler et al. 1981; Russel, 1979). The W37 mutation is a point mutation within the cytoplasmic kinase domain of c-kit (Reith et al. 1990; Nocka et al. 19906) that completely abolishes Kit kinase activity and the activation of downstream signaling steps (Reith et al. 1991). Wv/Wv homozygotes have completely white coats, and while viable to maturity, are severely anemic and sterile (Little and Cloudman, 1937). The Wv allele, which is identical to the W55 mutation, is a point mutation in the c-kit kinase domain resulting in diminished but detectable kinase activity (Reith et al. 1990; Nocka et al. 19906). The Sl allele is a complete deletion of the region coding for steel (Copeland et al. 1990; Zsebo et al. 1990a). S/SF1 homozygotes are black-eyed white, viable to maturity, severely anemic and sterile (Bernstein, 1960). The Sr allele has recently been shown to encode a protein which lacks the intracellular and transmembrane domains (Flanagan et al. 1991; Brannan et al. 1991). Hence, cells of genotype Sld/Sld appear to be completely devoid of the membrane form of Steel factor.

Expression of c-kit and steel in phenotypically affected lineages

1. Melanogenesis

The c-kit signal transduction pathway is essential for the viability and/or migration of neural crest derived melanoblasts (for review see Russell, 1979). Migration of these melanoblasts begins at about 9 dpc from the dorsal surface of the neural tube and at about 12–13 dpc they are incorporated into the epidermal hair follicles. The c-kit gene is expressed in skin from about 12.5 dpc onwards (Orr-Urtreger et al. 1990). Steel transcripts are found in a continuous wide layer adjacent to the sites of melanocyte migration and destination (Matsui et al. 1990). As expected, in all mutant embryos examined, c-kit expression was not observed in any of the tissues normally colonized by neural crest-derived melanoblasts (Figs 1C, 5A,C and not shown). In contrast, cells expressing steel were present in their normal patterns in the skin of embryos homozygous for the W37, Wv and Sld mutations (Figs 1D, 5B,D and not shown). The levels of steel expression in W37/W37 and Wv/Wv embryos were at least as high as in the wild-type embryos, and lower but detectable in Sld/Sld embryos.

Fig. 1.

In situ localization of c-kit and steel RNAs in normal and W37/W37 late gestation embryos. (A,B) Dark-field micrograph of 17dpc sagittal section through normal and (C,D) 16dpc W37/W37 embryo hybridized with c-kit (A,C) or steel (B,D) probes. Note the lack of labeling with c-kit over the dermis/epidermis in W37/W/37 embryos (C), and the strong labeling with steel in this area (D). A and B are not exactly in the same level (B more lateral), (cbl) cerebellum; (cer) cerebrum; (c.p.) choroid plexus; (d.r.g.) dorsal root ganglia; (g.v.) trigeminal ganglion; (hyp) hypothalamus; (liv) liver; (lun) lung; (o.e.) olfactory epithelium; (str) striatum; (tes) testis.

Fig. 1.

In situ localization of c-kit and steel RNAs in normal and W37/W37 late gestation embryos. (A,B) Dark-field micrograph of 17dpc sagittal section through normal and (C,D) 16dpc W37/W37 embryo hybridized with c-kit (A,C) or steel (B,D) probes. Note the lack of labeling with c-kit over the dermis/epidermis in W37/W/37 embryos (C), and the strong labeling with steel in this area (D). A and B are not exactly in the same level (B more lateral), (cbl) cerebellum; (cer) cerebrum; (c.p.) choroid plexus; (d.r.g.) dorsal root ganglia; (g.v.) trigeminal ganglion; (hyp) hypothalamus; (liv) liver; (lun) lung; (o.e.) olfactory epithelium; (str) striatum; (tes) testis.

2. Hematopoiesis

The hematopoietic system is the second major pathway affected by mutations at the W and Sl loci. During development, hematopoiesis occurs first in the yolk sac and then, between days 12-17, is found in the fetal liver. We compared the fetal livers of 13.5d +/+ and Sl/Sl embryos to determine whether cells expressing c-kit could be found in this organ in the absence of any steel factor. As shown in Fig. 2B, there was a marked reduction in the number of c-kit+ cells in the fetal livers of Sl/Sl embryos relative to that observed in their +/+ or Sl/+ littermates. The fetal livers of embryos homozygous for the W37/W37 mutation also showed a reduction in the number of c-kit+ cells (Fig. 1C). This reduction was not as marked as that seen for Sl/Sl fetal livers, an observation consistent with the relative severity of the two alleles. From the intensity of the in situ hybridization signals, those cells that expressed c-kit had approximately equivalent levels of c-kit transcripts to those observed in fetal liver cells of normal embryos. These in situ hybridization results are in agreement with previous observations demonstrating a marked, but not complete, reduction in the numbers of hematopoietic progenitor cells in the fetal livers of W or Sl mutant embryos (McCulloch et al. 1964; Iscove, 1978; Bennett, 1956; Russell, 1979; Nocka et al. 1989).

Fig. 2.

In situ localization of c-kit and steel RNAs in 13.5 dpc normal and Sl/Sl embryos. (A) Bright-field micrograph of sagittal section through 13.5 dpc +/? (+/+ or SI/+) (left) and Sl/Sl (right) embryo. The two embryos are from the same litter and were processed simultaneously. (B) Dark-field micrograph of c-kit and (C) steel expression in these embryos. Note the lack of any signal with steel probe over Sl/Sl embryo and the reduced hybridization with c-kit probe over the liver (liv) of Sl/Sl embryo compared to +/? embryo, c-kit labeled the ventricular zones and the labeling was higher over the fourth ventricle (IV), choroid plexuses (marked by arrow in B), and dorsal root ganglia (d.r.g.) (not shown in the +/? section), steel labeled heavily the ventricular zone of rostral telencephalon (tel), the anterior nuclei of the thalamus (thal) and the bed nucleus of stria terminalis and faintly the corpus striatum, (lun) lung; (o.e.) olfactory epithelium.

Fig. 2.

In situ localization of c-kit and steel RNAs in 13.5 dpc normal and Sl/Sl embryos. (A) Bright-field micrograph of sagittal section through 13.5 dpc +/? (+/+ or SI/+) (left) and Sl/Sl (right) embryo. The two embryos are from the same litter and were processed simultaneously. (B) Dark-field micrograph of c-kit and (C) steel expression in these embryos. Note the lack of any signal with steel probe over Sl/Sl embryo and the reduced hybridization with c-kit probe over the liver (liv) of Sl/Sl embryo compared to +/? embryo, c-kit labeled the ventricular zones and the labeling was higher over the fourth ventricle (IV), choroid plexuses (marked by arrow in B), and dorsal root ganglia (d.r.g.) (not shown in the +/? section), steel labeled heavily the ventricular zone of rostral telencephalon (tel), the anterior nuclei of the thalamus (thal) and the bed nucleus of stria terminalis and faintly the corpus striatum, (lun) lung; (o.e.) olfactory epithelium.

3. Gametogenesis

Gametogenesis is the third major lineage affected by mutations at the W or Sl loci. Primordial germ cells can first be detected at 7 dpc at the base of the allantois (Ginsburg et al. 1990), from where they commence migration toward the genital ridges. Colonization of the genital ridges takes place between 10 and 13.5 dpc and proliferation of germ cells follows this migration process. In W and Sl mice, primordial germ cells either fail to migrate (Mintz and Russell, 1957) or to proliferate (McCoshen and McCallion, 1975) and thus fail to colonize the gonad. By in situ RNA analysis, Orr-Urtreger et al. (1990) showed that c-kit is expressed in germ cells at 12.5 dpc. Recently, Matsui et al. (1990) reported that from 9 dpc onward steel is expressed along the migratory pathways of the primordial germ cells and in the genital ridges. We were unable to detect c-kit expressing cells in the gonads of W and Sl mutant embryos (data not shown), whereas steel expression in the gonads of embryos homozygous for the Wv, W37 or Sld mutations was as high as in +/+ embryos (e.g. lódpc W37/W37 testis, Fig. 1D).

In the adult female, c-kit transcripts are present at all stages of oocyte development (Orr-Urtreger et al. 1990), in the interstitial tissue and in some theca cells (Manova et al. 1990). steel RNA was found at high levels in the granulosa (follicular) cells that immediately surround oocytes at all stages of maturation (Fig. 3B). The strikingly reciprocal patterns of c-kit and steel expression in the adult ovary, together with the differentiation block associated with some Sl alleles, provide evidence that successful oocyte maturation requires stimulation of the c-kit signaling pathway via follicular cell-oocyte interaction.

Fig. 3.

In situ localization of c-kit and steel in adult gonads. (A) Dark-field micrograph of c-kit and (B) steel expression in ovary, (gran) granulosa (follicular) layer; (oo) oocyte. (C) Bright-field micrograph of seminiferous tubules of normal mouse and dark-field micrograph of c-kit (D) and steel expression (E). (Ley) Leydig cells; (Ser) Sertoli cells; (sp) spermatocytes. (FJ Bright-field micrograph of testis from 2 month old +/+ mouse (left) and his Sr/SF* brother, and dark-field micrograph of c-kit (G) and steel (H) expression. Note the lack of spermatocytes in SP/SP testis (F) and the higher levels of steel expression in SP/St1 testis (H).

Fig. 3.

In situ localization of c-kit and steel in adult gonads. (A) Dark-field micrograph of c-kit and (B) steel expression in ovary, (gran) granulosa (follicular) layer; (oo) oocyte. (C) Bright-field micrograph of seminiferous tubules of normal mouse and dark-field micrograph of c-kit (D) and steel expression (E). (Ley) Leydig cells; (Ser) Sertoli cells; (sp) spermatocytes. (FJ Bright-field micrograph of testis from 2 month old +/+ mouse (left) and his Sr/SF* brother, and dark-field micrograph of c-kit (G) and steel (H) expression. Note the lack of spermatocytes in SP/SP testis (F) and the higher levels of steel expression in SP/St1 testis (H).

c-kit is expressed in the adult testis in the interstitial Leydig cells (Orr-Urtreger et al. 1990). Manova et al. (1990) demonstrated that c-kit is also expressed in germ cells between the stages of type A2 spermatogonia and preleptotene spermatocytes (Manova et al. 1990). In agreement with predictions based on the behavior of cells of mutant Sl genotype in aggregation chimeras (Nakayama et al. 1988) and in transplantation assays (Kuroda et al. 1989), we detected steel RNA in Sertoli cells (Fig. 3E). We also observed low levels of steel expression in what appeared to be germ cells; however, Sertoli cells have cytoplasmic processes which surround developing germ cells, which could account for the weak signal observed (Fig. 3E). The analogy between the expression in the ovary and the testis is evident. In both cases, the steroid-responsive epithelial cells (granulosa and Sertoli cells, respectively) express steel while germ cells and mesenchymal (stromal) steroid-producing cells (theca and interstitial cells in the ovary and Leydig cells in the testis) express c-kit.

Markedly elevated levels of steel transcripts were observed in Sertoli cells of both Sld/Sld and Wv/Wv testes, while the levels of c-kit expression in the Leydig cells was not significantly different (Fig. 3G,H and data not shown). Wv/Wv testes have few spermatogonia and are composed mainly of Sertoli cells (Coulombre and Russell, 1954). These results suggest a possible role of germ cells in down-regulating the steel gene in Sertoli cells.

Expression of c-kit and steel in embryonic cell lineages not known to be affected by W and S1 mutations

Reciprocal expression of c-kit and steel was evident as early as 7.5 (dpc) in the presomitic embryo. At this stage, steel is highly expressed in the embryonic endodermal cells while significant labeling with c-kit probe was observed over the adjacent embryonic ectodermal cells (Fig. 4A,B,E,F). Complementary patterns of expression were also observed in the maternal decidua and the extraembryonic tissue. As reported previously, high levels of c-kit mRNA were found in the mesometrial decidual cells (Orr-Utreger et al. 1990 and Fig. 4D,H). At 7.5 dpc steel was also expressed in the decidua, but primarily in endothelial cells. At 9.0 dpc, sites of steel expression in the decidua were more clearly defined as the newly forming blood vessels (data not shown).

Fig. 4.

In situ localization of c-kit and steel RNAs in 7.5 dpc embryo and in extraembryonic tissue. (A) Bright-field and (E) dark-field micrograph of c-kit expression in 7.5 dpc embryo. (B) Bright-field and (F) dark-field micrograph of steel expression in 7.5 dpc embryo, (am) amnion; (ec) embryonic ectoderm; (m) embryonic mesoderm. The arrow head marks the embryonic endoderm. (C) Bright-field and (G) dark-field micrograph of c-kit expression in 9.0dpc placenta. (D) Brightfield and (H) dark-field micrograph of steel expression in 9.0dpc placenta, (ch) chorionic side and (ec) ectopiacental side of the diploid placenta; (de) maternal decidua; (gt) giant trophoblastic cells; (pe) parietal endoderm; (ys) visceral yolk sac. (I,K) Dark-field micrograph of c-kit and (J,L) steel expression in the 13.5 dpc chorioallantoic placenta of +/? (I,J) and Sl/Sl (K,L) embryos from the same litter. In the chorioallantoicplacenta (especially in labyrinth) part of the labeling is due to erythrocytes and is not a hybridization signal, (de) maternal decidua; (la) labyrinthine trophoblast; (sp) spongiotrophoblast; (pe) parietal endoderm.

Fig. 4.

In situ localization of c-kit and steel RNAs in 7.5 dpc embryo and in extraembryonic tissue. (A) Bright-field and (E) dark-field micrograph of c-kit expression in 7.5 dpc embryo. (B) Bright-field and (F) dark-field micrograph of steel expression in 7.5 dpc embryo, (am) amnion; (ec) embryonic ectoderm; (m) embryonic mesoderm. The arrow head marks the embryonic endoderm. (C) Bright-field and (G) dark-field micrograph of c-kit expression in 9.0dpc placenta. (D) Brightfield and (H) dark-field micrograph of steel expression in 9.0dpc placenta, (ch) chorionic side and (ec) ectopiacental side of the diploid placenta; (de) maternal decidua; (gt) giant trophoblastic cells; (pe) parietal endoderm; (ys) visceral yolk sac. (I,K) Dark-field micrograph of c-kit and (J,L) steel expression in the 13.5 dpc chorioallantoic placenta of +/? (I,J) and Sl/Sl (K,L) embryos from the same litter. In the chorioallantoicplacenta (especially in labyrinth) part of the labeling is due to erythrocytes and is not a hybridization signal, (de) maternal decidua; (la) labyrinthine trophoblast; (sp) spongiotrophoblast; (pe) parietal endoderm.

c-kit and steel transcripts were also highly expressed in extraembryonic tissues. At 7.5 dpc, steel RNA was present in the ectoplacental cone (data not shown) and at 9.0 dpc, both c-kit and steel were expressed in the diploid trophoblast of the developing placenta (chorion and ectoplacental cone Fig. 4C,D,G,H). At this stage, the limited sensitivity of the in situ method did not allow a clear distinction between the population of cells expressing the two genes. However, by 14.5 dpc, placental expression of c-kit and steel was clearly complementary, with c-kit expression high in maternal decidual cells, and low in the spongiotrophoblastic layer, and steel expression confined to the labyrinthine trophoblast (Fig. 4I,J).

The high expression levels of c-kit and steel in the placenta prompted us to look for changes in placenta morphology in Sl/Sl embryos. As expected, no signal was observed with the steel probe in Sl/Sl embryos (Fig. 4L). However, the pattern of c-kit expression in the decidua and placenta and the morphology of the placenta were indistinguishable from those of normal mice (Fig. 4K).

This same absence of altered tissue morphology or patterns of expression of c-kit or steel was observed in other tissues not known to be affected in W or Sl mutant mice when mutant embryos were compared with their matched +/+ controls. For example, Fig. 1C,D shows expression of both c-kit and steel RNAs in the lung, gut and epithelium of the nasal cavities of a 16 dpc W37/W37 embryo. Close inspection of the fine morphology and of c-kit and steel expression patterns revealed no detectable alterations from wild-type controls (data not shown), c-kit (in agreement with observations by Orr-Urtreger et al. 1990), and steel expression were detected in neural crest-derived cranial and dorsal root ganglia from 12.5 and 15.5 dpc respectively, and the expression of c-kit was unaffected in 13.5 dpc Sl/Sl (Fig. 2B), Sld/Sld and W37-W37 (Fig. 5) mutant embryos. Similar results and conclusions were reached for the expression in the trigeminal ganglion (which is derived mainly from neural crest) of W37/W37 and Sl/Sl embryos (Fig. 1 and not shown).

Fig. 5.

In situ localization of c-kit and steel RNAs in dorsal root ganglia of mutant mice. (A,C) Dark-field micrograph of c-kit and (B,D) steel in dorsal root ganglia of (A,B) 16dpc W37/W37 and (C,D) 16.5dpc Sld/Sld embryos. Notice the lack of signal with c-kit probe in the skin (marked by arrow).

Fig. 5.

In situ localization of c-kit and steel RNAs in dorsal root ganglia of mutant mice. (A,C) Dark-field micrograph of c-kit and (B,D) steel in dorsal root ganglia of (A,B) 16dpc W37/W37 and (C,D) 16.5dpc Sld/Sld embryos. Notice the lack of signal with c-kit probe in the skin (marked by arrow).

Both c-kit and steel are expressed in the embryonic CNS (Orr-Urtreger et al. 1990; Matsui et al. 1990). The anatomical and functional complexity of the brain, together with the fact that there is no phenotype connected with this organ in W or Sl mutants, made it of interest to determine the relationship between the compartments expressing the W and Sl genes and to search for any anatomical changes in the brains of mutant mice. Therefore, we performed a detailed comparative analysis of c-kit/steel expression patterns in normal and mutant mid- and late-gestation embryonic brains. Two major conclusions can be drawn from this analysis. First, c-kit and steel are expressed in distinct cell groups in complementary fashion. However, there is no obvious common function to all of the structures expressing either one of these genes. Second, the patterns and numbers of cells expressing c-kit were not affected in any obvious way in embryos homozygous for Wor Sl mutations. At 13.5 dpc, high levels of c-kit RNA were observed in the choriod plexuses and the ventricular zone of the fourth ventricle and lower levels were observed over the ventricular zone of the other ventricles (Fig. 2B left). At this stage, the highest levels of steel were detected in the thalamus, in the bed nucleus of stria terminalis and in the rostral portion of the telencephalon (Fig. 2C left). As expected, we did not observe any signal with the steel probe on 13.5 dpc Sl/Sl embryos (Fig. 2C right). However, c-kit expression patterns in Sl/Sl brains were indistinguishable from those of normal embryos (Fig. 2B right and not shown).

In late gestation normal embryos (around 16.5 dpc), high levels of both c-kit and steel RNAs were detected in overlapping patterns of expression in the olfactory bulb, the cortical plate and rhombencephalic lip (Fig. 1A,B). However, within these structures in the adult brain, the sites of c-kit and steel expression were clearly in distinct but contiguous groups of cells (see below). In addition, the c-kit probe labeled the hippocampus (in presumptive pyramidal cells), the hypothalamus, and choroid plexuses (Fig. 1A and not shown). High levels of steel expression were observed in the dorsal thalamus and in bed nucleus of stria terminalis, while lower levels of steel expression were found in the dentate gyrus and striatum (Fig. 1B and not shown). The same patterns were observed in embryos homozygous for the W37, Wv or Sld mutations. For example, Fig. 1C,D illustrates the patterns in 16dpc W37/W37 embryonic brain.

The patterns in the developing normal brain are in agreement with a recent study by Keshet et al. (1991) in which they report that c-kit is expressed mainly in early postmitotic regions in the neural tube and brain and steel in the floor plate and thalamus. In addition they report that c-kit and steel are expressed in complementary manner in the embryonic olfactory bulb and in some unidentified nuclei of the brain.

Expression of c-kit and steel in normal and mutant adult brain

The complicated patterns of c-kit and steel expression in the embryonic brain prompted us to study the relationships between the sites expressing the receptor and its ligand in the mature brain, and to search for any disturbances of their expression patterns in W and Sl adult mice. The patterns of c-kit and steel expression in transverse sections of the brain are shown in Fig. 6 and enlargements from similar sections highlighting the main sites of expression are shown in Figs 7 and 8. Very high levels of steel expression were found in the thalamus whereas both steel and c-kit transcripts were localized in different sites within the olfactory bulb, cerebral cortex, hippocampal region and cerebellum. The distribution of c-kit and steel transcripts in different compartments of the adult brain is summarized in Table 1. Wherever possible, the identification of the expressing cells as neuronal (N) or glial (G) is indicated. Two main conclusions can be drawn from these data. First, most of the structures that express c-k.it and steel from mid-gestation embryonic stages still express these genes in the adult, arguing for a (additional) role such as neural differentiation, migration and aggregation that may not be connected directly to earlier developmental events. The second conclusion is, once again, the reciprocal expression pattern of the two genes. In the nervous system, neurotransmission from the axon of one neuron to the dendrite (or cell body) of a target neuron give an obvious polarity to cell-cell associations. We were, therefore, particularly interested in determining whether cells that expressed steel, and therefore were involved in presenting this ligand to cells expressing the Kit receptor, established the same polarity as neurotransmitter-based information flow, or the opposite polarity, like the target derived trophic factor, NGF.

Table 1.

Distribution of c-kit and steel RNAs in adult mouse brain

Distribution of c-kit and steel RNAs in adult mouse brain
Distribution of c-kit and steel RNAs in adult mouse brain
Fig. 6.

In situ localization of steel and c-kit RNAs in the adult brain. (A) Dark-field micrograph of transverse section through the brain of 2 month old C57B1/6 mouse hybridized with steel and (B) c-kit. (ctx) cerebral cortex; (dg) dentate gyrus; (mol) molecular layer of the cerebellum; (ob) olfactory bulb; (pyr) pyramidal layer of hippocampus; (sub) subiculum; (sp) septum; (st) striatum.

Fig. 6.

In situ localization of steel and c-kit RNAs in the adult brain. (A) Dark-field micrograph of transverse section through the brain of 2 month old C57B1/6 mouse hybridized with steel and (B) c-kit. (ctx) cerebral cortex; (dg) dentate gyrus; (mol) molecular layer of the cerebellum; (ob) olfactory bulb; (pyr) pyramidal layer of hippocampus; (sub) subiculum; (sp) septum; (st) striatum.

Fig. 7.

In situ localization of c-kit and steel in the olfactory bulb and in cerebrum cortex. (A) Dark-field micrograph of transverse section through the olfactory bulb hybridized to c-kit and (B) steel probes. (C) Bright-field micrograph of the same area, c-kit positive cells are located in the areas reported to be occupied by superficial short axon neurons. The band just superficial to mitral layer (mit) marked with steel probe corresponds to internal tufted neurons and the band over the deep side of the periglomerular region probably corresponds to the external tufted cells, (ep) external plexiform layer; (glom) glomerular layer; (gran) granular layer; (ip) internal plexiform layer; (mit) mitral cell layer. (D) Dark-field micrograph of transverse section through the cerebrum cortex hybridized to c-kit and (E) steel probes. (F) Bright-field micrograph of the same area. The numbers of the layers are marked at the top. The border of layer 1 was identified by the lack of neurons in this layer, and the border of layer 4 by the higher density of neurons in this layer. Note the existence of c-kit positive cells in layer 1, and the restriction of steel positive cells to layers 2 and 3.

Fig. 7.

In situ localization of c-kit and steel in the olfactory bulb and in cerebrum cortex. (A) Dark-field micrograph of transverse section through the olfactory bulb hybridized to c-kit and (B) steel probes. (C) Bright-field micrograph of the same area, c-kit positive cells are located in the areas reported to be occupied by superficial short axon neurons. The band just superficial to mitral layer (mit) marked with steel probe corresponds to internal tufted neurons and the band over the deep side of the periglomerular region probably corresponds to the external tufted cells, (ep) external plexiform layer; (glom) glomerular layer; (gran) granular layer; (ip) internal plexiform layer; (mit) mitral cell layer. (D) Dark-field micrograph of transverse section through the cerebrum cortex hybridized to c-kit and (E) steel probes. (F) Bright-field micrograph of the same area. The numbers of the layers are marked at the top. The border of layer 1 was identified by the lack of neurons in this layer, and the border of layer 4 by the higher density of neurons in this layer. Note the existence of c-kit positive cells in layer 1, and the restriction of steel positive cells to layers 2 and 3.

Fig. 8.

In situ localization of steel and c-kit in adult cerebellum and hippocampus region. (A,C,E) expression of steel and c-kit (B,D,F) in the cerebellum of about 2 month old C57B1/6 normal (A,B), Wv/Wv (C,D) and Sld/Sld (E,F) mice, (gran) granular layer; (mol) moleculaFlayer; (Pur) Purkinje cell layer. (G,I,K) Expression of steel and (H,J,L) c-kit in hippocampus region of about 2 month old C57B1/6 normal (G,H), Wv/Wv (I,J) and Sld/Sld (K,L) mice, (dg) dentate gyrus; (en) enthorhinal cortex; (hp) pyramidal neurons of hippocampus; (thal) thalamus. abundant population of cells, perhaps neurons, in layers 2 and 3 (Fig. 7E).

Fig. 8.

In situ localization of steel and c-kit in adult cerebellum and hippocampus region. (A,C,E) expression of steel and c-kit (B,D,F) in the cerebellum of about 2 month old C57B1/6 normal (A,B), Wv/Wv (C,D) and Sld/Sld (E,F) mice, (gran) granular layer; (mol) moleculaFlayer; (Pur) Purkinje cell layer. (G,I,K) Expression of steel and (H,J,L) c-kit in hippocampus region of about 2 month old C57B1/6 normal (G,H), Wv/Wv (I,J) and Sld/Sld (K,L) mice, (dg) dentate gyrus; (en) enthorhinal cortex; (hp) pyramidal neurons of hippocampus; (thal) thalamus. abundant population of cells, perhaps neurons, in layers 2 and 3 (Fig. 7E).

Fig. 7 shows the profiles of c-kit and steel expression in the olfactory bulb and cerebrum cortex. As shown in Fig. 7A, c-kit transcripts were observed in cells located close to the junction between the external plexiform layer and the glomerular layer and in the superficial half of the external plexiform layer, the positions reported to be occupied by superficial short axon cells (Schneider and Macrides, 1978), but they could also be glial cells. In contrast, as shown in Fig. 7B, the steel probe hybridized to two narrow bands of cells, one of them just superficial to the mitral cell layer corresponding to the internal tufted neurons and the other one, in the deeper side of the periglomerular region, probably corresponding to the external tufted cells (Schneider and Macrides, 1978; Pinching and Powell, 1971). As the axonal collaterals of tufted cells project to the dendrites of the superficial short axon neurons (Pinching and Powell, 1971), steel could act in the olfactory bulb in the same direction as the information flow. In addition, the output axons of tufted neurons (which express steel) project to the paleocortex, a site where c-kit is also highly expressed.

In the adult cerebral neocortex, c-kit transcripts were detected in punctate cells distributed mainly in layers 1 to 3 and to a lesser extent in layers 4 to 6 (Fig. 7D). The hybridization to cells in the outermost neuron-free layer 1 (molecular layer) suggests that glial cells are responsible for the c-kit signal (at least in this layer). In contrast, steel expression was localized to a much more abundant population of cells, perhaps neurons, in layers 2 and 3 (Fig. 7E).

Perhaps the most striking example of the complementary patterns of c-kit and steel expression is shown in the hippocampus and regions into which it is connected, as shown in Fig. 8G,H. The classic primary synaptic connectivity through this region involves enthorhinal cortex neurons projecting to dentate gyrus neurons, which then project to hippocampal CA3 pyramidal neurons, which in turn project to hippocampal CAI pyramidal neurons, with CAI neurons finally projecting to subicular cortex neurons (Swanson and Cowan, 1977). High levels of steel RNA were found in the first two structures (enthorhinal cortex and dentate gyrus) whereas c-kit was found in the last three (CA3 and CAI pyramidal neurons of the hippocampus and subiculum). Accordingly, only steel expression in dentate gyrus and c-kit receptor expression in the CA3 region fits simply a model in which the ligand is active in the same direction as the information flow. However, if the minor connections in this region are also taken into account, the patterns of steel and c-kit expression are consistent with this model. In addition to projecting to dentate gyrus, the enthorhinal cortex is known to be projecting to CAI region (Swanson and Cowan, 1977) (which expresses c-kit). The subiculum (which expresses c-kit) is one of the targets of the thalamus (Shipley and Sorensen, 1975; Herkenham, 1978) and the thalamus has, probably, the highest levels of steel transcripts in the brain (Fig. 6). The thalamus projects to several layers throughout the cerebral cortex. Thus, Steel factor produced in the thalamus could also act on the dispersed c-kit expressing glial or neuronal cells in the cortex.

Interestingly, although CA3 and CAI express high levels of c-kit, much lower expression was observed in the CA2 region, c-kit is therefore one of the first molecular markers that give clear boundaries to the CA2 pyramidal cells.

In the cerebellum, very high levels of steel were confined to the Purkinje cells (Fig. 8A) and the deep cerebellar nuclei which receive afferents from Purkinje cells expressed c-kit (data not shown). The high levels of c-kit in dispersed glial cells in the molecular layer (Fig. 8B) suggest that the steel/c-kit pathway in the cerebellum, as in the cerebral neocortex discussed above, may be performing a signaling function from neurons to glial cells. We were unable to observe any change in the histology or the profiles of expression of c-kit and steel in the brains of adult Wv/Wv and Sld/Stdmice (compare Fig. 8 C-F to 8 A-B and 8 I-L with 8 G-H and not shown). Although both the Wv and Sldalleles have residual function, these observations on adult brains, together with similar findings with embryonic brains from embryos carrying either the severe W37 or the null Sl mutations, suggest that Kit receptor function may not be required for proliferative or developmental functions in the CNS.

The experiments presented in this paper demonstrate that the genes for both the Kit receptor and its ligand are expressed in a wide range of strikingly complementary regions throughout the body, from the early presomite stage to the mature adult. Mild or severe mutations at the W or Sl loci affect the patterns of c-kit and steel expression in the three known affected cell lineages, but do not appear to affect the patterns or levels of expression of these genes in cell types and organs that do not display a correspondingly mutant phenotype.

Diversity of cells expressing c-kit and steel

The broad diversity of cells expressing c-kit and steel transcend both common developmental origin or function. For example, c-kit is expressed in germ cells, in various tissues derived from mesoderm and endoderm, in neural crest-derived melanoblasts, sensory ganglia and craniofacial structures, and in neuroectoderm. In the neuroectoderm, c-kit is expressed in both neurons and glial cells (Table 1), and its distribution does not correlate with any known neurotransmitter or receptor. With respect to function, c-kit is expressed in both limbic (e.g. hypothalamus and hippocampus) and sensory motor regions (e.g. neocortex), in deep cerebellar neurons which receive synaptic input from inhibitory neurons (Purkinje cells) and in CA3 pyramidal neurons which receive synaptic input from excitatory neurons (dentate gyrus neurons). Despite this wide range of cell types that express these genes, the in situ RNA hybridization analysis indicates that the expression of both genes is restricted to distinct cells and anatomical sites.

Complementary patterns of c-kit and steel expression

The in situ RNA hybridization of consecutive tissue sections with either the c-kit or steel probes revealed the strikingly complementary patterns of expression of these genes and the close physical proximity between cells expressing c-kit (frequently seen as individual positive cells) and the band of cells expressing steel. In some instances, different groups of cells in the same area expressed either c-kit or steel (e.g. neocortex). At other sites, for example, the dorsal root ganglia, expression of one gene (c-kit at 12.5 dpc) was followed later by expression of the other (steel at 15.5dpc). Finally, we observed c-kit expression in cells of the parietal endoderm with no detectable neighbouring cells expressing steel.

With the exception of this last example, the results of the RNA in situ hybridization analysis support earlier conclusions that steel acts locally by affecting the microenvironment in which other cells develop, migrate and proliferate. This conclusion was initially based on the phenotype of chimeras, established at the embryonic stage (Nakayama et al. 1988) or by the transplantation of splenic fragments between wild-type and Sl mutants (Harrison and Russell, 1972). A similar conclusion has been reached from the observations that the protein product of the steel locus is membranebound (Flanagan and Leder, 1990; Anderson et al. 1990) and that the mild Steel allele SC1 does not make the membrane-bound form of Steel factor (Flanagan et al. 1991; Brannan et al. 1991). Taken together, these observations, derived from three entirely different experimental approaches, all suggest that activation of the Kit receptor by binding of the Steel factor normally occurs via cell-cell interactions in which the membranebound form of Steel factor on one cell binds to the extracellular domain of the Kit receptor on a neighboring cell. This binding then initiates a series of intracellular signaling events that include tyrosine autophosphorylation of the Kit receptor and association with various downstream signaling molecules, including phosphotidylinositol 3’-kinase and phospholipase-Cγ1 (Rottapel et al. 1991; Reith et al. 1991).

The complementary patterns of c-kit and steel expression are observed in both embryos and adult animals. These observations suggest that the Kit pathway has an ongoing function in the adult, a conclusion consistent with the phenotypic analyses of viable W and Sl mutants. For example, W/Wv adult mice are severely anemic, have reduced numbers of erythroid progenitor cells, and defective mast cell proliferation. Similarly, although germ cells fail to proliferate and migrate from the hindgut to the gonadal ridge in embryos homozygous for the severe W or Sl alleles, oocytes are present in Sl/Slt females but they fail to mature (Kuroda et al. 1988). The observation that follicle cells express high levels of steel in the adult female and that oocytes express c-kit is consistent with a role for the Kit pathway in oocyte maturation. Similarly, the ongoing expression of steel in Sertoli cells in the adult testes, together with the impaired differentiation of germ cells in Sl/ + adult males (Nishimune et al. 1984), and the ability of injected antic-kit monoclonal antibodies to block spermatogonial maturation (Yoshinaga et al. 1991), suggests that activation of the Kit receptor is required for spermatogenesis.

Possible role of the Kit pathway outside the three known affected lineages

The highly contiguous patterns of expression of c-kit and steel in a broad array of distinct anatomical sites make it likely that the c-kit signaling pathway has important biological functions in these diverse tissues and cell types. Nevertheless, mutations at the W or Sl loci do not have any obvious phenotypic consequences outside of their profound effect on melanogenesis, hematopoiesis and gametogenesis. In an attempt to resolve this paradox, we determined the patterns of expression of both genes in embryos homozygous for the W37, WSl, Sl or Sld mutations. With the exception of a reduction or absence of c-kit-positive germ cells, melanocytes, fetal liver cells and mast cells, there was no detectable change in the levels of c-kit expression elsewhere nor were any changes evident in the organization of cells expressing c-kit. These data suggest that the Kit signaling pathway is not essential for cellular proliferation and organogenesis in those sites that are not phenotypically affected by mutation at the W or Steel loci.

There are several possible explanations for these results. First, phenotypic abnormalities may not have been observed because these morphological changes were too subtle to be detected in our studies. Second, the c-kit signaling pathway may be redundant with other signal transduction pathways, particularly in the brain where a large number of both receptor and non-receptor tyrosine kinases are highly expressed (see for examples Ross et al. 1988; Klein et al. 1990a, 1990h; Wanaka et al. 1990; Reid et al. 1990; Yeh et al. 1991; Lhotak et al. 1991; Lai and Lemke, 1991). Functional redundancy with another RTK pathway could also explain the absence of placental defects in Sl/Sl embryos. In this regard, it is interesting to note that both PDGF-R and CSF-1R, two RTKs which belong to the same subfamily as the Kit receptor, and their respective ligands, are also expressed in extraembryonic tissues (Mercóla et al. 1990; Regenstreif and Rossant, 1989; Arceci et al. 1989). The sites of PDGF-R and PDGF expression within extraembryonic tissues have not yet been reported. However, in the case of CSF-1R (c-fms) and its ligand CSF-1, there is little overlap with the patterns of c-kit/steel expression. Both c-kit and c-fms are expressed in the decidua at the presomite stage; however, while c-kit expression is restricted to the mesometrial side, c-fms transcripts surround the embryo (Regenstreif and Rossant, 1989; Arceci et al. 1989). In the mature placenta, c-fms RNA is found mainly in trophoblastic giant cells and in the spongiotrophoblast, whereas c-kit is predominantly expressed in the decidua. Similarly, the ligands for these two RTKs are expressed in distinct locations: steel is first found in the blood vessels (and ectopiacental cone) and later in the labyrinth layer whereas CSF-1 is present in the uterine epithelium (Regenstreif and Rossant, 1989; Arceci et al. 1989).

Third, for some of the mutant W or Sl alleles examined, particularly Wv and Sld it could be argued that the residual levels of signaling via the Kit pathway in these mutants are sufficient for function outside of the three cell lineages affected by these mutations. However, both the severe W37 allele and the Sl allele, which is a deletion of the entire coding region of the Sl locus, are null alleles, and yet, as shown here, the brains of W37/W37 and Sl/Sl embryos still contained apparently normal numbers and organization of cells expressing c-kit.

And fourth, the absence of a functional c-kit pathway may affect post-developmental and post-mitotic cell signaling events, causing phenotypic abnormalities not readily detectable by morphology alone. In the brain, Kit pathways may participate in the processes of axonal pathfinding and synaptogenesis. The formation, maintenance and breakage of synapses is a continuous process throughout life (Cotman, 1978), potentially explaining the continuous expression of c-kit and steel in the brain from embryogenesis into adulthood. Mutations in genes affecting these processes might not be expected to affect overall viability. Studies in Drosophila have shown that the cytoplasmic tyrosine kinase encoded by D-abl is involved in growth cone guidance (Elkins et al. 1990). Only double mutants, in abl and in the adhesion molecule fasciclin I, show a dramatic defect in axonal guidance, suggesting the existence of redundant genetic pathways. Of particular relevance is the fact that even the double mutants (which have an obvious defect in axonal guidance) do not display any decrease in viability under laboratory conditions (Elkins et al. 1990). Another possible role for Kit/Steel factor in the CNS is direct participation in synaptic transmission, either by modulating neurotransmitter release or by affecting neurotransmitter receptor activation or desensitization (for recent review, see Huganir and Greengard, 1990). The expression of c-kit in a small portion of the glial cell population could reflect its function in unidentified subgroup(s) of glial cells or at certain stages in the cell cycle. In contrast, the ubiquitous expression of PDGF-A in CNS and PNS neurons, together with its known effect on glial cells, suggests a much more widespread role for the PDGF system in glial cell regulation (Yeh et al. 1991, and references therein). In vitro elucidation of Steel factor influence on diverse neuron and glial cell populations should provide valuable information about the possible role of the Kit signaling pathway in the nervous system. Direct and detailed examination of axon bundles and synapses, as well as rigorous tests of behavioral capabilities, are necessary in order to examine how normal W and Sl mutant mice really are.

We thank D. E. Williams, S. Lyman and D. M. Anderson from Immunex for generously providing us with the steel cDNA probe. Work from the authors’ laboratory is supported by grants from the National Institute of Health (U.S.), the National Cancer Institute of Canada (NCIC) and the Medical Research Council of Canada. B.M. is supported by a fellowship from the Leukemia Research Fund. J.R. is a Terry Fox Cancer Research scientist of the NCIC. A.B. and J.R. are International Research Scholars of the Howard Hughes Medical Institute.

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