trkC, a receptor for neurotrophin-3, is widely expressed in the developing nervous system and in non-neuronal tissues

The Trk gene family encodes receptor tyrosine kinases (RTKs) that include trk, trkB and trkC (for review see: Chao, 1992). The best characterized of these genes is human trk, which was originally described as a dominantly acting oncogene (Martin-Zanca et al., 1986) and recognized to encode a RTK (Martin-Zanca et al., 1989). However, the normal biological role of gp145^prototrk remained enigmatic until expression studies provided the first clues regarding possible function (Martin-Zanca et al., 1990). Biochemical analysis demonstrated that the gp140^prototrk RTK interacts directly with Nerve Growth Factor (NGF; Kaplan et al., 1991a,b; Klein et al., 1991), is required for NGF function (Loeb et al., 1991) and forms part of the high affinity NGF receptor (Klein et al., 1991; Hempstead et al., 1991). Furthermore, the NGF-related neurotrophins have been identified as ligands for the Trk-related RTK-encoding genes trkB and trkC (Berkemeier et al., 1991; Ip et al 1992; Soppet et al., 1991; Squinto et al., 1991; Lamballe et al., 1991; Tsoulfas et al., 1993). The interaction between neurotrophins and their respective receptors stimulates receptor tyrosine kinase activity and elicits different biological responses depending on the cellular environment of receptor expression. For example, in the PC12 rat pheochromocytoma cell line, neurotrophic factors promote neuronal survival and differentiation upon binding to the different members of the Trk receptor family (Kaplan et al., 1991a; Loeb et al., 1991; Squinto et al., 1991; Tsoulfas et al., 1993; D. Soppet and L. F. P., unpublished data). In contrast, stimulation of Trk receptors by neurotrophins induces proliferation in transfected NIH-3T3 fibroblasts (Glass et al., 1991; Barbacid et al., 1991). Other examples of RTKs exhibiting pleiotropic effects depending on the particular site of expression are the let-23 and c-kit genes. In Caenorhabdi tis elegans, genetic evidence indicates that the let-23 gene, which encodes a putative RTK of the epidermal growth factor (EGF) receptor subfamily, can control two opposing pathways in vulval differentiation and functions in at least five others tissues (Aroian and Sternberg, 1991). In the mouse, the c-kit RTK acts differently in cells originating from diverse embryonic lineages such as primordial germ cells, melanocytes and hematopoietic stem cells (Geissler et al., 1981, 1988). Similarly, findings that NGF has specific effects in the rat seminiferous epithelium (Parvinen et al., 1992) and B-lymphocytes (Otten et al., 1989) suggest that, in addition to its neurotrophic actions, this factor and presumably its related neurotrophins, brain-derived neurotrophic factor (BDNF; Leibrock et al., 1989), neurotrophin-3 (NT-3; Enfors et al., 1990; Hohn et al., 1990; Jones and Reichardt, 1990; Kaisho et al., 1990; Maisonpierre et al., 1990a; Rosenthal et al., 1990), Xenopus NT4 (xNT-4, Hallböök et al., 1991; Ip et al., 1992) and human NT-5 (hNT-5; Berkemeier et al., 1991), may have broader physiological effects.

The rat trkC locus is complex, encoding multiple distinct receptors (Tsoulfas et al., 1993). These trkC isoforms bind NT-3, a widely expressed neurotrophin (Maisonpierre et al., 1990b), in chemical cross-linking and equilibrium binding analyses (Lamballe et al., 1991, Tsoulfas et al., 1993).

In this study, we have investigated the expression of trkC in the mouse embryo and adult CNS. We find trkC transcripts throughout the nervous system but also in non-neural tissues. Furthermore, we contrast the expression of the trkC gene with that of trk and trkB. Finally, we have mapped the chromosomal location of the three Trk loci and show that they are unlinked and map in the vicinity of previously existing neurological mutations. This information may lead to a direct association of Trk genes with the phenotypes elicited by known spontaneous mutations in the mouse.

RNA was extracted using RNAzol (Cinna/Biotecx) following the manufacturer’s recommendations. RNAse protection experiments were performed as previously described (Tessarollo et al.,1992) using the RPA kit (Ambion). A genomic trkC-specific probe that spans 196 nucleotides of the extracellular domain (aa 336-401 of the rat sequence; Tsoulfas et al., 1993) and including 64 downstream intronic nucleotides was used to generate an antisense RNA probe employed in RNAse protection analysis. A 250 base β-actin c-RNA probe (Alonso et al., 1986) was included in the same reaction as a means of assessing relative levels of RNA present in each hybridization.

In situ hybridization protocols were as described (Martin-Zanca et al. 1990) with the following modifications. Dissected embryos were fixed overnight in 4% paraformaldehyde, dehydrated with alcohols and xylenes, and embedded in paraffin. Embryos were sectioned at 5 μm thickness and mounted on gelatin-coated slides. Slides were deparaffinized in xylene and rehydrated in graded (100-30%) ethanol solutions. After fixing in 4% paraformaldehyde, the tissues were pretreated with proteinase K (20 μg/ml) (Boehringer Mannheim), refixed and immersed in triethanolamine buffer containing acetic anydrate and dehydrated. Sections were hybridized with antisense cRNA probes (5×10₅ cts/minute) in a buffer containing 50% formamide, 0.3 M NaCl, 20 mM Tris-Cl (pH 7.4), 1× Denhardt’s solution, 0.5 mg/ml yeast tRNA and 10 mM DTT at 50°C for 20 hours. After hybridization, washes were performed in 4× SSC and 10 mM DTT at 50°C. The slides were then incubated for 30 minutes at 37°C with RNase A (20 μg/ml) and RNase T₁ (2 μg/ml) followed by a 30 minute incubation at 55°C in 50% formamide, 0.2× SSC, 10 mM DTT, washed twice for 30 minutes in 0.2× SSC, 1% sodium pyrophosphate (w/v), 10 mM DTT and dehydrated. The slides were dipped in Kodak emulsion NTB-2 and exposed for up to 10 days at 4°C. The slides were then developed in Kodak D-19, fixed as recommended by manufacturer and stained in 0.2% toluidine blue. All photomicroscopy was done on a Zeiss Axiophot microscope. Sense and anti-sense probes labeled with ³⁵S were prepared by standard procedures (Krieg and Melton, 1987) by using UTP as the labeled nucleotide. The trkC antisense probe was synthesized from a ∽2 kb rat trkC cDNA (NRT-8) as described by Tsoulfas et al. (1993). The trk and trkB probes are described in detail by Martin-Zanca et al. (1990) and Klein et al. (1989), respectively.

Interspecific backcross progeny were generated by mating (C57BL/6J × Mus spretus)F₁ females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 N₂ progeny were obtained; a random subset of these N₂ mice were used to map the Trk loci (see text for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer and hybridization were performed as described (Jenkins et al., 1982). All blots were prepared with Zetabind nylon membrane (AMF-Cuno). Hybridization probes were labeled with [α³²P] dCTP using a nick translation labeling kit (IL-9) (Boehringer Mannheim) or random priming (trk, trkB, trkC) (Amersham Corporation). Washing was done to a final stringency of 0.2-0.8 × SSCP, 0.1% SDS, 65°C.

The three mouse Trk loci, trk, trkB and trkC, have been designated Ntrk1, Ntrk2 and Ntrk3, respectively, to conform with the current human locus designations for these genes. The trk probe was a 1.5 kb EcoRI genomic fragment from the mouse trk extracellular domain (Martin-Zanca et al., 1990). The trk probe hybridization to a 6.4 kb fragment in BglII-digested C57BL/6J (B6) DNA and 5.0 and 1.3 kb fragments in M. spretus (S) DNA. The two M. spretus-specific fragments cosegregated and were followed in backcross mice. The trkB probe was a 938 bp corresponding to amino acid 1-308 of mouse trkB sequences (Klein et al., 1989). The trkB probe hybridized to 3.8, 2.5, 1.1, 0.9 and 0.35 kb fragments in TaqI-digested B6 DNA and 3.5, 3.1, 2.5, 1.4, 1.1 and 0.35 kb fragments in S DNA. The 3.5, 3.1 and 1.4 kb M. spre tus-specific fragments cosegregated and were followed in backcross mice. The trkC probe was a 288 bp cDNA corresponding to amino acid 282-378 of the mouse trkC gene (Tsoulfas et al., 1993). The TrkC probe hybridized to a 1.3 kb fragment in EcoRIdigested B6 DNA and a 3.6 kb fragment in S DNA. The 1.3 kb M. spretus-specific fragment was followed in backcross mice.

A description of most of the probes and RFLPs for loci used to position the Trk loci on the interspecific map have been reported. The loci in include: fibrinogen, gamma polypeptide (Fgg), connexin-40 (Cnx-40) and nerve growth factor beta (Ngfb) on chromosome 3 (Mucenski et al., 1988; Cox et al., 1991; Haefliger et al., 1992); neuroendocrine convertase-1 (Nec-1) on chromosome 13 (Copeland et al., 1992); and insulin-like growth factor 1 receptor (Igflr), feline sarcoma oncogene (Fes) and tyrosinase (Tyr) on chromosome 7 (Lunsford et al., 1990; Copeland et al., 1992). One locus, interleukin-9 (IL-9) on chromosome 13 has not been previously reported for our panel. The IL-9 probe (pP40.2B4) was a 550 bp mouse cDNA (Van Snick et al., 1989). The IL-9 probe hybridized to an 18.0 kb fragment in SphI-digested B6 DNA and a 5.4 kb fragment in S DNA. The 5.4 kb M. spretus-specific fragment was followed in backcross mice.

Recombination distances were calculated as described (Green, 1981) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.

To determine the temporal expression pattern for trkC during murine development, a trkC-specific probe was used in RNAse protection assays employing a β-actin internal control as described in Materials and methods. 10 μg of total mouse embryo RNA were assayed and, although a faint protected band is first observed on day 7.5, trkC gene expression is clearly detected at embryonic day 8.5 coinciding with the timing of neural tube formation (Fig. 1). Relative to the internal β-actin control, a substantial increase in trkC mRNA expression occurs around embryonic day 10.5 (25–35 somites).

The trk and trkB genes are expressed predominantly in the nervous system (Martin-Zanca et al., 1990; Klein et al., 1990a). Having determined that trkC transcripts are present in the embryo from the earliest stages of neural induction, we next performed RNA in situ hybridization of postimplantation embryo sections from day 7.5 through day 17.5 and in neo-nate and adult central nervous system (CNS; see Materials and methods). For comparison, in the course of this study, we hybridized adjacent embryo sections with trk, trkB and trkC probes.

The results obtained from our in situ experiments on trkC expression are summarized in Table 1. The expression of this gene is highly complex. We first observed trkC hybridization as a weak signal in 7.5 day egg cylinders with a pattern indicating expression in the early neuroectoderm (not shown). Whole-mount in situ experiments are in progress to extend these observations. trkC gene expression is evident throughout the neuroepithelium of 9.5 (12–30 somites) day embryos (Fig. 2A-D). At these early stages, we also observe trkC transcripts in regions where neural crest cells are known to localize. These include forming dorsal root ganglia (DRG) and regions adjacent to the neural tube and the dorsal aorta (Fig. 2C,D; arrows), locations identified as migratory routes used by cells of neural crest origin (see below).

During organogenesis, additional sites of active trkC are found. Fig. 2E-F shows a sagittal section from an 11.5 day embryo (35 somites). trkC expression is exhibited throughout the CNS (telencephalon, diencephalon, mesencephalon, rhombencephalon and neural tube) and comparison with an adjacent trkB hybridized section (not shown) indicates overlapping expression in the mantle (postmitotic) layer of the spinal cord, whereas the ependymal (mitotic) layer expresses only trkB. The trkB and trkC genes are coexpressed in the branchial arch arteries; however, more caudal trkB transcripts are present in the mesonephric region while trkC is expressed in the midgut and genital ridge associated mesenchyme (see Fig. 2E-F).

At 13.5 days of development, the trkC gene is expressed throughout the neural network (Fig. 2G-I). At this stage, trkC mRNA is present in neural tissues where trk and trkB gene activity has not been previously detected (MartinZanca et al., 1990; Klein et al., 1989, 1990a). One striking example is the expression of trkC in neural-crest-derived cells of the developing enteric nervous system (Figs 2H, 7). In contrast, trkC and trkB transcripts are present in the trigeminal (V^th cranial) ganglion (Fig. 2H,I) where punctate hybridization reflects expression in a small subset of cells (Fig. 2I). The three afferent branches of the trigeminal ganglion (ophthalmic, maxillary and mandibular) can be readily traced by abundant trkC hybridization, presumably reflecting expression in the neural crest-derived Schwann cells that migrate and differentiate along the trigeminal processes (Fig. 2G).

Several insights can be drawn from a direct comparison of trk, trkB and trkC expression at later stages in development. Fig. 3 shows in situ hybridization of day 17.5 sections, from a single fetus, which were hybridized with probes to the three related genes. trk expression is distinctive in its tight regulation primarily limited to trigeminal (V), superior cervical (C), sympathetic (S) and dorsal root ganglia (arrows; Fig. 3A; Martin-Zanca et al., 1990). In comparison, trkB is expressed in these ganglia and at high levels in the CNS and additional structures including the whisker pad (W), tongue (T) and tooth buds (white arrowhead; Fig. 3B). This complex expression pattern is consistent throughout mid-gestation (days 14-18; L. F. P., unpublished observations). Expression of the trkC gene mirrors that of trkB in many regions of the embryo (Fig. 3B,C). Like the trkB gene (Klein et al 1990a; Parada et al., 1992), trkC is expressed throughout the nervous system including many of the same cranial and spinal ganglia (V, VII, VIII, X, DRG). The trkC and trkB genes are also apparently coexpressed in several non-neural structures including the tongue, the whisker pad (vibrissae) mesenchyme and forming teeth (Fig. 3B,C). In the tooth, trkB is expressed in the outer dermal papilla while trkC transcripts appear confined to the core of the dermal papilla (compare Fig. 3B and C). trkC transcripts are also found in several additional neural crest-derived structures including enteric ganglia (see Fig. 7) and in other non-neural cells (see below).

The pattern of trkC gene expression is summarized in Table 1 and examples of in situ hybridization in the embryonic CNS are shown in Figs 2, 3C and 4. Interestingly, this gene shows both overlapping and exclusive expression profiles in the cephalic CNS when compared to the trkB gene. In the telencephalic cortical plate, both trkC and trkB transcripts are highly represented whereas in the intermediate layer only trkC mRNA was observed. In the ventricular layer, trkB expression is high and trkC expression is low (Figs 3B,C, 4A-D). In the striatum (Sr), trkC mRNA is most abundant centrally while trkB transcripts predominate along the ependymal layer. Finally, the thalamic nuclei (t) appear to express either trkB or trkC (Fig. 3).

A cross section through the caudal spinal cord of a 15.5 day embryo reveals trkC transcripts throughout the dorsalventral axis of the mantle layer including the ventral horn where motor neurons are located (Fig. 4E,F). No evidence of expression was seen in the floor plate at any developmental stages examined.

trkC transcripts are also abundant in the embryonic metencephalon (cerebellum). Fig. 5 provides examples of trkC expression in cerebellum at embryonic days 13.5, 17.5 and postnatal day 10. Transcripts are seen throughout embryonic stages with high levels in the immature but postmitotic Purkinje cells (Fig. 5A-D; Altman and Bayer, 1985). No expression was observed in the external germinal layer (egl). In all postnatal stages examined, including adult, trkC mRNA levels are very high in the cerebellar granule layers and in Purkinje neurons (Fig. 5E,F and not shown).

The neural crest gives rise to a large component of the peripheral nervous system (PNS). trkC transcripts are found in many PNS structures and also in cells that appear to be migratory, neural crest cells (Table 1, Fig. 2A,C,E, and unpublished results). As previously noted (Figs 2, 3), all Trk genes are expressed to a greater or lesser extent in neural crest-derived embryonic cranial, spinal and sympathetic ganglia. Fig. 6 shows in situ hybridization of three adjacent DRG sections from a 17.5 day embryo. These sections (5 μm) provide direct comparison of Trk gene expression and, in agreement with a report by Carroll and co-workers (1992), show that a majority of neurons express trk (Fig. 6A), while trkB (Fig. 6B) and trkC (Figs 6C, 2I) transcripts are present in a reduced subset of neurons. Very similar distribution of the three genes is seen in trigeminal ganglion (Fig. 3; see also Martin-Zanca et al., 1990; and Klein et al., 1990a).

The ganglia of the enteric nervous system are formed from migratory neural crest cells originating in the vagal crest region (Le Douarin and Teillet, 1973). These cells hybridize with trkC probes (Fig. 7A) as exemplified by hybridization of embryonic day 13.5 stomach where trkCexpressing migratory neural crest cells can be observed intercalated within the mesenchyme. Several days later, trkC transcripts can be localized to the forming ganglia that appear throughout the muscle and submucosal layers of the intestine (Fig. 7B). We do not observe trkC expression in the epithelium (Fig. 7). Thus, trkC expression is found in diverse PNS structures some of which do not appear to express other Trk-family receptors (Martin-Zanca et al., 1990; Klein et al., 1990a).

trkC gene expression remains abundant in the adult mouse CNS. The cerebral cortex exhibits high levels of transcripts particularly in the superficial layers and in the deep layers with reduced expression in the intermediate layers (Fig. 8). TrkC transcripts are also abundant in the caudate putamen from embryonic stages (Fig. 4A,B), a pattern that remains unchanged in the adult CNS (Fig. 8B). Similarly, thalamic nuclei maintain trkC expression throughout development and in the adult (Fig. 8A,B). Thalamic nuclei do not appear to express trkB transcripts in adult brain (Klein et al., 1990a).

In the hippocampus of adult brain, the pyramidal cell layer as well as the neurons of the dentate gyrus exhibit high levels of trkC mRNA (Fig. 8A-C). In the cerebellum, both the Purkinje and the granule cell layer express high levels of trkC transcripts. Thus, expression of the trkC gene observed in the mature brain is consistent with that exhibited during embryonic development.

As can be appreciated from Figs 2 and 3, trkC-encoding transcripts are abundant throughout the embryo including sites that are not necessarily consistent with expression in neural cells (see Table 1). Note the presence of trkC transcripts in vibrissae and dental papillae (Fig. 3). We also find trkC transcripts in cells of the submandibular gland (Fig. 9A,B) and in the mesenchyme surrounding mesonephric and urogenital ducts (Fig. 9C,D). High levels of trkC transcripts can also be localized to the brown adipose tissue dorsal to the cervical spinal cord (Fig. 9D,E); the cortex of the metanephros; and adrenal gland (Fig. 9F,G). Finally, abundant trkC expression is found along the subendothelial mesenchyme of arteries throughout the developing embryo (Figs 9H,I and 2, 3).

We next wished to identify the chromosomal location of the three Trk genes to determine whether they are linked or map to separate chromosomal locations and to assess the possibility that these three genes might be associated with previously reported neurological mutations in the mouse. The chromosomal location of trk, trkB and trkC was determined by interspecific backcross analysis using progeny derived from matings of [(C57BL/6J × Mus spretus)F₁ × C57BL/6J] mice that have been typed for over 1100 loci distributed among all mouse chromosomes (Copeland and Jenkins, 1991; see Materials and methods).

Each of the Trk genes mapped to a different mouse autosome (Fig. 10), indicating that they have become well dispersed during chromosome evolution. To determine whether any of the Trk genes mapped near a known mouse mutation with a phenotype that might be consistent with a defect in a Trk gene, we aligned our interspecific maps of chromosomes 3, 13 and 7 with composite linkage maps that report the map location of many uncloned mouse mutations (compiled from GBASE, a computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Interestingly, all three genes mapped near loci that exhibit neurological phenotypes.

trk mapped in the vicinity of the spontaneous neurological mutation spastic (spa) located on mouse chromosome 3 (Chai, 1961; Van Heyningen et al., 1975; Wilson et al., 1986; reviewed in Green, 1989). trkB mapped in the vicinity of the spontaneous neurological mutation Purkinje cell degeneration (pcd) located on chromosome 13 (Mullen et al., 1976; O’Gorman, 1985; reviewed in Green, 1989). Finally, trkC mapped in the vicinity of a gene on mouse chromosome 7 that has been shown to affect susceptibility of inbred mice to audiogenic seizures. (Neumann and Collins, 1991).

Recent studies from a number of laboratories, showing that trk and trkB are expressed mainly in the nervous system and encode functional receptors for the neurotrophins, have been important for advancing our understanding of the mechanisms that regulate neural development.

In the present study, we show that trkC, a third member of the Trk gene family (Lamballe et al., 1991; Tsoulfas et al., 1993), is expressed in a broad component of the nervous system and in non-neural tissues, including cell types where other Trk genes are not active.

trkC expression was first observed in the gastrulating embryo, coordinate with the temporal expression pattern of its ligand NT-3, the more abundant neurotrophin detected early in development (Maisonpierre et al., 1990b). This is the earliest example of an active Trk gene reported to date, raising the possibility that the trkC RTK may mediate important functions in early neuroepithelium formation.

Kalcheim and co-workers (1992) have reported a proliferative activity of NT-3 in migratory neural crest cells. In the present study, we provide evidence for NT-3 receptor expression in migratory neural crest cells, thus supporting the notion that Trk receptors and their ligands may mediate alternative functions to trophic signals during development (for review, see Barbacid et al., 1991).

The trkC gene is expressed in many neural crest derivatives including cranial, dorsal root and sympathetic ganglia. In trigeminal and dorsal root ganglia, all Trk genes are active. trk mRNA is perhaps most abundant and confined to neurons in these ganglia. We note that in addition to expression in sympathetic ganglia late in development, trk expression can also be detected in isolated neurons of the basal forebrain, hindbrain and in other locations of the late embryonic and adult brain (Holtzman et al., 1992; L. F. P., unpublished observations). Like trkB, trkC is expressed in fewer neurons in sensory ganglia. However, in the trigeminal ganglion, the afferent projections are outlined by expression of these genes suggesting that trkB and trkC are expressed in neural crest-derived Schwann cells that migrate along the axonal projections to their sites of differentiation.

Another notable site of trkC-specific expression is the enteric neural crest and ganglia. Through trkC hybridization, it is possible to trace the migration of vagal neural crest cells into the gut region, suggesting that neurotrophins may have important functions in the migratory and trophic potential of enteric neurons.

trkC is also expressed in a group of non-neural tissues that, in quail-chick studies (Le Douarin, 1982) or in radioisotopic labeling experiments in the amphibian embryo (Chibon, 1964), have suggested neural crest origin. Among these are the artery walls derived from aortic arches, tooth papillae (Chibon, 1970) and salivary glands. Using RNAse protection and northern analyses, we also observe trkC transcripts in the thymus (Tsoulfas et al., 1993), another tissue whose connective tissue component is derived from the neural crest.

Brown adipose tissue, particularly in the cervical region dorsal to the spinal cord, also expresses high levels of trkC transcripts (Fig. 9E,F). Because of the high silver grain density caused by strong expression, we are unable to identify precisely the expressing cell types within this tissue. Electron microscopy studies indicate that brown adipose is particularly well innervated and Schwann cells have been observed sheathing unmyelinated nerve fibers in the lobules, close to the vessels, and between adipocytes (Bargmann et al., 1968; Linck et al., 1973). Therefore, trkC expression in this tissue may reflect the presence of transcripts in neural cells and not in adipocytes. However, in chick, the subcutaneous adipose of the face and the ventral neck region is derived from the neural crest (Le Douarin, 1982), while the precise origin of the brown adipose tissue has not been well established (Néchad, 1986). Thus, it is also possible that this adipose tissue may be of neural crest origin and retain trkC expression.

Trk genes are expressed in a broad spectrum of neural crest derivatives implying that neurotrophins may function in cells where activity has not previously been associated.

The trkB and trkC expression profiles exhibited in migratory neural crest cells along the descending aorta suggest that trkB and trkC products may represent good markers for subsets of neural-crest-derived cells.

In the CNS, the trkB and trkC genes are coexpressed and differentially expressed in intriguing patterns. We observed coexpression of these genes in the hippocampus (pyramidal and granule neurons), in the cerebellum (Purkinje cells), in the ventral spinal cord and in regions of the cerebral cortex (L. T. and L. F. P., unpublished data). In contrast, thalamic nuclei appear to express trkC transcripts preferentially in the adult. Similarly in the cerebellum, trkC expression is abundant in the granular layer where little or no trkB transcripts could be detected (see Klein et al., 1990b).

The neurotrophin theory has been well substantiated in the sensory nervous system through the study of NGFrelated neurotrophins and through the availability of welldefined primary culture systems (Barde, 1989). The search for trophic molecules acting on motor neurons has proven more elusive. Culturing of motor neurons is technically difficult and, although tissue extracts have been reported to promote motor neuron survival, only one molecule in particular, CNTF (Sendtner et al., 1992a) has been identified to promote survival of these cells in vivo. Until recently, efforts to identify a motor neuron response to the NGF family of neurotrophins have not been successful. Very recently BDNF has been shown to rescue motor neurons in vivo in rat and chick (Yan et al., 1992; Oppenheim et al., 1992; Sendtner et al., 1992b). The present results indicate the existence of trkC and trkB (Klein et al., 1990a) transcripts during embryogenesis in the ventral horn of the spinal cord and in other regions where motor neurons are localized. These results therefore suggest the need for a renewed effort at understanding neurotrophin function in motor neurons.

In mouse, the three Trk family members map to different mouse autosomes and are located in regions containing known neurological mutations. For example, the mouse trk gene maps near the spa mutation on chromosome 13. spa homozygotes show spastic symptoms although no anatomical abnormalities have been described. Glycine receptor deficiencies have been reported in these mice (White and Heller, 1982; White, 1985), but it remains to be determined whether these deficiencies are the cause or a secondary effect of the spa mutation.

Likewise, trkB maps on chromosome 3, in a region where the pcd mutation has previously been mapped. pcd homozygotes show a moderate ataxia beginning at 3 to 4 weeks of age. They are smaller than their normal littermates but live a fairly normal life span although adult males are infertile. In these mice, Purkinje cells begin to degenerate by 15 to 18 days of age followed by a slower degeneration of the photoreceptor cells of the retina and mitral cells of the olfactory bulb. Later (50-60 days of age) discrete populations of thalamic neurons also degenerate. Studies of fusion chimeras between pcd/pcd and +/+ embryos suggest that pcd acts directly within the Purkinje cells themselves (Mullen, 1977). The trkB gene is expressed in Purkinje cells, in retinal ganglion cells, in motor neurons and in a spectrum of other neural tissues. Thus the pcd phenotype could be accounted for by mutations in the trkB locus.

The trkC locus maps in a region of chromosome 7 that has been associated with increased tendency to exhibit auditory defects in mice (Neumann and Collins, 1991). These auditory defect susceptibilities are ill-defined at this time but it is possible that the trkC locus may contribute to such abnormalities since the NT-3 gene and its receptor (data not shown) is expressed in the sensory components of the developing ear during embryogenesis (Pirvola et al., 1992). As germline mutations are induced in the Trk genes via homologous recombination in embryonic stem cells, it should be possible to perform allelism studies to determine whether any of these mutations do in fact result from defects in Trk genes.

The present mouse mapping studies can also be used to predict where the Trk genes will map in humans. For example, trkB maps between Il-9 and Nec-1 which have both been mapped to the long arm of human chromosome 5 (Fig. 10), suggesting that trkB will reside on the long arm of human chromosome 5 as well. Likewise, trkC does not recombine with Fes, which has been mapped to 15q25-qter. Finally, trk has been mapped by two groups to the long arm of human chromosome 1 (Miozzo et al., 1990; Morris et al., 1991). This placement is consistent with the mouse mapping studies shown in Fig. 10.

We have studied the expression of the trkC gene during development and in the adult CNS, and compared these data with that of the other identified Trk gene family members (Martin-Zanca et al., 1990; Klein et al., 1990a). These genes map to unlinked chromosomal locations and are primarily, though not exclusively, expressed in the nervous system. Understanding the expression patterns of Trk genes provides important information regarding potential sites of neurotrophin action and identification of cells that preferentially coexpress combinations of Trk receptors or which uniquely express one specific receptor. These data suggest that experimental strategies must be defined to explore the physiological significance of Trk receptor coexpression and of their possible function in previously unidentified sites of activity including motor neurons and non-neural cells. Finally knowledge of the sites of Trk-gene expression will provide important insights for the analysis of mice that lack Trk-gene function due to mutations generated by homologous recombination in embryonic stem cells.

We thank Dan Soppet for the mouse trkB sequence and for many helpful discussions. We are grateful to the members of the Parada lab for their support and, in particular, to James Pickel and Dan Soppet for their advise and critical reading of the manuscript. We also thank B. Cho and M. Barnstead for technical assistance and Richard Fredrickson for his skillful assistance with artwork and Cindy Fitzpatrick for manuscript preparation. This research was supported by the National Cancer Institute, DHHS, under contract NO1-CO-74101 with ABL.