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The Journal of Neuroscience, January 15, 1999, 19(2):775-782
Evidence for a Role of Truncated trkC Receptor Isoforms in
Mouse Development
Mary Ellen
Palko,
Vincenzo
Coppola, and
Lino
Tessarollo
Neural Development Group, Advanced Bioscience Laboratories-Basic
Research Program, National Cancer Institute-Frederick Cancer
Research and Development Center, Frederick, Maryland 21701
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ABSTRACT |
The trkC locus encodes several receptors for
neurotrophin-3, including the well studied full-length tyrosine kinase
isoform, in addition to receptor isoforms lacking the kinase active
domain. TrkC receptors are widely expressed throughout mouse
development in many different organs. To investigate the function of
truncated receptors in vivo and to identify cell types
that are biologically responsive to this gene product, we have
overexpressed a physiological truncated trkC isoform in the mouse. Mice
overexpressing this receptor develop to term but die in the first
postnatal days. High levels of transgene expression result in severe
developmental defects in the peripheral nervous system and in the
heart. The severity of neuronal losses observed in these animals
suggests that truncated receptors may act by sequestering neurotrophin, thus, closely relating this mouse model to the
neurotrophin-3-deficient one. Lower levels of exogenous
truncated receptor in transgenic mice result in a more modest phenotype
and, in some neuronal populations, do not cause neural deficits. Taken
together, these data suggest that truncated trkC receptor isoforms may
have modulatory functions in development.
Key words:
trkC; truncated receptor; development; neurotrophin; trk
receptors; transgenic
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INTRODUCTION |
Neurotrophins and their receptors
are highly expressed throughout mouse ontogenesis in both neuronal and
non-neural cell types (for review, see Tessarollo, 1998 ). These
families of genes play crucial roles in the development and function of
the nervous system (Korsching, 1993; Snider, 1994; Bothwell, 1995;
Lewin and Barde, 1996). Neurotrophin activation of trk tyrosine kinase
receptors results in the initiation of signal transduction cascades,
including the ras pathway, and the induction of proliferation,
survival, and differentiation of peripheral nervous system (PNS) and
CNS neurons. Many functions of these tyrosine kinase receptors
and their signaling mechanisms have been described (Segal and
Greenberg, 1996). The trkB and trkC loci additionally encode, by
alternative splicing, receptor isoforms that lack the catalytic kinase
domain (Klein et al., 1990; Middlemas et al., 1991; Tsoulfas et al., 1993; Valenzuela et al., 1993; Garner and Large, 1994). The unique intracellular domains of truncated forms of both trkB and trkC are
highly conserved in human, mouse, rat, and chicken (Garner and Large,
1994; Baxter et al., 1997). The high conservation of these
intracellular domains has raised the notion that truncated isoforms may
have signaling ability.
Indeed, recent data obtained in vitro have shown that
truncated trkB receptors can induce a ligand-mediated increase in the release of cellular acidic metabolites (Baxter et al., 1997), and that
activated truncated trkC receptors can promote differentiation of
chicken neural crest cells (Hapner et al., 1998). Mice lacking all trkC
isoforms, including the truncated ones, have a more severe phenotype,
including reduced viability and a more pronounced neuronal deficit,
compared with mice lacking only the full-length tyrosine kinase
receptors, suggesting a positive role for truncated trkC receptors
in vivo (Klein et al., 1994; Minichiello et al., 1995; Tessarollo et al., 1997). However, no signaling molecules downstream of
truncated receptors have been identified to date.
The presence of abnormalities in mice heterozygous for a specific
neurotrophin null allele has demonstrated that precise levels of
neurotrophins are critical for normal mouse development (Ernfors et
al., 1994; Korte et al., 1995; Donovan et al., 1996; Erickson et al.,
1996; Chen et al., 1997). Expression analysis of truncated trkB
receptors and results obtained from coexpressing full-length and
truncated trkB receptors have suggested that truncated receptors could
act by sequestering excessive neurotrophins or as naturally occurring
dominant negative elements of the full length receptor (Biffo et al.,
1995; Eide et al., 1996; Ninkina et al., 1996). However, there are as
yet no in vivo data available in support of such
nonsignaling functions of truncated receptors.
To investigate functions of truncated trkC receptors in vivo
and to identify cell types that respond to these receptors, we have
overexpressed a truncated trkC isoform in mouse. Ectopic expression of
this truncated receptor results in perinatal lethality. Severe
deficiencies were observed in sites that normally express endogenous
truncated trkC receptors, such as the heart and the PNS (Hiltunen et
al., 1996; Menn et al., 1998). The effects on PNS development vary with
the amount of transgenic mRNA expressed, suggesting that truncated trkC
receptors may have pleiotropic modulatory functions in
vivo.
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MATERIALS AND METHODS |
Generation of transgenic mice. A trkC truncated
fusion gene containing the human B-actin promoter and the SV40 poly(A)
signal was constructed by inserting a 2.2 kb rat trkC truncated cDNA (Tsoulfas et al., 1993) into the pHB APr-1 vector (Gunning et al.,
1987). This gene was microinjected into fertilized (C57BL/6J × C3H/HeJ) F2 mouse eggs as previously described (Osborn et al., 1987).
RNase protection analysis. 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 a ribonuclease protection assay kit
(Ambion). A genomic TrkC-specific probe that spans nucleotides 1238-1424 of the extracellular domain (trkC-all) of the rat
sequence (Tsoulfas et al., 1993) was used to generate an antisense RNA probe used in RNase protection analysis to detect all trkC isoforms. The trkC truncated (TK )-specific probe was obtained by subcloning a
region of the rat trkC truncated receptor (nucleotides 1732-1909; Tsoulfas et al., 1993). The glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)-specific probe (Ambion) was included in
the same reaction as a mean of assessing relative levels of RNA present
in each hybridization.
Histological techniques and neuronal cell counts. After
microinjection, transgenic mice were checked for the presence of the transgene. Mutant and wild-type littermates were killed by
decapitation, and the bodies and heads were immediately fixed in
Bouin's fixative overnight. The next day, after rinsing for a few
hours in water, the heads and spinal cords were transferred to 70%
ethanol and processed for paraffin embedding. For neuronal counts, the
heads of one transgenic littermate and one-wild type littermate were embedded in the same block, sectioned sagitally at 5 µm, and
Nissl-stained with 0.1% cresyl violet. To assure unbiased analysis,
neuronal counts were performed in a blinded manner.
Immunohistochemical neurofilament staining. For
immunohistochemistry, embryos fixed in 4% paraformaldehyde were
bleached for 1 hr on ice in 3:1 methanol/hydrogen peroxide (30%
solution in water), rehydrated, washed in PBS containing 0.1%
Tween 20 (PBT), treated with proteinase K (20 µg/ml) for 5 min, and
refixed. Embryos were then incubated overnight at 4°C in 1% serum
and mouse monoclonal anti-neurofilament 160 antibody (1:1000; Sigma
Chemical Co., St. Louis, MO), washed for 1 d in PBT, and incubated
overnight at 4°C in PBT containing 1% serum and
peroxidase-conjugated rabbit anti-mouse Ig (1:1000; Sigma). The color
reaction was developed by incubation in 0.3 mg/ml diaminobenzidine,
0.03% H2O2, and 0.05% NiCl2 and stopped by washing in PBT.
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RESULTS |
Generation of truncated trkC transgenic mice
The trkC gene is expressed in many tissues and at almost all
stages of development (Tessarollo et al., 1993; Tsoulfas et al., 1993;
Lamballe et al., 1994). The specific pattern of expression of the
different isoforms encoded by this locus is not known. However,
truncated trkC receptors are expressed in both the PNS and CNS and,
although at lower levels, in non-neural tissues, including the
cardiovascular system (Tsoulfas et al., 1993; Hiltunen et al., 1996;
Menn et al., 1998) (Figure
1A). Thus, we have used the human  -actin promoter (Gunning et al., 1987) to drive the expression of a rat truncated trkC receptor that has a 36 amino acid
domain that is 100% conserved between rat and chicken (Tsoulfas et
al., 1993). Mouse and rat truncated trkC receptors are very conserved
and are biologically indistinguishable; therefore, we used a rat
truncated isoform to distinguish between the endogenous and the
transgenic truncated receptor (P. Tsoulfas, personal
communication) (Menn et al., 1998). RNase protection experiments
showed that indeed transgenic truncated trkC (Tg trkC TK) RNA could
be detected at high levels in both neural and non-neural tissues (Fig.
1A,B).
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Figure 1.
Expression of trkC receptors in truncated isoform
transgenic animals. A, RNase protection analysis
performed with a probe specific for the truncated trkC receptor
(trkC TK) reveals higher expression of endogenous trkC
TK in the brain compared with the leg in wild-type animals. Levels of
expression of trkC TK gene in transgenic animals are also shown.
B, RNase protection analysis performed with an
extracellular rat trkC antisense cDNA probe (all-trkC)
that detects all isoforms of trkC RNA including the transgenic one
(Tg). A polymorphism allows the distinction between the
transgenic (bottom trkC band) from the wild-type mRNA
(top band). C, High levels of expression
of the transgene are associated with early postnatal lethality. RNA
from legs of mice that were moribund at the indicated age (P0,
P1, P3) was analyzed by RNase protection for the
presence of the transgene (Tg TrkC TK) by using the
all-trkC probe. The endogenous trkC transcripts are also detected
(Wt TrkC). Note that the time of death correlated with
particular levels of expression of the transgene. In all experiments, a
GAPDH antisense probe was included in the hybridization mixture to
control for RNA loading.
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Expression analysis of transgenic animals indicated that overexpression
of this gene, even at high levels, did not impair embryo viability,
because embryos could develop to term. However, transgenic mice fail to
thrive when compared with their wild-type littermates, and several
newborn pups died immediately after birth (Fig.
2). Tissue analysis from mice that died
immediately after birth revealed that they had the highest copy number
of transgenic DNA (data not shown) and that they had the highest levels
of the Tg trkC TK transcript when compared with other transgenic
animals that could live up to 9 d [postnatal day 9 (P9); Fig.
1C]. Although we do not know the cause of death of these
mice, their dusky appearance and immediate death suggested cardiac
deficits as a potential cause. Histological evaluation of trkC TK
transgenic mouse hearts revealed abnormalities in the valvular
architecture and cardiac septation. Some of the defects, including
abnormally thickened aortic and semilunar valves and atrial septal
defects, are shown in Figure 3. These
findings strongly suggest that cardiac dysfunction similar to that
observed in the neurotrophin-3 (NT-3)- or trkC-deficient animals could
play a role in the perinatal lethality associated with overexpression
of the trkC truncated receptor (Donovan et al., 1996; Tessarollo et
al., 1997).
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Figure 2.
Mice overexpressing a truncated trkC receptor
fail to thrive and display a behavioral phenotype characteristic of
proprioception deficiency. A, A transgenic animal
(left) is compared with a wild-type littermate
(right). B, Transgenic mice display
severe movement impairments.
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Figure 3.
Cardiac defects in truncated trkC transgenic
animals. A, Section of a newborn trkC TK transgenic
heart demonstrating an atrial septal defect (asd),
markedly dilated atria with decreased trabeculations, and large septum
primum. Magnification, 50×. B, Higher magnification of
A with asd, septum primum (sp), and
septum secundum (ss) indicated by arrows.
Magnification, 100×. C-F, Sections through the
pulmonic (C, D) and aortic (E, F)
valves of a normal (C, E) and a transgenic (D,
F) mouse heart. Magnification, 100×. Note the
thickening of the pulmonic (pv) and aortic
(av) valve leaflets (arrows) of the
mutant animal compared with the control littermate. rv,
Right ventricle; lv, left ventricle; ra,
right atrium; la, left atrium; pa,
pulmonic artery; ao, aorta; tv, tricuspid
valve; mv, mitral valve.
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Peripheral nervous system deficiencies in mice expressing high
levels of trkC truncated receptor
Newborn transgenic mice exhibited deficiencies in their movements
and postures that resembled the proprioception defects observed in trkC
and NT-3 mutant animals (Fig. 2). To address whether these animals had
PNS defects, we evaluated selected sensory and sympathetic neuronal
populations of mice with the most severe phenotype (moribund at P0)
compared with wild-type littermates. Severe reductions were observed in
the trigeminal, geniculate, vestibular, and cochlear ganglia, whereas
the petrosal-nodose ganglia displayed only a modest reduction. Also
the superior cervical ganglion showed substantial neuronal losses when
compared with controls. Thus, neuronal deficits in the transgenic
animals are present in both sensory and sympathetic neuronal
populations similar to the NT-3 mutant mouse (Table
1). The development of an NT-3-like
phenotype suggests that truncated receptors act by sequestering soluble
neurotrophins. If truncated trkC receptor would act exclusively as
dominant negative inhibitors of the full-length kinase active isoforms,
the phenotype should be more similar to trkC-deficient mice. However,
in contrast to mice lacking either the kinase active or all trkC
receptors, the trkC TK-overexpressing mice develop severe sympathetic
neuronal losses like the NT-3 knock-out mice (Table 1) (Tessarollo et al., 1997).
PNS deficiencies arise before neuronal target innervation
We then asked at what stage of development the PNS of transgenic
mice expressing high levels of the truncated trkC gene exhibited cell
losses. Whole-mount analysis using an antibody that detects neurofilament 160 protein suggested normal development of the PNS in
embryonic day 11 (E11) transgenic embryos when compared with wild-type
littermates (Fig.
4A,B). However, by E13,
reductions in the size of dorsal root ganglia (DRG) could be observed
(Fig. 4C,D). These results suggest that the deficits
observed in newborn animals develop between E11 and E13 before neuronal
target innervation occurs. Interestingly, NT-3 mutant mice also display
reductions in trigeminal and DRG neurons at this time of development
(Tessarollo et al., 1994; Fariñas et al., 1996; Wilkinson et al.,
1996). Therefore, the onset of neuronal deficits in the DRG of our
transgenic mice is comparable with that observed in NT-3 null mutant
mice.
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Figure 4.
Neuronal losses in mice overexpressing a truncated
trkC receptor occur between embryonic days 11 and 13. The PNS of
control (A) and transgenic
(B) mice at day E11 of embryonic development was
visualized by immunohistochemistry using an anti-neurofilament 160 antibody. No significant differences were detected between the PNS of
the two embryos at this developmental stage. By E13, transgenic
(D) dorsal root ganglia (drg,
arrow) are reduced in size when compared with wild-type drg
(C, arrow) in sections at the same
thoracic level. Cranial ganglia are indicated: TG,
trigeminus with ophtalmic (op), maxillary
(mx), and mandibular (md) branches;
g, geniculate; V, vestibulocochlear;
p, petrosal; n, nodose; O,
optic nerve; SC, spinal cord; lu,
lung.
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PNS in mice expressing low levels of the trkC
truncated receptor
Truncated trkC receptor expressed at high levels may act by
neutralizing neurotrophins, as suggested by the fact that these transgenic mice develop a phenotype similar to that caused by NT-3
deficiency. As a result, this may mask the potential effects of a
signaling function by the overexpressed truncated receptor in
responsive cells. Thus, we analyzed mice with lower levels of the
transgene. Such animals displayed a less-severe behavioral phenotype
and prolonged viability (up to 9 d). Histopathological analysis of
a variety of organs from mice that died at P9 showed no obvious
abnormalities (data not shown). Neuronal counts of four sensory ganglia
from this group of animals revealed reductions in the geniculate and
the cochlear ganglia, although less severe than in mice expressing
higher levels of the transgene. However, neuronal losses were higher in
the petrosal-nodose ganglion. Surprisingly, we even observed a mild
increase in the vestibular neuronal population (Table
2). To investigate the specificity of
this effect, we analyzed additional transgenic litters. Mice that died
at P0-P1 expressed high levels of the truncated trkC transgene as
reported above. However, by P3 we were able to identify animals that
displayed weight losses but only mild behavioral phenotypes. To avoid
losing these mice, we killed them for analysis of their peripheral
nervous system at P3 (Table 3). We again
observed neuronal losses in almost all investigated ganglia compared
with controls. However, they were less severe than in the transgenic
pups analyzed at P9. Most importantly, we observed again an increase in
neuronal counts of the vestibular ganglia. This increase is not
statistically significant; however, the reproducibility of this result
suggests that this trkC truncated effect on the vestibular ganglia may be biologically significant.
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Table 2.
Neuronal cell counts in sensory populations of wild-type
and transgenic Tg trkC TK mice at postnatal day 9
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Table 3.
Neuronal cell counts in sensory populations of wild-type
and transgenic Tg trkC TK mice at postnatal day 3
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DISCUSSION |
Ectopic expression of truncated trkC receptors interferes with
normal mouse development
The function of truncated trk neurotrophin receptor
isoforms (lacking a functional cytoplasmic kinase domain) is still
unknown. Expression of truncated trkC receptors in cell lines, e.g., of the sympathoadrenal (PC12) and mesoderm (NIH3T3) lineages, failed to
induce any apparent change in the cell cycle or cellular morphology (Tsoulfas, personal communication). This result suggested that either
these cells are lacking the signal transduction machinery downstream of
this receptor, or the mechanism of action cannot be revealed
because of the limitations of these in vitro systems. One
goal of this study was to identify cell types responsive to truncated
receptor functions in vivo. The truncated trkC receptor isoform that we used to generate transgenic mice is widely expressed in
normal mouse development (Tsoulfas et al., 1993; Valenzuela et al.,
1993; Hiltunen et al., 1996; Menn et al., 1998). In particular, it has
been found throughout the CNS and PNS, including the DRGs and cranial
ganglia (Menn et al., 1998). Furthermore, its expression has also been
detected in several organs outside the nervous system, including the
cardiovascular system and mesodermal derivatives such as muscles
(Tsoulfas et al., 1993; Hiltunen et al., 1996; Menn et al., 1998) (Fig.
1A). Here we have shown that mice overexpressing this
truncated trkC receptor at high levels die perinatally because of
severe cardiac defects and PNS abnormalities. All the tissues for which
we report defects normally do express endogenous truncated trkC
receptors, suggesting specific functions for these receptors in the
development of those organs (Hiltunen et al., 1996; Menn et al., 1998).
Despite the ubiquitous expression of trkC TK in the transgenic
animals, histological analysis to date has failed to detect
abnormalities in other organs (data not shown), suggesting that
truncated receptors do not affect cell survival, proliferation, or
differentiation per se.
NT-3-deficient mice and trkC TK transgenic mice develop similar
PNS and cardiac deficiencies
Expression analysis in rodents and chicken during development has
shown the presence of truncated trkB and trkC receptors in non-neuronal
tissues, and it has been suggested that these receptors act by limiting
neurotrophic factors available to bind kinase active receptors (Klein
et al., 1990; Tsoulfas et al., 1993; Valenzuela et al., 1993; Garner
and Large, 1994; Biffo et al., 1995). In fact, truncated trkB receptors
in the ventricular zone of the spinal cord and the ependimal layer of
ventricles in the brain provide a barrier to the diffusion of
intraventricular injections of brain-derived neurotrophic factor,
consistent with the idea that this type of receptor could remove
neurotrophins (Yan et al., 1994; Armanini et al., 1995; Biffo et al.,
1995). However, no data are available supporting such function during development. Here, we show that ubiquitous high-level expression of a
truncated trkC isoform in mouse causes cardiac defects that resemble
those of mice lacking the NT-3 or trkC gene (Donovan et al., 1996;
Tessarollo et al., 1997). However, NT-3-deficient mice develop more
severe neuronal losses than trkC-deficient mice (Tessarollo et al.,
1997; Table 1). Overexpression of the trkC TK transgene in the mouse
results in PNS neuronal losses that most resemble the NT-3 null
phenotype (Table 1). Furthermore, the establishment of neuronal
deficits occurs between E11.5 and E13.5, as reported for the NT-3
mutant mice (Fariñas et al., 1996; Wilkinson et al., 1996). These
results support the hypothesis that truncated receptors function in
neutralizing NT-3. However, we cannot rule out that the truncated
receptor also inhibits the full-length tyrosine kinase receptor
directly by acting as a dominant negative receptor (Eide et al., 1996;
Ninkina et al., 1996). In fact, the observed co-expression of truncated
and full-length trkC receptors in developing peripheral sensory neurons
suggests such a role (Menn et al., 1998). However, if this putative
dominant negative function was the only mechanism of action by
truncated isoforms, the phenotype of the transgenic mice would never
be more severe than the one seen in trkC-deficient mice, which
is not the case. Additionally, truncated trkC receptors could function as dominant negative inhibitors of trkA or trkB receptors. However, heterodimerization of different trk receptors has not been demonstrated in vivo, and none of the defects observed in the trkC TK
transgenic animal is characteristic of trkA or trkB null mutants (Klein
et al., 1993; Smeyne et al., 1994). For example, the proprioception deficits observed in the transgenic mice (Fig. 2) suggest that NT-3-trkC is the main ligand-receptor system affected in the trkC TK overexpressing mice (Tessarollo, 1998). Furthermore, trkA and trkB
full-length receptors are not expressed in the heart during development
(Hiltunen et al., 1996). Thus, only inhibition of the full-length trkC
receptors by truncated isoforms could account for the defects observed
in the transgenic animals.
Modulatory effects of trkC TK isoforms on PNS development
A noncatalytic receptor that controls NT-3 access to specific
neuronal targets and that regulates the level of neurotrophins interacting with kinase active trk receptors can provide the organism with additional ways to increase functional diversity. Such a mechanism
is even more relevant for a neurotrophin such as NT-3, which may have
the ability to activate all trk family members and which is widely
distributed during development (Bothwell, 1995; Lewin and Barde, 1996;
Tessarollo, 1998). However, it seems unlikely that an organism would
need different types of truncated receptors just to regulate
neurotrophin levels during ontogenesis. The high degree of conservation
of intracellular domains of truncated receptors among species suggests
other functions, such as signaling, for these receptors (Klein et al.,
1990; Middlemas et al., 1991; Valenzuela et al., 1993; Garner and
Large, 1994).
Indeed recently, Hapner et al. (1998) have shown that ectopic
overexpression of truncated trkC receptors in neural crest cells in vitro, although lacking any mitogenic effect, induces
neural differentiation with participation of p75 LANR. This
differentiation response is elicited only by a small subset of cells
and is induced exclusively by truncated trkC receptors, which contain
the evolutionarily conserved 39 amino acid intracellular domain,
suggesting that specific intracellular domains of truncated receptors
are required for activation of the differentiation pathway (Hapner et
al., 1998). Furthermore, it has been reported that truncated trkB
receptors can induce a ligand-mediated increase in the rate of acidic
metabolites released from the cells, a common physiological consequence
of many signaling pathways. Again, a conserved, truncated,
isoform-specific domain is required to induce this response (Baxter et
al., 1997). These data, although not providing information on the type
of pathway activated by truncated receptors, do represent very
intriguing results and suggest that both trkB and trkC truncated
receptors activate intracellular signaling pathways.
The variations observed in the PNS neuronal populations of trkC TK
transgenic mice, including a tendency to an increase in the number of
neurons in the vestibular ganglia, are reciprocally related to the
level of expression of the transgene, suggesting a modulatory role of
truncated trkC on ganglia of the PNS (Tables 2, 3). The data are also
consistent with the finding that mice lacking the truncated trkC
isoforms, in addition to the full-length receptors, are missing 15%
more DRG and 20% more cochlear ganglia neurons compared with mice
lacking only the tyrosine kinase isoforms (Minichiello et al., 1995;
Tessarollo et al., 1997). The observation that changes in neuron
numbers are limited in both transgenic (only a 10% increase in neurons
in the vestibular ganglia) and knock-out animal models [20% fewer
cochlear and DRG neurons in mice lacking both kinase and truncated trkC
receptors compared with the ones lacking only the kinase isoforms
(Minichiello et al., 1995; Tessarollo et al., 1997)] indicates that
only a subset of neurons is responsive to the presence of truncated
receptors. Indeed, the expression of endogenous truncated trkC
receptors is restricted to a subset of DRG neurons at some stages of
embryonic development. The identity and functional role of these
neurons remain to be determined (Menn et al., 1998).
Different mechanisms, such as the ability of truncated receptors to
remove ligand, inhibit the kinase active isoforms, or promote survival
and/or differentiation, may not be mutually exclusive. The cellular
environment may determine the mechanisms by which truncated receptors
will exert their functions. Mice with conditional targeted alleles for
truncated trkC receptor mutations may serve as tools to address
specific signaling functions in vivo. Furthermore, such
mouse strains would enable the investigation of a role of truncated
receptors in the maturation process of the nervous system, an analysis
that is now precluded by the limited life span of mice in which NT-3
and trkC receptor levels have been altered during development.
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FOOTNOTES |
Received July 13, 1998; revised Oct. 7, 1998; accepted Oct. 28, 1998.
This work was supported by the National Cancer Institute under contract
with Advanced Bioscience Laboratories. We thank Esta Sterneck, Eileen
Southon, and Barbara Hempstead for critical reading of this manuscript;
Frances Lefcort and Pantelis Tsoulfas for sharing unpublished results
and for useful discussions; Debbie Swing, Susan Reid, and Janet
Blair-Flynn for excellent technical assistance; and Carmen Birchmeier
and Alistair Garratt for the whole-mount immunohistochemistry protocol.
Correspondence should be addressed to Dr. Lino Tessarollo, ABL-Basic
Research Program, National Cancer Institute-Frederick Cancer Research
and Development Center, P.O. Box B, Frederick, MD 21702.
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Abstract
Introduction
