1027
Development 120, 1027-1033 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
p75-deficient embryonic dorsal root sensory and neonatal sympathetic
neurons display a decreased sensitivity to NGF
Kuo-Fen Lee1, Alun M. Davies2 and Rudolf Jaenisch1
1Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Nine Cambridge
Center, Cambridge, MA 02142, USA
2School of Biological and Medical Sciences, Bute Medical Buildings, University of St. Andrews, St. Andrews, Fife KY16 9AJ,
Scotland, UK
SUMMARY
To understand the role of low-affinity neurotrophin
receptor p75 in neural development, we previously
generated mice carrying a null mutation in the p75 locus
(Lee, K. F., Li, E., Huber, L. J., Landis, S. C., Sharpe, A.
H., Chao, M. V. and Jaenisch, R. (1992) Cell 69, 737-749).
To elucidate the mechanisms leading to deficits in the
peripheral nervous system in p75 mutant mice, we have
employed dissociated cultures to examine the responses of
p75-deficient dorsal root ganglion (DRG) and superior
cervical ganglion (SCG) neurons to different neurotrophins. We found that p75-deficient DRG and SCG
neurons displayed a 2- to 3-fold decreased sensitivity to
NGF at embryonic day 15 (E15) and postnatal day 3 (P3),
respectively, ages that coincide with the peak of naturally
occurring cell death. Furthermore, while p75-deficient E15
DRG neurons did not change their response specificity to
BDNF, NT-3, and NT-4/5, P3 SCG neurons became more
responsive to NT-3 at higher concentrations (nanomolar
ranges). These results may help explain the deficits in the
peripheral nervous system in p75 mutant mice and provide
evidence that p75 can modulate neurotrophin sensitivity in
some neurons.
INTRODUCTION
affinity binding sites for NGF and other neurotrophins
(Godfrey and Shooter, 1986; Rodrigeuz-Tébar and Barde,
1988; Rodriguez-Tébar et al., 1992; Sutter et al., 1979). Two
classes of cell surface receptors have been described: all neurotrophins bind to p75 with low affinity (Johnson et al., 1986;
Radeke et al., 1987; Ernfors et al., 1990; Rodriguez-Tébar et
al., 1990; Hallböök et al., 1991; Rodriguez-Tébar et al., 1992)
and to specific tyrosine receptor kinases (trkA, trkB, and trkC)
with higher affinity (Barbacid et al., 1991; Cordon-Cardo et
al., 1991; Kaplan et al., 1991; Klein et al., 1991a,b; Lamballe
et al., 1991; Soppet et al., 1991; Squinto et al., 1991; Chao,
1992). Experiments utilizing fibroblasts expressing the individual trks indicate that trkA is the cognate receptor for NGF,
trkB for BDNF and NT-4/5, and trkC for NT-3, although there
is a certain degree of ligand-receptor promiscuity (Chao, 1992;
Meakin and Shooter, 1992). In contrast to the wide expression
of p75 (Bothwell, 1991), trks have a more restricted pattern of
expression (Klein et al., 1990; Martin-Zanca et al., 1990;
Ernfors et al., 1992) and are essential for neurotrophin signal
transduction. Recently, Klein et al. (1993) showed that mice
homozygous for a mutation in the trkB receptor gene exhibit
deficits in central and peripheral nervous system.
The contribution, if any, of p75 to neurotrophin signal transduction in neurons is not clear. There is a major controversy
over whether high-affinity NGF binding and signal transduction are dependent soley on the expression of trkA or whether
co-expression of trkA and p75 is required. While Klein et al.
Nerve growth factor (NGF; Levi-Montalcini, 1987) is a
member of a gene family of neurotrophic factors (neurotrophins) that includes brain-derived neurotrophic factor
(BDNF; Barde et al., 1982; Leibrock et al., 1989), neurotrophin-3 (NT-3; Hohn et al., 1990; Maisonpierre et al.,
1990; Rosenthal et al., 1990; Ernfors et al., 1990; Jones and
Reichardt, 1990) and neurotrophin-4/5 (NT-4/5; Berkemeier et
al., 1991; Hallböök et al., 1991; Ip et al., 1992). In vitro, the
neurotrophins promote the survival of distinct populations of
neurons from the peripheral and central nervous systems
(Barde, 1989). Treatment of embryos with NGF and BDNF
prevents naturally occurring cell death in certain populations
of neurons that would otherwise occur due to the limited availability of NGF and BDNF in the target fields of these neurons
(Hofer and Barde, 1988; Levi-Montalcini, 1987). In addition
to their survival activity, NGF and probably other neurotrophins exert diverse effects on the nervous system
including promoting neural crest and neural tube cell proliferation (Cattaneo and McKay, 1990; Kalcheim et al., 1992),
sensory neuron differentiation and maturation (Wright et al.,
1992), synaptogenesis (Garofalo et al., 1992), dendritic
arborization (Snider, 1988), axonal extension (Campenot,
1977) and modulation of neuropeptide gene expression
(Lindsay and Harmar, 1989).
Neurotrophin-responsive neurons possess both low and high
Key words: p75, neurotrophins, survival, mouse
1028 K.-F. Lee, A. M. Davies and R. Jaenisch
(1991a) and Cordon-Cardo et al. (1991) have provided
evidence that trkA alone is sufficient for both high-affinity
binding and NGF signaling, results from Hempstead et al.
(1991) suggest that both p75 and trkA are required for the
formation of high-affinity binding. Unlike the trks, p75 does
not contain a tyrosine kinase domain although sequence
analysis has identified a G-protein binding-like domain in the
intracellular region of p75 (Feinstein and Larhammar, 1990).
Whether p75 can mediate signal transduction alone through
this domain remains to be tested. Furthermore, although all
neurotrophins bind p75 with similar affinity, their dissociation
rates are markedly different; NGF and NT-3 being the fastest
and slowest, respectively (Rodriguez-Tébar et al., 1992). This
difference raises the possibility that p75 plays a role in discriminating among neurotrophins, thereby tuning the specificity of neurotrophin binding.
In addition to its potential role in neurons, p75 may serve a
variety of functions in non-neuronal cells. The observation that
p75 is expressed on Schwann cells in regenerating peripheral
nerves (Johnson et al., 1988) and in target fields prior to and
during their innervation (Wyatt et al., 1990) has led to the suggestion that it may function to concentrate and present the neurotrophins to the high-affinity receptors on the growing axons.
Moreover, in vitro experiments using antisense oligonucleotides to prevent translation of p75 mRNA suggest that p75
plays a role in kidney morphogenesis (Sariola et al., 1991) and
recent results suggest a role for p75 in neuronal patterning
through its influence on the migration of developing Schwann
cells (Anton and Matthew, 1992).
To elucidate the role of p75 in neural development, we previously generated mutant mice carrying a null mutation in the
p75 locus by gene-targeting in embryonic stem cells (Lee et
al., 1992). Mutant mice are viable and exhibit markedly
decreased cutaneous sensory innervation by calcitonin-gene
related peptide and substance P immunoreactive fibers in the
periphery, which is associated with decreased heat sensitivity
and the development of skin ulcerations in the extremities (Lee
et al., 1992). Mutant animals also display tissue-specific
deficits in the sympathetic innervation (Lee et al., unpublished
results). For example, while the sympathetic innervation from
SCG to iris is normal, the pineal gland lacks sympathetic innervation. Furthermore, sympathetic innervation of the sweat
glands is selectively depleted in some footpads.
The phenotype of the peripheral nervous system in p75
mutant animals could result from deficits in the neurons,
Schwann cells, or target tissues. As a first step in elucidating
the role of p75 in the neurotrophin signal transduction in
neurons, we examined neurotrophin sensitivity in p75-deficient
DRG and SCG neurons. Our results show that p75-deficient
DRG and SCG neurons display a decreased sensitivity to NGF
during the period of naturally ocurring cell death. Because the
concentration of NGF in the target field is limiting, decreased
sensitivity would result in excessive loss of DRG and SCG
neurons in vivo. This finding indicates that p75 plays a role in
mediating neurotrophin function in some neurons and can
partially explain the phenotype of p75 mutant mice.
MATERIALS AND METHODS
Animals
Embryos were obtained from timed-pregnant mice and staged by the
criteria of Theiler (1972; day of the presence of vaginal plug = E0).
In most cases, embryos homozygous for the p75 mutation were
obtained from crosses between outbred mutant animals on a mixed
background of inbred strains 129 and Balb/c (for details, see Lee et
al., 1992). Control embryos were obtained from crosses from inbred
Balb/c mice. To determine whether the response of neurons was
dependent on p75 gene dosage, mice heterozygous for the p75
mutation were intercrossed. The genotypes of individual embryos
were determined by Southern blotting analysis (Lee et al., 1992).
Neuron cultures
DRGs dissected from embryos were collected in L15 medium (Gibco)
and were incubated for 7 minutes at 37oC with 0.05% trypsin (Worthington) in calcium- and magnesium-free Hanks balanced salt
solution (HBSS). Trypsin was neutralized by adding 10 ml of F12
medium containing 10% heat-inactivated horse serum (Gibco). After
washing once with the same medium, the ganglia were gently triturated with a fire-polished Pasteur pipette to obtain a suspension of
single cells and were plated at a density of 200-400 neurons per 35
mm dish (Nunc), which had been coated overnight at room temperature with poly-DL-ornithine (Sigma; 0.5 mg/ml, in 0.15 M borate
buffer, pH 8.3) and subsequently with laminin (BRL; 20 µg/ml) for
4 hours at 37°C. The neurons were cultured in F14 medium (Imperial
Laboratories Ltd., UK) supplemented with 2 mM glutamine, 0.35%
bovine albumin (Pathocyte-4, ICN), 60 ng/ml progesterone, 16 µg/ml
putrescine, 400 ng/ml l-thyroxine, 38 ng/ml sodium selenite, 340
ng/ml tri-iodo-thyronine, 60 µg/ml penicillin and 100 µg/ml streptomycin.
For postnatal SCG neurons, ganglia were dissected and cleaned free
of connective tissue using tungsten needles and were incubated for 30
minutes with a solution of collagenase (Worthington; 1 mg/ml) and
dispase (Boehringer-Mannheim; 5 mg/ml) in HBSS. The ganglia were
washed twice with HBSS and were incubated with 0.25% of trypsin
(Gibco) in HBSS for 30 minutes at 37°C. The ganglia were triturated
and cultured as described above for DRG neurons.
In each experiment, triplicate cultures were established for all conditions. 6-12 hours after plating, the number of attached neurons
within a 12×12 mm square in the centre of each dish was counted to
determine the initial number of neurons for the experiment. After 48
hours incubation, the percentage of neurons surviving in the presence
or absence of different neurotrophins was determined by counting the
neurons that were phase-bright and neurite-bearing in the same 12×12
mm area in each dish. The results are expressed as a percentage of
initial number of attached neurons.
RESULTS
p75-deficient DRG neurons have a decreased
sensitivity to NGF
To examine whether p75-deficient DRG neurons have an
altered response to NGF during the mid to late embryonic
period when naturally occurring neuronal death occurs, dosereponse experiments were performed on cultures of DRG
neurons isolated from E15, E17, and E19 embryos. Virtually
no neurons survived in the absence of neurotrophins after 48
hours in culture. The NGF dose-response of wild-type DRG
neurons normally shifts to higher NGF concentrations with age
(as seen in Fig. 1; A. M. Davies, unpublished results). The concentration of NGF required to promote the survival of half of
the wild-type neurons increased from approximately 10 pg/ml
at E15 to 100 pg/ml at E19 (Fig. 1). When p75-deficient DRG
neurons from the same stage were examined, no difference in
response at saturating concentrations of NGF was observed (2
or 10 ng/ml). However, at sub-saturating concentrations of
p75-deficient DRG and SCG neurons 1029
E15 DRG
100
100
80
Percentage Survival
Percentage Survival
80
60
40
60
+/+
40
+/-/-
20
20
0
0.1
1
10
100
1000
10000
0
0.1
E17 DRG
10
100
1000
10000
NGF Concentration (pg/ml)
Fig. 2. Effect of gene dosage on NGF sensitivity in DRG neurons.
E14 DRG neurons of embryos from a cross between two mice
heterozygous for the p75 mutation were isolated for culture.
Genotypes of individual embryos were determined and were
matched with the survival results. The NGF dose responses of
trigeminal neurons from wild-type (filled circle), heterozygous (filled
triangle) and homozygous (open circle) embryos are shown. Cultures
were set up in triplicate for the neurons from each embryos, and the
combined mean and standard error of the mean are plotted.
100
80
Percentage Survival
1
60
40
20
0
0.1
1
0.1
1
10
100
1000
10000
10
100
1000
10000
E19 DRG
100
Percentage Survival
80
60
40
20
individual embryos obtained from a cross between mice heterozygous for the p75 mutation and were subjected to the
survival assay with NGF. The main advantage of this experiment is that the responses of neurons from the three different
genotypes could be compared at the same time. Moreover,
because the genotypes of individual embryos were not known
before obtaining the results of the neuronal cultures, these
results were obtained free of any potential observer bias. When
genotypes and survival results were compared, only a small
shift to higher concentrations was seen with neurons from heterozygous embryos (Fig. 2). In contrast, the dose-response
curve of p75-deficient DRG neurons was significantly shifted
towards higher concentrations compared with wild-type
neurons (Fig. 2), confirming the results in Fig. 1. These results
demonstrate that p75 plays a role in the survival response of
embryonic DRG neurons to NGF.
0
NGF Concentration (pg/ml)
Fig. 1. p75-deficient DRG neurons have decreased sensitivity to
NGF. Dissociated DRG neurons were obtained from E15, E17 and
E19 embryos and were cultured as described in Methods and
Materials. The percentage survival of wild-type (filled circles) and
p75-deficient neurons (open circles) is plotted against a range of
NGF concentrations (on a log scale). The mean and standard error of
triplicate cultures are shown for each concentration.
NGF, p75-deficient DRG neurons were two- to three-fold less
sensitive to NGF compared with wild-type neurons (Fig. 1AC). The most pronouced shift in the NGF dose-response was
seen in E15 cultures; the half-maximal NGF concentration was
10 pg/ml for wild-type neurons and 30 pg/ml for p75-deficient
neurons (P<0.05, t-test).
To determine if p75 gene dosage affects the response of
DRG neurons to NGF, E14 DRG neurons were isolated from
Neurotrophin specificity is unchanged in p75deficient DRG neurons
Experiments using avian DRG neurons have demonstrated that
distinct populations of neurons depend on different neurotrophins for survival in vitro (Davies et al., 1986; Hory-Lee
et al., 1993). In situ hybridization studies have revealed that
while individual trks are expressed in subsets of DRG neurons,
most, if not all, DRG neurons express p75 (Yan and Johnson,
1988). Recently, Rodriguez-Tébar et al. (1992) have suggested
that p75 plays a role in disciminating among neurotrophins
because different neurotrophins, although having similar
affinity, exhibit significantly different dissociation rates with
p75. For this reason we examined whether neurotrophin specificity was altered in p75-deficient DRG neurons. E15 DRG
neurons were isolated and cultured in the presence of NGF,
BDNF, NT-3, or NT4/5. As illustrated in Fig. 3, saturating concentrations of all four neurotrophins (NGF, BDNF, NT-3, or
NT-4/5) promoted the survival of a similar percentage of DRG
1030 K.-F. Lee, A. M. Davies and R. Jaenisch
100
120
100
Percentage Survival
Percentage Survival
80
60
40
20
80
60
40
20
0
0
BDNF
NT-3
NT-4/5
Fig. 3. p75-deficient DRG neurons have unaltered neurotrophin
specificity. E15 DRG neurons were cultured in the presence of
saturating concentrations (2 or 10 ng/ml) of NGF, BDNF, NT-3 or
NT-4/5. The mean and standard error of the neuron survival counts
in triplicate culture dishes are plotted.
neurons from both control and p75-deficient neurons. Thus,
p75 does not play a role in neurotrophin discimination in E15
DRG neurons at saturating concentrations.
p75-deficient postnatal SCG neurons have a
decreased sensitivity to NGF
SCG neurons undergo naturally occurring cell death during the
first week of postnatal life with the peak from P3 to P7 (Wright
et al., 1983). Most, if not all, SCG neurons express p75 and
trkA (Yan and Johnson, 1988; Ernfors et al., 1992; Schecterson and Bothwell, 1992) and depend on NGF for survival in
vitro (Levi-Montalcini, 1987). Because sympathetic innervation by SCG in some target tissues was decreased, we
examined the response of p75-deficient SCG neurons to
different neurotrophins. For this, P3 or P4 SCG neurons from
wild-type and p75 mutant animals were cultured in parallel in
the presence of NGF. Like p75-deficient embryonic DRG
neurons, postnatal SCG neurons from mutant animals
exhibited a small but significantly decreased sensitivity to NGF
(Fig. 4). Interpolation of the survival curves indicated that the
half-maximal concentration was significantly greater for p75deficient SCG neurons (150 pg/ml) than for wild-type neurons
(50 pg/ml) (P<0.05, t-test). This finding shows that p75 also
plays a role in enhancing the NGF sensitivity in postnatal SCG
neurons in contrast to SCG neurons isolated from embryos
(Davies et al., 1993).
p75-deficient SCG neurons are more sensitive to
NT-3
While most SCG neurons are dependent on NGF, NT-3 has
been shown to elicit neurite-outgrowth and support the survival
of small percentage of SCG neurons (Dechant et al., 1993).
Interestingly, low levels of trkC, the cognate receptor for NT3, were detected by in situ hybridization in all SCG neurons
although it was not known whether the signals represented the
full-length or truncated receptor (Ernfors et al., 1992). We
therefore studied if responsiveness to NT-3 was altered in p75deficient SCG neurons. Consistent with previous findings
(Dechant et al., 1993), the results in Fig. 5 show that a very
small percentage of SCG neurons was supported by NT-3 at 2
or 10 ng/ml. This is in marked contrast to the survival of the
10
100
1000
NGF Concentration (pg/ml)
10000
Fig. 4. p75-deficient SCG neurons become less sensitive to NGF.
SCG neurons were isolated from animals at postnatal day 3 or 4 (P3
or P4) and cultured as described in Methods and Materials. The
percentage survival of wild-type (filled circles) and p75-deficient
neurons (open circles) is plotted against a range of NGF
concentrations (on a log scale). The mean and standard error of
triplicate cultures are shown for each concentration.
120
100
Percentage Survival
NGF
80
60
40
20
0
0.01
0.1
1
10
100
NT-3 Concentration (ng/ml)
1000
Fig. 5. p75-deficient SCG neurons are more sensitive to NT-3. P3
SCG neurons were cultured in the presence of different
concentrations of NT-3. The percentage survival of wild-type (filled
circles) and p75-deficient neurons (open circles) is plotted against a
range of NT-3 concentrations (on a log scale). The mean and
standard error of triplicate cultures are shown for each concentration.
majority of SCG neurons with 10 ng/ml NGF (compare Figs 4
and 5). Although the survival of SCG neurons increased with
higher concentrations of NT-3, it had not reached a maximum
with NT-3 concentrations as high as 250 ng/ml. Surprisingly,
p75-deficient SCG neurons were more responsive to NT-3 than
control neurons at concentrations of NT-3 higher than 10 ng/ml
(Fig. 5). These results indicate that the p75 mutation results in
an increased sensitivity of postnatal SCG neurons at high concentrations of NT-3.
DISCUSSION
p75 mutant mice were used to investigate the role of p75 in the
p75-deficient DRG and SCG neurons 1031
survival of sensory and sympathetic neurons. Our results show
that p75-deficient embryonic DRG and post-natal SCG
neurons are 2- to 3-fold less sensitive to sub-saturating concentrations of NGF than wild-type neurons. This may help
explain the phenotype of p75 mutant mice. Because the concentration of NGF is limiting in the target fields of sensory and
sympathetic neurons during development, the decreased sensitivity of p75-deficient sensory and sympathetic neurons to
NGF may lead to excessive loss of neurons in DRG and SCG
in vivo. Consistent with this possibility, p75 mutant mice have
substantially reduced numbers of CGRP and SP immunoreactive nerve fibres in the periphery (Lee et al., 1992) and it has
been shown that SP-positive and CGRP-positive DRG neurons
respond to NGF (Kessler and Black, 1980; Otten et al., 1980;
Lindsay and Harmar, 1989). p75 mutant mice also have
decreased sympathetic innervation in sweat glands and the
pineal gland (Lee, unpublished results) and it is known that the
in vivo survival of SCG neurons is dependent on a supply of
NGF in the postnatal period (Levi-Montalcini, 1987).
Previously, we have shown that embryonic p75-deficient
trigeminal ganglion sensory neurons, like DRG neurons, are
less sensitive to NGF than wild-type neurons (Davies et al.,
1993). Trigeminal ganglion neurons are born in the midembryonic period and undergo naturally occurring cell death
between E12 and E18 in vivo (Davies and Lumsden, 1984).
Although the period of naturally occurring cell death for DRG
neurons in mouse has not been documented, these neurons are
also born in the mid-embryonic period (Sims and Vaughn,
1979) and likely undergo naturally occurring cell death during
the same period as trigeminal neurons. Therefore, the shift in
the survival response of p75-deficient DRG neurons to higher
NGF concentrations is observed at the time of naturally
occurring cell death. This shift may be correlated with the
expression of p75 and trkA mRNAs, both of which increase at
the onset of NGF responsiveness at E12 (Wyatt and Davies,
1993) and are maintained at similar levels throughout
embryonic development. In contrast to our present finding that
postnatal p75-deficient SCG neurons are less sensitive to NGF
than aged-matched wild-type neurons, our previous results
showed that embryonic SCG neurons from p75 and wild-type
mice respond similarly to NGF (Davies et al., 1993). This agerelated difference in the response of SCG neurons from p75deficient and wild-type mice may be related to the late development of sympathetic neurons and developmental changes in
the levels of p75 and trkA mRNAs in these neurons. SCG
neurons are born before birth but undergo naturally occurring
cell death only during the first postnatal week (Wright et al.,
1983). The level of trkA mRNA in embryonic SCG neurons
begins to increase with acquisition of NGF responsiveness at
E14, but in contrast to sensory neurons the level of p75 mRNA
remains low until the late fetal period before any substantial
increase is observed; at E17 the ratio of trkA mRNA to p75
mRNA is over six to one and by P3 this ratio has decreased to
less than two (S. Wyatt and A. M. Davies, unpublished data).
Thus, the decreased NGF responsiveness of sensory and sympathetic neurons in p75-deficient mice is observed only during
the stage of development when the level of p75 is normally
elevated, i.e., in sensory neurons during the mid to late
embryonic period and in SCG neurons during the postnatal
period.
TrkA is essential for the NGF signal transduction pathway
which is probably initiated by ligand-induced homodimerization (Jing et al., 1992). The hallmark of this pathway is phosphorylation of a succession of proteins that includes phospholipase C-g1 and MAP-2 kinase (Gomez and Cohen, 1991; Loeb
et al., 1992; Miyasaka et al., 1991; Vetter et al., 1991; Ohmichi
et al., 1992; Thomas et al., 1992; Wood et al., 1992). Activation of this pathway results in the expression of a variety of
gene products that are required for neuronal survival and morphological differentiation. Because p75 does not contain a
tyrosine kinase domain, its role in neurotrophin signal transduction is not clear. Our results, however, indicate that p75 is
involved in NGF sensitivity in DRG and SCG neurons. The
mechanism(s) by which p75 increases NGF sensitivity in these
neurons is not clear. There are at least three possibilities that
are not mutually exclusive. First, it is known that NGFsensitive DRG and SCG neurons possess a 10-fold higher
number of low-affinity receptors than high-affinity receptors.
The low-affinity receptors may function to present the neurotrophin more efficiently to the high-affinity receptor trkA,
thereby increasing NGF sensitivity. Second, p75 may interact
directly with trkA for the formation of high-affinity binding
and the signalling response as suggested by Hempstead et al.
(1991). Third, p75 may interact with other proteins. For
example, when PC12 cells are treated with NGF, two protein
kinases, p130 and 6-thioguanine-sensitive protein kinase N
(PKN), become phosphorylated and associated with p75
(Ohmichi et al., 1991; Volonte et al., 1993). Therefore, it is
possible that the activation of p130 and PKN and possibly the
activation of other unidentified proteins may increase the efficiency of the trk-mediated intracellular protein phosphorylation cascade.
It was surprising that p75-deficient SCG neurons were more
sensitive to higher concentrations of NT-3 than wild-type
neurons, raising the possibility that p75 might modulate the
response of neurons to neurotrophins other than NGF. Developing sympathetic neurons are known to express both trkA
(Enfors et al., 1992; Schecterson and Bothwell, 1992) and trkC
(Enfors et al., 1992). Although trkC is the cognate receptor for
NT-3 (Lamballe et al., 1991), NT-3 is capable of eliciting a
mitogenic response in fibroblast cells expressing trkA in the
absence of p75 (Ip et al., 1993). Thus, it is possible that the
effect of high concentrations of NT-3 on the survival of SCG
neurons may be mediated by either trkA or trkC. Perhaps p75
may contribute to the specificity of NT-3 binding and, in its
absence, high levels of NT-3 may promiscuously interact with
trkA and enhance the survival of SCG neurons. Consistent with
this interpretation, NT-3 displays a rather limited effect on
PC12 cells (which express high levels of p75) as compared to
that on trkA-expressing fibroblast cells. Moreover, expression
of a truncated p75 receptor in PC12 cells results in decreased
expression of wild-type p75 and increased neurite out-growth
in response to NT-3 (Benedetti et al., 1993). Alternatively, in
the absence of p75, NT-3 may activate trkC more efficiently.
It should be noted, however, that the concentration of NT-3
used in our experiment is probably above physiological levels
(above nanomolar ranges; 50 ng/ml equal to 1.9×10-9 M).
Because the concentration of NT-3 in the target field is
unknown but likely to be quite low, this result may have no
physiological relevance and may be of pharmacological
interest only. At present, we do not understand why DRG
neurons do not exhibit a similar change in their response to
1032 K.-F. Lee, A. M. Davies and R. Jaenisch
NT-3. Perhaps, there are p75 homologues expressed in DRG
neurons that can substitute for the function of p75, as suggested
by the discovery of a number of receptors bearing homology
to the extracellular domain of p75 (reviewed by Mallett and
Barclay, 1991; Itoh et al., 1991; Durkop et al., 1992).
Several lines of evidence demonstrate that cell type as well
as the physiological context of cells are important for elucidating neurotrophin signal transduction pathway. For example,
100-fold higher concentrations of NT-3 are required to elicit
cellular responses in trkB-expressing PC12 cells than fibroblasts expressing the same receptor (Berkemeier et al., 1991;
Cordon-Cardo et al., 1991; Squinto et al., 1991; Ip et al., 1993).
In addition to cell type, the developmental stage of neurons
may influence their response to neurotrophins. The availability of p75 mutant mice provides an opportunity to examine the
responses of different types of neurons to neurotrophins at
different stages of development, as illustrated in this report.
Generating mice carrying mutations in individual trks and
intercrossing these mutant mice with p75 mutants will
undoubtedly further facilitate our understanding of the neurotrophin signaling pathway in an appropriate physiological
context.
We thank Ruth Curry for technical assistance, James Miller and
Quan Yan of Amgen, Inc. for the BDNF and NT-3, Bill Mobley of
UCSF for the NGF, Gene Burton, John Winslow and Arnon Rosenthal
of Genentech for the NT-4/5. We are also grateful to Story Landis,
Brian Bates and Patrik Ernfors for critical comments on the
manucript. This work was supported by a grant from The Wellcome
Trust to A.M. Davies and by NIH grant no. 5 R35 CA44339-05 to R.
Jaenisch. K.-F. Lee was supported by a Fellowship from Amgen, Inc.
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(Accepted 7 January 1994)