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635
Development 119, 635-647 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
Regulation of expression of mRNAs encoding the nerve growth factor
receptors p75 and trkA in developing sensory neurons
Sean Wyatt and Alun M. Davies
School of Biological and Medical Sciences, Bute Medical Buildings, University of St. Andrews, St. Andrews, Fife KY16 9AJ,
Scotland
SUMMARY
We have used a quantitative reverse transcription/polymerase chain reaction amplification technique to study
the regulation of p75 mRNA and trkA mRNA expression
in developing NGF-dependent trigeminal neurons.
Before becoming NGF dependent, these neurons express
low levels of p75 and trkA mRNAs in vivo. At this stage
in vitro, the level of p75 mRNA is maintained and upregulated by BDNF, whereas the level of trkA mRNA is
sustained independently of neurotrophins and is downregulated by BDNF. With the acquisition of NGF dependence, p75 and trkA mRNA levels increase markedly in
vivo. At this stage in vitro, the level of p75 mRNA is upregulated by NGF, but this response is lost at later stages.
INTRODUCTION
A family of small homodimeric proteins termed neurotrophins plays a key role in the development of the vertebrate nervous system. Some of the most extensive information on the function of these proteins has come from work
on sensory neurons and their progenitors. At an early developmental stage, there is evidence that brain-derived neurotrophic factor (BDNF) directs pluripotent neural crest cells
to differentiate along the sensory neuron lineage (SieberBlum, 1991). Before dorsal root ganglion neurons innervate
their targets, BDNF and neurotrophin-3 (NT-3) promote an
early maturational change in these neurons (Wright et al.,
1992). When their axons reach their targets, sensory neurons
become dependent for survival on one or more neurotrophins produced by these targets (Davies and Lumsden,
1984; Lindsay et al., 1985; Davies et al., 1986a,b; Davies,
1987b; Hohn et al., 1990; Vogel and Davies, 1991), and
certain sensory neurons change their neurotrophin requirements during the early stages of target field innervation
(Buchman and Davies, 1993). After stabilisation of neuronal
numbers, neurotrophins play an ongoing role in regulating
neuropeptide expression in sensory neurons (Lindsay et al.,
1989).
The timing of neurotrophin responsiveness is tightly controlled in developing sensory neurons. For example, the
onset of BDNF dependence in placode-derived cranial
sensory neurons is coordinated with the onset of target field
innervation by an intrinsic developmental program (Davies
The level of trkA mRNA is sustained in neurons grown
with NGF but is not up-regulated by concentrations of
NGF above those required to support survival. At no
stage during the early development of trigeminal
neurons do depolarising levels of potassium ions affect
the expression of either p75 mRNA or trkA mRNA.
These findings suggest that the expression of p75 and
trkA mRNAs are differentially regulated by BDNF and
NGF at successive early stages of neuronal development.
Key words: mouse, neurotrophin, mRNA, nerve growth factor,
sensory neuron
and Vogel, 1991; Vogel and Davies, 1991) that is initiated
in neuron progenitor cells (Vogel and Davies, 1993). To
understand how the timing of neurotrophin responsiveness
in developing sensory neurons is controlled, it is necessary
to determine the time course of neurotrophin receptor
expression and elucidate the signals that control the
expression of these receptors.
Neurotrophins specifically bind to two kinds of transmembrane glycoproteins: p75 (Chao et al., 1986; Johnson et
al., 1986; Radeke et al., 1987) and members of the trk family
of receptor tyrosine kinases (Hempstead et al., 1991; Kaplan
et al., 1991a,b; Klein et al., 1991a,b, 1992; Berkemeier et
al., 1991; Cordon-Cardo et al., 1991; Glass et al., 1991;
Lamballe et al., 1991; Nebreda et al., 1991; Soppet et al.,
1991; Squinto et al., 1991; Meakin et al., 1992). Whereas
p75 binds NGF, BDNF, NT-3 and NT-4 with similar lowaffinity (Sutter et al., 1979; Rodriguez-Tébar and Barde,
1990; Hallbook et al., 1991; Rodriguez-Tébar et al., 1992),
trk tyrosine kinases exhibit a greater degree of specificity.
Cell lines expressing trkA bind NGF, NT-3 and NT-5 but
not BDNF (Hempstead et al., 1991; Kaplan et al., 1991a,b;
Klein et al., 1991a), trkB-expressing cells bind BDNF, NT3, NT-4 and NT-5 but not NGF (Berkemeier et al., 1991;
Glass et al., 1991; Klein et al., 1991b, 1992; Soppet et al.,
1991; Squinto et al., 1991; Ip et al., 1992) and trkC-expressing cells bind NT-3 but not other neurotrophins (Lamballe
et al., 1991).
The demonstration that neurotrophins promote rapid
transphosphorylation of trk tyrosine kinases (Kaplan et al.,
636
S. Wyatt and A. M. Davies
1991a,b; Klein et al., 1991a,b; Soppet et al., 1991) and elicit
a response from oocytes (Nebreda et al., 1991), cell lines
(Cordon-Cardo et al., 1991; Glass et al., 1991; Lamballe et
al., 1991; Loeb et al., 1991; Squinto et al., 1991) and
embryonic neurons (Allsopp et al., 1993) transfected with
trk cDNAs shows that trk tyrosine kinase receptors play a
role in neurotrophin signal transduction. Although p75 is
probably not a functional receptor alone (Hempstead et al.,
1989), several findings suggest that p75 is required for
certain responses to neurotrophins. Membrane fusion and
cell transfection experiments suggest that both p75 and trkA
have to be present for the formation of functional, highaffinity NGF receptors (Hempstead et al., 1989, 1991; Matsushima and Bogenmann, 1990; Pleasure et al., 1990). PC12
cells transfected with chimeric receptors consisting of the
extracellular domain of the EGF receptor and the transmembrane and intracellular domains of p75 extend neurites
in response to EGF (Yan et al., 1991). Antisense p75
oligonucleotides interfere with kidney morphogenesis
(Sariola et al., 1991) and retard an early maturational change
in developing sensory neurons (Wright et al., 1992). Null
mutation of the p75 gene in transgenic mice causes a sensory
deficit in homozygotes due to loss of cutaneous sensory
nerve fibres (Lee et al., 1992) and results in the shift in the
dose-response curve of sensory neurons to higher NGF concentrations (Davies et al., 1993). In contrast, the finding that
preventing NGF binding to p75 by anti-p75 antiserum
(Weskamp and Reichardt, 1991) or NGF mutation (Ibanez
et al., 1992) does not interfere with the action of NGF and
that 3T3 fibroblasts proliferate in response to neurotrophins
after transfection with trkA, trkB or trkC cDNAs (CordonCardo et al., 1991; Glass et al., 1991; Lamballe et al., 1991;
Klein et al., 1992) suggest that certain responses to neurotrophins may occur without p75.
Developmental studies of the time course of neurotrophin
receptor expression have been restricted to binding of
iodinated NGF to neurons, which does not distinguish
between p75 and trkA expression, and to the detection of
p75 mRNA. In situ hybridisation (Hallbook et al., 1990;
Heuer et al., 1990) and northern blotting (Wyatt et al., 1990)
have shown that p75 mRNA is expressed in developing
sensory neurons before their start innervating their targets.
Shortly after contacting their peripheral targets, NGFdependent sensory neurons express much higher level of p75
mRNA (Wyatt et al., 1990), are labelled by iodinated NGF
(Davies et al., 1987) and start responding to NGF (Davies
and Lumsden, 1984). After the period of naturally occurring
cell death, there is a reduction in the capacity of sensory
neurons to bind iodinated NGF (Herrup and Shooter, 1975;
Raivich et al., 1985, 1987) and a decrease in the levels of
p75 mRNA (Buck et al., 1987) and p75 protein (Yan and
Johnson, 1987). Although several studies have shown that
trk receptor tyrosine kinases are expressed in the developing nervous system (Klein et al., 1989, 1990a,b; MartinZanca et al., 1990), there has been no detailed study of the
time course of trk expression in relation to neurotrophin
responsiveness.
Most of the studies of the regulation of neurotrophin
receptor expression have been carried out in cell lines and
in postnatal or adult neurons. The level of p75 mRNA is upregulated by NGF in PC12 cells (Doherty et al., 1988),
postnatal sympathetic neurons (Miller et al., 1991), adult
basal forebrain cholinergic neurons (Cavicchioli et al., 1989;
Higgins et al., 1989) and adult DRG neurons (Lindsay et al.,
1990; Verge et al., 1992). The effect of NGF on trkA mRNA
expression is controversial. In PC12 cells, NGF has been
reported to either have no effect on trkA mRNA (Kaplan et
al., 1991b) or to increase its level (Holtzman et al., 1992).
Prolonged in vivoexposure of forebrain cholinergic neurons
to exogenous NGF causes a modest increase in the grain
density in sections hybridised with a trkA probe (Holtzman
et al., 1992). Although NGF infusion does not apparently
alter the trkA mRNA grain density over adult DRG neurons,
this treatment partly restores the reduced grain density that
occurs after peripheral nerve section (Verge et al., 1992). In
MAH cells, a retrovirally immortalised sympathoadrenal
progenitor cell line, trkA mRNA is induced by depolarising
levels of KCl but not by NGF (Birren et al., 1992).
To ascertain the normal developmental time course of p75
and trkA gene expression and determine how the expression
of these genes is regulated at different stages of neuronal
development, it is essential to study a well-characterised
population of neurons that is accessible from the earliest
stages of its development. For this reason, we studied the
mouse embryo trigeminal ganglion, a population of NGFdependent sensory neurons that innervates the facial region.
In vivo studies have documented the timing of axonal
outgrowth, target encounter and naturally occurring
neuronal death for these neurons (Davies and Lumsden,
1984, 1986; Davies, 1987a). In vitro studies have revealed
the changing survival requirements of these neurons and
have provided some data on the expression of NGF
receptors. Trigeminal neurons survive independently of neurotrophins when their axons are growing to their targets.
With the onset of target field innervation, the neurons
display a transitory survival response to BDNF and NT-3
that is lost as they become NGF-dependent shortly before
the onset of naturally occurring neuronal death (Buchman
and Davies, 1993). The onset of NGF responsiveness
appears to be related to an increase in the level of p75
mRNA (Wyatt et al., 1990) and labelling of the neurons by
iodinated NGF (Davies et al., 1987). In our current study,
we have used a quantitative PCR amplification technique to
study the regulation of p75 and trkA mRNA levels in lowdensity dissociated trigeminal neuron cultures set up at
intervals throughout the early stages of neuronal development. We show that, although the normal developmental
time courses of p75 and trkA mRNA expression are similar,
neurotrophins affect the expression of these mRNAs in
different ways that change during the early stages of target
field innervation.
MATERIALS AND METHODS
Neuronal cultures
Mouse embryos were obtained from overnight matings of CD1
mice. Pregnant females were killed by cervical dislocation and the
precise stage of development of the embryos was determined by
the criteria of Theiler (1972). Electrolytically sharpened tungsten
needles were used to dissect trigeminal ganglia from E9.5 to E15
embryos. Ganglia were incubated for 5 minutes at 37°C with
0.05% trypsin (Worthington) in calcium- and magnesium-free
Hanks Balanced Salt Solution (HBSS). After removal of the trypsin
Regulation of p75 and trkA expression
solution, the ganglia were washed twice with 10 ml of Hams F12
medium containing 10% HIHS and were gently triturated with a
fire-polished, siliconised Pasteur pipette to give a single cell suspension. The cells were plated at a density of 200 to 500 neurons
per dish in 35 mm plastic tissue culture dishes (Nunc) that had been
precoated with polyornithine (0.5 mg/ml, overnight) and laminin
(20 µl/ml for 4 hours). The neurons were incubated at 37.5°C in a
humidified 3.5% CO2 incubator in a defined medium consisting of
Hams F14 supplemented with 2 mM glutamine, 0.35% bovine
serum albumin (Pathocyte-4, ICN), 60 ng/ml progesterone, 16
µg/ml putrescine, 400 ng/ml L-thyroxin, 38 ng/ml sodium selenite,
340 ng/ml tri-iodo-thyronine, 60 mg/ml penicillin and 100 mg/ml
streptomycin.
Neurons were recognised by their bipolar morphology under
phase-contrast optics. The total number of neurons surviving in
each culture dish was estimated by counting the number of neurons
in a 12×12 mm grid in the centre of the dish and multiplying this
number by the quotient of the total growth area of the dish and the
grid area.
Neurotrophins were added to the culture medium prior to plating
the neurons. Mouse submandibular salivary gland NGF was a gift
of Bill Mobley (UCSF) and purified recombinant full-length BDNF
and NT-3 were gifts of John Winslow and Gene Burton (Genentech
Inc.).
Measurement of trkA mRNA and p75 mRNA levels by
quantitative PCR
A quantitative reverse transcription/polymerase chain reaction
(RT/PCR) amplification technique was used to measure the very
low levels of trkA mRNA and p75 mRNA in trigeminal cultures
and dissected whole ganglia. The reverse transcription and PCR
reactions were calibrated by the inclusion of control RNA
templates in the reverse transcription reaction. The control RNA
templates were transcribed in vitro from trkA and p75 cDNA clones
that had been modified by the insertion between the PCR primer
sites of 3 bp in the case of trkA cDNA and 4 bp in the case of p75
cDNA. To generate the p75 cDNA control template from which
the control RNA template was transcribed, a 597 bp fragment of
the mouse p75 cDNA corresponding to nucleotides 426 to 1023 of
the rat p75 cDNA was isolated from mouse brain total RNA using
RT/PCR and was cloned into pGEM 3Z (Promega). The 601 bp
p75 cDNA control template was constructed by cleaving the cloned
p75 cDNA at a single internal AvaI site to generate DNA with 4
bp of 5′ overhang at each end. These overhangs were filled in with
the Klenow fragment of DNA polymerase I in the presence of 5
mM dNTPs and ligating the resulting blunt ends. The control p75
cRNA template was synthesised by in vitro transcription of the
control p75 cDNA from the Sp6 RNA polymerase of the pGEM
vector. To generate the trkA cDNA control template, a 452 bp
fragment of the mouse trkA cDNA corresponding to nucleotides
838 to 1290 was cloned into pGEM 3Z and was cleaved at a single
internal PpUMI site to generate DNA with 3 bp of 5′ overhang at
each end that were filled in and ligated as before. The resulting 455
bp cDNA was transcribed from the Sp6 RNA polymerase of the
pGEM vector to produce the trkA RNA control template.
Total RNA (Chomczynski and Sacchi, 1987), spiked with
known amounts of the appropriate control RNA, was reverse transcribed for 45 minutes at 37°C with Gibco BRL Superscript
enzyme in a 10 µl reaction containing the manufacturer's buffer
supplemented with 0.5 mM dNTPs and 10 µM random hexanucleotides. Each reverse transcription reaction was then gently
mixed with a 40 µl PCR solution comprising 1× NBL Taq DNA
polymerase buffer (with an additional 0.4 mM MgCl2 in the case
of p75), 1 unit of NBL Taq DNA polymerase, 40 ng of 5′ endlabelled primers and 0.1 mM dNTPs. The primers for p75 were:
(5′) 5′-CCGATACAGTGACCACTGTGATG-3′and (3′) 5′AGCAGCCAAGATGGAGCAATAGAC-3′. These hybridise 97
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bp apart in the sequence of mouse p75 cDNA and 101 bp apart in
the p75 control cDNA. The primers for trkA were: (5′) 5′-CGTCATGGCTGCTTTTATGG-3′ and (3′) 5′-ACTGGCGAGAAGGAGACAG-3′. These hybridise 75 bp apart in the sequence of
mouse trkA cDNA and 78 bp apart in trkA control cDNA.
p75 cDNA was amplified by 8 cycles of 94°C for 60 seconds,
58°C for 60 seconds and 72°C for 60 seconds, followed by 15
cycles of 91°C for 60 seconds, 56°C for 45 seconds and 72°C for
60 seconds. trkA cDNA was amplified by 8 cycles of 94°C for 60
seconds, 56°C for 45 seconds and 72°C for 45 seconds followed
by 12 cycles of 90°C for 60 seconds, 55°C for 60 seconds and 72°C
for 60 seconds. These conditions were optimal for reverse transcription and amplification of 1 fg of trkA and p75 control transcripts, respectively, such that the rate of reaction does not plateau.
If reactions were allowed to reach the plateau phase, the formation
of heteroduplex DNA products during this phase altered the ratio
between homoduplex products, leading to inaccuracies.
The PCR products of the control and native cDNA templates
were resolved on 7% non-denaturing polyacrylamide gels that were
dried and autoradiographed. Reactions were set up such that the
autoradiographic signals from the PCR products of the native and
control cDNA templates were approximately equal. The autoradiographs were scanned with a Molecular Dynamics Personal Laser
Densitometer and the intensity of the respective signals was ascertained using ImageQuant software (Molecular Dynamics). These
values enabled the level p75 mRNA or trkA mRNA in the initial
total RNA sample to be calculated.
Measurement of trkA mRNA levels by quantitative
northern blotting
Total RNA from dissected ganglia (Chomczynski and Sacchi,
1987) was glyoxylated, electrophoresed in 0.8% agarose gels and
vacuum blotted onto nitrocellulose filters in 15× SSC. After
baking, the filters were hybridised with a 32P-labelled, trkA cRNA
probe made by in vitro transcription from the SP6 promoter of a
pGEM riboprobe vector containing 530 bp of mouse trkA (corresponding to the extracellular domain and part of the transmembrane domain). Filters were prehybridised at 60°C for 3 hours in
the following solution: 50% formamide, 6× SSC, 50 mM sodium
phosphate pH 6.5, 5 mM EDTA, 1.25% SDS, 6.25 Denhardt's
solution, 250 µg/ml salmon sperm DNA and 250 µg/ml E coli
tRNA. The filters were hybridised at 60°C for 15 hours in the above
solution containing 1.25× 106 cpm/ml of the 32P-labelled cRNA
probe. Filters were washed twice for 15 minutes in 1× SSC with
0.1% SDS at 60°C and twice for 30 minutes in 0.1× SSC with 0.1%
SDS at 60°C before exposure to Fuji X-ray film at −70°C with
intensifying screens. trk mRNA was detected as a 3.2 kb band on
autoradiograms.
The amount of trkA mRNA in tissue extracts was quantified by
reference to a series of calibration standards that were glyoxylated
and run on the agarose gel alongside the tissue RNA samples. The
calibration standards were known molar quantities of an unlabelled, sense trkA 0.6 kb RNA transcript. Band intensities were
measured using a Molecular Dynamics densitometer. These figures
were corrected for RNA losses during extraction, electrophoresis
and blotting by reference to the band intensity of a known quantity
of the same sense trkA 0.6 kb RNA transcript used as a recovery
standard (0.6 kb) that was added to tissue samples prior to RNA
extraction (Heumann et al., 1984).
RESULTS
Specific, quantitative detection of p75 and trkA
mRNAs by RT/PCR
To measure the extremely low levels of p75 and trkA
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S. Wyatt and A. M. Davies
mRNAs in total RNA extracted from low-density neuronal
cultures, each of these target mRNAs together with a slightly
larger control RNA (made by in vitro transcription from p75
cDNA with a 4 bp insert and trk cDNA with a 3 bp insert,
respectively) were co-reverse transcribed and co-amplified
by PCR. Because the target mRNA and its control RNA
were present in the same reactions and used the same
primers, the variables affecting amplification efficiency
were nullified. This was confirmed by carrying out reactions
with known quantities of target and control RNA transcripts.
The ratio between the reaction products during the log-phase
of the PCR reaction was identical to the initial ratio between
target and control RNAs irrespective of the starting levels
of these RNAs (data not shown).
Fig. 1 shows the closely spaced bands of reaction products
resulting from the reverse transcription and amplification of
p75 and trkA mRNAs and their corresponding control
RNAs. The amplification of total RNA that was not reverse
transcribed produced no PCR products with either set of
primers, demonstrating that RNA samples contained no contaminating DNA. As little as 0.1 fg of either control RNA
template could be reliably detected by the RT/PCR assay,
effectively allowing the quantification of p75 and trkA
mRNA levels in less than 10 neurons. The extreme sensitivity of this technique has allowed us to study the regulation of expression of p75 and trkA mRNAs in very low
density cultures of early trigeminal neurons that are essentially free of the complication of significant levels of contamination by neurotrophins produced by non-neuronal cells.
Developmental time course of p75 and trkA
mRNAs in trigeminal neurons
The level of trkA mRNA in the trigeminal ganglion was
determined at closely staged intervals from E9.5 to E15 by
hybridising northern blots of ganglion total RNA with a 32P-
Fig. 1. Sensitivity and specificity of the RT-PCR technique.
Autoradiograph showing the products of RT-PCR reactions
amplified with either trkA-specific primers (trkA) or p75-specific
primers (p75). (1) Reactions containing 1 fg of either the trkA or
p75 control RNA templates, showing the 78 bp and 98 bp
amplification products, respectively. (2) Reactions containing total
RNA from ten E12 trigeminal neurons, showing the 75 bp trkA
mRNA and 94 bp p75 mRNA amplification products,
respectively. (3) Reactions containing 1 fg of the respective
control RNA template plus total RNA from ten E12 trigeminal
neurons, showing both sets of the respective amplification
products. (C) No products resulted from control reactions that
contained the respective control RNA template plus total RNA
from ten E12 trigeminal neurons, but from which the reverse
transcription step was omitted.
labelled trkA cRNA probe. The presence of trkA mRNA was
revealed by a single band on autoradiograms corresponding
to 3.2 kb (Fig. 2A). trkA mRNA was clearly detected at E9.5,
the stage at which the earliest axons start growing towards
their targets. The level of trkA mRNA increased from 0.6 pg
per ganglion at E9.5 to over 70 pg per ganglion at E14 and
E15 (Fig. 2B). Measurement of the level of trkA mRNA in
E9.5 to E15 ganglia by quantitative PCR gave results that
were not significantly different from those obtained by
northern blotting (data not shown).
To ascertain the identity of the cells that express trkA
mRNA in the trigeminal ganglion, the level of trkA mRNA
was measured in purified preparations of neurons and
satellite cells obtained by low-temperature, differential sedimentation (Davies, 1986). At E14, the earliest age at which
this technique reliably separates the cells in the trigeminal
A
B
Fig. 2. Northern blot analysis of trkA mRNA in the trigeminal
ganglion. (A) Autoradiogram of a northern blot of E9.5 to E12
trigeminal ganglion total RNA hybridised with the 32P-labelled
trkA cRNA probe. The 3.2 kb band of trkA mRNA and the 0.6 kb
bands of the recovery and calibration standards are indicated. In
this example, total RNA was extracted from 22 E9.5 ganglia, 18
E10 ganglia, 18 E11 ganglia and 10 E12 ganglia. 80 pg of
recovery RNA standard was added to each sample of tissue prior
to RNA extraction. The amount of calibration standard added to
first four lanes is given in pg. (B) Graph of the level of trkA
mRNA per ganglion from E9.5 to E15. The mean ± SEM of
between 3 and 8 separate measurements at each age are shown.
Regulation of p75 and trkA expression
ganglion, northern blot hybridisation and quantitative PCR
amplification revealed that trkA mRNA, like p75 mRNA
(Wyatt et al., 1990), was present only in neurons. No trkA
mRNA signal was observed in satellite cells even when a 5fold larger number of these cells was used (Fig. 3A).
Restriction of trkA mRNA expression to neurons in the
ganglion enabled the mean level of trkA mRNA per neuron
to be calculated because the number of neurons in the
ganglion from E10 onwards is known (Davies and Lumsden,
1984; Davies, 1987b). Fig. 3B shows that the mean level of
trkA mRNA per neuron was low and unchanged from E10
to E11 (mean level equivalent to approximately 250
molecules of trkA mRNA per neuron). There was a small
A
B
Fig. 3. Expression of p75 mRNA and trkA mRNA in trigeminal
neurons. (A) Autoradiogram of products of a PCR reaction for the
detection of trkA mRNA in purified neurons (N) and satellite cells
(S) obtained from E14 trigeminal ganglia. Both reactions included
total RNA from approximately 100 cells plus 25 fg of control
RNA template. There was less than 1% contamination of neurons
by satellite cells and vice versa. Whereas the 78 bp amplification
product of the control RNA template was present in both
reactions, the 75 bp amplification product of trkA mRNA was only
detectable in RNA extracted from neurons. A control reaction (C)
that contained 25 fg of control RNA template plus total RNA from
100 E14 neurons but no reverse transcriptase resulted in no
reaction products. (B) Graph of the mean levels of p75 mRNA
(filled circles) and trkA mRNA (open circles) per neuron in
trigeminal ganglia from E10 to E15, calculated by dividing the
data on the amount of p75 mRNA and trkA mRNA per ganglion
by the number of neurons in the ganglion at each age. The mean ±
SEM are shown.
639
rise in the mean neuronal level of trkA mRNA between E11
and E12, and a greater increase through later ages.
Previous estimation of the age-related changes in the
mean level of p75 mRNA in developing trigeminal neurons
by northern blotting suggested that the level was largely
unchanged from E10 to E12 and increased between E12 and
E16 (Wyatt et al., 1990). When we repeated these measurements of p75 mRNA using quantitative PCR, we observed
a similar developmental trend with the exception of a small
gradual increase in p75 mRNA during the early stages of
trigeminal ganglion development (Fig. 3B). The levels of
p75 mRNA per neuron determined by quantitative PCR
were, however, higher than the previously reported values.
This quantitative discrepancy can be attributed to the greater
efficiency of the RNA extraction method used in our current
study (Chomczynski and Sacchi, 1987) because measurement of p75 mRNA by quantitative PCR using total RNA
extracted by the method that we used previously (Melera
and Rusch, 1973) gave results that were similar to those previously obtained by northern blotting (data not shown). Fig.
3B shows that the mean neuronal levels of p75 mRNA and
trkA mRNA were similar and underwent similar developmental changes.
Differences and developmental changes in the
regulation of p75 mRNA and trkA mRNA
expression by neurotrophins
We have studied the influence of neurotrophins on receptor
gene expression in the context of the changing responsiveness of developing trigeminal neurons to neurotrophins.
Previous work has shown that the survival of trigeminal
neurons is independent of neurotrophins when cultured at
the stage when their axons are growing to their targets. In
E10 and E11 cultures, over 80% of these neurons survive
for 24 hours in the absence of neurotrophins, but all die by
48 hours (Buchman and Davies, 1993). During the earliest
stages of target field innervation, trigeminal neurons are
supported BDNF. They then lose the BDNF response and
become dependent on NGF for survival during the phase of
naturally occurring cell death in the trigeminal ganglion. In
E10 and E11 cultures, BDNF promotes the survival of all
neurons for at least 48 hours incubation, whereas NGF
supports only 5 and 25%, respectively. By E12 and E13,
virtually all of the neurons are supported by NGF and only
40% and 5% are supported by BDNF, respectively
(Buchman and Davies, 1993). Thus, the E11/E12 transition
coincides with a switch in responsiveness from BDNF to
NGF.
Investigation of the influence of neurotrophins on
receptor gene expression is complicated by the need to supplement cultures with neurotrophins to promote the survival
of neurons beyond 24 hours incubation. For this reason, our
initial studies focused on the effect of neurotrophins at concentrations greater than those required to promote neuronal
survival. We set up at least 5 separate dose responses at each
age to determine the lowest concentration of BDNF that
promoted maximal survival in E10 and E11 cultures and the
lowest concentration of NGF that promoted maximal
survival in E12, E13 and E14 cultures. These concentrations
were 3.2 pg/ml for BDNF and 7.2, 16 and 80 pg/ml for NGF
at E12, E13 and E14, respectively. We then grew E10 to E14
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S. Wyatt and A. M. Davies
neurons with these and higher concentrations of neurotrophins and measured the p75 and trkA mRNAs levels in
these cultures after 24, 36 and 48 hours incubation. The data
from 5 to 10 separate experiments at each age are summarised in Fig. 4.
With increasing concentrations of BDNF in E10 and E11
cultures and increasing concentrations of NGF in E12, E13
and E14 cultures, the mean level of p75 mRNA per neuron
increased to reach a maximum at each age. The increase in
the p75 mRNA level from the low to the maximally effective
neurotrophin concentration was two-fold in E10 cultures, it
increased over three-fold in E12 cultures and increased twofold in E14 cultures. Thus, although neuron number was
unchanged by concentrations of neurotrophins above those
required for maximal neuronal survival, the level of p75
mRNA increased in cultured neurons with increasing neurotrophin concentration. In all cultures, there was no significant difference in the p75 mRNA levels after 24, 36 and 48
hours incubation with a given concentration of neurotrophin
(P<0.02, t-test).
Although our previous work has shown that p75 mRNA
is not detectable by northern blotting in non-neuronal cells
freshly isolated from embryonic trigeminal ganglia (Wyatt
et al., 1990), it is possible that these non-neuronal cells
might express p75 mRNA in culture. Because the cultures
that we used to study the regulation of p75 mRNA
expression contained some non-neuronal cells in addition to
neurons, p75 mRNA expression in non-neuronal cells might
have affected the results. To determine if trigeminal nonneuronal cells express significant levels of p75 mRNA in
culture, highly enriched preparations of neurons and nonneuronal cells obtained by differential sedimentation from
E14 trigeminal ganglia were grown in low-density culture.
The level of p75 mRNA in non-neuronal cells after 24 and
48 hours incubation was less than 5% of the level in neurons.
Moreover, the very low level of p75 mRNA expression in
non-neuronal cells was unaffected by NGF, BDNF or NT3 (data not shown). Thus, the contribution of non-neuronal
cells to the overall levels of p75 mRNA in our cultures is
negligible and is unaffected by neurotrophins.
Our findings suggest that concentrations of BDNF above
those required for neuronal survival increase the level of p75
mRNA in E10 and E11 neurons and that concentrations of
NGF above those required for neuronal survival increase the
level of p75 mRNA in E12 and older cultures. To determine
if NGF influences the level of p75 mRNA in neurons before
they become NGF-responsive, E11 cultures were grown
with the lowest concentration of BDNF needed for survival
(low BDNF) together with NGF at the concentration that is
maximally effective in up-regulating the level of p75 mRNA
in E12 cultures (high NGF). At 24, 36 and 48 hours in vitro,
there was no significant difference in the level of p75 mRNA
in E11 cultures containing low BDNF alone and cultures
containing low BDNF plus high NGF (Fig. 5). To determine
Fig. 4. Levels of p75 mRNA and trkA mRNA in trigeminal neurons grown with different concentrations of neurotrophins. Graphs of the
levels of p75 mRNA and trkA mRNA per neuron in cultures of E10 to E14 neurons grown with either BDNF (E10 and E11 neurons) or
NGF (E12, E13 and E14 neurons) at concentrations ranging from the lowest required to promote maximal neuronal survival for at least 48
hours to a concentration that was over three orders of magnitude higher. From 5 to 10 separate experiments were carried out at each age.
At each concentration in all experiments, there were no significant differences in the levels of either p75 mRNA or trkA mRNA at 24, 36
and 48 hours. For this reason, the graphs show the overall mean ± the standard error of 24, 36 and 48 hour measurements in all
experiments.
Regulation of p75 and trkA expression
641
Fig. 5. Levels of p75 mRNA and trkA mRNA in trigeminal neurons grown with BDNF and NGF alone and combined. Bar charts of the
levels of p75 mRNA and trkA mRNA per neuron in cultures of E11 and E12 neurons grown with low and high concentrations of BDNF
and NGF alone and combined. LB, BDNF at the lowest concentration (3.2 pg/ml) required to promote maximum survival in E11 cultures.
LN, NGF at the lowest concentration (7.2 pg/ml) required to promote maximum survival in E12 cultures. HB, BDNF at the higher
concentration (2 ng/ml) that promotes the greatest increase in the level of p75 mRNA in E11 trigeminal neurons. HN, NGF at the higher
concentration (2 ng/ml) that promotes the greatest increase in the level of p75 mRNA in E12 trigeminal neurons. Three separate
experiments were carried out at each age. In all experiments there were no significant differences in the levels of either p75 mRNA or
trkA mRNA at 24, 36 and 48 hours. For this reason, the graphs show the overall mean ± the standard error of 24, 36 and 48 hour
measurements in all experiments.
if BDNF influences the level of p75 mRNA in NGFdependent neurons, E12 cultures were grown with the
lowest concentration of NGF needed for survival (low NGF)
together with BDNF at the concentration that is maximally
effective in up-regulating the level of p75 mRNA in E11
cultures (high BDNF). At 24, 36 and 48 hours in vitro, there
was no significant difference in the level of p75 mRNA in
E12 cultures containing low NGF alone and cultures containing low NGF plus high BDNF (Fig. 5). In both E11 and
E12 cultures, there was no significant difference between the
number of neurons surviving with a low level of either
BDNF or NGF and the number of neurons surviving with
both factors (data not shown), indicating that p75 mRNA
levels were measured in the same population of neurons
under both experimental conditions. Taken together, these
findings suggest that BDNF and NGF up-regulate the level
of p75 mRNA in developing trigeminal neurons at stages
when the survival of these neurons is promoted by BDNF
and NGF, respectively.
There was no increase in the level of trkA mRNA over
the range of neurotrophin concentrations that up-regulated
the level of p75 mRNA (Fig. 4). In E10 and E11 cultures
containing BDNF, the level of trkA mRNA was lower than
the corresponding in vivo level and showed little change
over the broad range of BDNF concentrations used. In E12
and E13 cultures, there was a small decrease in the level of
trkA mRNA with increasing concentrations of NGF. In E14
cultures, there were relatively large fluctuations in the level
of trkA mRNA but neither an upward nor a downward trend
with increasing NGF concentration was evident (Fig. 4).
When E11 neurons were grown with BDNF plus NGF, as
described for p75 mRNA experiments, there was no significant difference in the level trkA mRNA in these cultures
compared with neurons grown with BDNF alone. Likewise,
the level of trkA was not affected by growing E12 neurons
with NGF plus BDNF (Fig. 5). These results were not
affected by the expression of trkA mRNA in small number
of non-neuronal cells in these cultures because measurements of trkA mRNA in highly enriched cultures of E14
trigeminal non-neuronal cells showed that the level of trkA
mRNA in these cells is less than 5% of the level in purified
E14 neurons and is unaffected by neurotrophins (data not
shown).
In addition to the different changes in p75 and trkA
mRNA levels caused by neurotrophins, the in vitro levels of
these mRNAs differed from the corresponding in vivo levels
in several contrasting ways. In Fig. 6, the levels of p75
mRNA and trkA mRNA in E10 to E14 neurons cultured with
low and high concentrations of neurotrophins are compared
with the in vivo levels of these mRNAs over the same period
of development. In E10 and E11 cultures supplemented with
the lowest concentration of BDNF needed for survival, the
mean neuronal level of p75 mRNA was similar to the in vivo
level during this period of development. In contrast, the
642
S. Wyatt and A. M. Davies
Fig. 6. Comparison of the in vitro and in vivo levels of p75 mRNA and trkA mRNA in trigeminal neurons. Bar charts of the levels of p75
mRNA and trkA mRNA per neuron in cultures of E10 to E14 neurons grown with either BDNF (E10 and E11 neurons) or NGF (E12, E13
and E14 neurons) at the lowest concentrations required to promote maximum neuronal survival (stippled bars) and at the higher
concentrations that cause the greatest increase in the level of p75 mRNA (hatched bars). The mean ± the standard error of all 24, 36 and
48 hour measurements are shown. The mean ± the standard error of the levels of p75 mRNA and trkA mRNA per neuron in E10 to E14
trigeminal ganglia are also shown (black bars).
mean neuronal level of trkA mRNA in E10 and E11 neurons
grown with BDNF was much lower than the corresponding
in vivo level. Thus, whereas the level of p75 mRNA was
maintained and up-regulated by BDNF in early trigeminal
neurons in culture, the level of trkA mRNA was neither
maintained nor up-regulated by BDNF. In E13 and E14
cultures, the level of p75 mRNA was not maintained at in
vivo levels. Even at the highest NGF concentration, the level
of p75 mRNA in vitro was less than a third of the corresponding in vivo level. In contrast, the level of trkA mRNA
in E14 neurons grown with NGF was much closer to the in
vivo level. Thus, whereas the level of trkA mRNA is largely
maintained in trigeminal neurons cultured with NGF during
the period of naturally occurring cell death, the level of p75
mRNA falls well below the in vivo level in these cultures.
Time course of the effects of neurotrophins on
p75 and trkA mRNA levels
To determine the time course of neurotrophin effects on p75
and trkA mRNA levels, early trigeminal neurons were
cultured with and without neurotrophins for periods of
between 3 and 24 hours in culture. E11 and E12 neurons
were used for these experiments because 80% and 60%,
respectively, survive in the absence of neurotrophins for 24
hours in vitro and because the great majority of neurons
switch their responsiveness from BDNF to NGF between
E11 and E12. The number of neurons was counted in each
culture dish prior to RNA extraction and the results are
expressed in p75 mRNA or trkA mRNA levels per neuron.
In E11 cultures, the level of p75 mRNA dropped in the
absence of BDNF during the first 3 hours, whereas in the
presence of BDNF the level of p75 mRNA increased
markedly during the first 12 hours and this elevated level
was maintained to 24 hours (Fig. 7A). The level of p75
mRNA in neurons grown with BDNF was between 6- and
20-fold higher than in neurons grown in control cultures
throughout the 24 hour culture period. In complete contrast,
the level of trkA mRNA in the same E11 cultures decreased
in presence of BDNF during the first 3 hours (Fig. 7B), and
throughout the 24 hour culture period the level of trkA
mRNA in control cultures was between 2- and 3-fold higher
than in cultures containing BDNF. These results clearly
demonstrate that BDNF up-regulates p75 mRNA and downregulates trkA mRNA in developing trigeminal neurons
during the period of BDNF dependence.
In E12 cultures, the level of p75 mRNA also fell in control
cultures (Fig. 7C). In contrast to E11 cultures, the level of
p75 mRNA also fell in the presence of BDNF during the
first 12 hours. The fall in the level p75 mRNA in the
presence of BDNF was not, however, as great as the fall in
control cultures. In the presence of NGF, the level of p75
mRNA increased rapidly during the first 3 hours and level
attained at this time was maintained until 24 hours. In
contrast to the marked fall in the level of p75 mRNA in E12
control cultures, the level of trkA mRNA gradually
increased in these cultures to reach a peak at 18 hours (Fig.
7D). Whereas in E11 cultures the level of trkA mRNA fell
in the presence of BDNF, in E12 cultures the level of trkA
mRNA increased in the presence of either BDNF or NGF
during the first three hours. By 18 hours, however, there was
no significant difference between the levels of trkA mRNA
in control and neurotrophin-supplemented cultures.
Regulation of p75 and trkA expression
643
ference in the levels of either p75 mRNA or trkA mRNA
between neurons grown with or without KCl at either low
or high concentrations of neurotrophins. Typical PCR gels
illustrating these results are shown in Fig. 8A and B. The
combined 24 and 48 hour data from the E12 experiments (at
least 7 measurements for each experimental condition
compiled from 3 separate experiments) are shown in Fig.
8C.
DISCUSSION
Fig. 7. Time courses of changes in the levels of p75 mRNA and
trkA mRNA in cultured trigeminal neurons. The levels of p75
mRNA and trkA mRNA per neuron in cultures of E11 and E12
neurons grown with 2 ng/ml BDNF (filled circles), 2 ng/ml NGF
(filled squares, E12 cultures only) and no added neurotrophins
(open circles) 3, 9, 18 and 24 hours after plating. The values
reported at 0 hour are the corresponding in vivo estimates in the
neurons of E11 and E12 ganglia. The mean ± the standard error of
3 separate experiments are shown.
Effect of depolarising levels of KCl on the
expression of p75 and trkA mRNA levels
To investigate if depolarisation affects the expression of
either p75 mRNA or trkA mRNA in developing sensory
neurons, E11, E12 and E13 trigeminal neurons were grown
for 72 hours with and without depolarising levels of KCl (40
mM) in the culture medium. To promote neuronal survival,
E11 neurons were grown with BDNF and E12 and E13
neurons were grown with the lowest concentration of neurotrophin that promotes maximal neuronal survival for 72
hours (3.2 pg/ml BDNF for E11 neurons; 7.2 pg/ml NGF
for E12 neurons; 16 pg/ml NGF for E13 neurons). In
addition, neurons were also grown with neurotrophins at
concentrations that promote the greatest increase in p75
mRNA (2 pg/ml BDNF for E11 neurons and 2 pg/ml NGF
for E12 and E13 neurons). The levels of p75 and trkA
mRNAs were measured after 24, 48 and 72 hours incubation. Two separate experiments were set up at E11, three at
E12 and one at E13. Overall, there was no significant dif-
Developmental time course of p75 mRNA and trkA mRNA expression
By direct measurement of trkA mRNA in purified neuron
and non-neuronal cell preparations from developing trigeminal ganglia, we have shown that trkA mRNA expression,
like that of p75 mRNA (Wyatt et al., 1990), is restricted to
neurons. Likewise, by in situ hybridisation, trkA mRNA
expression also appears to be restricted to neurons in developing sensory ganglia (Martin-Zanca et al., 1990). This
facilitates the interpretation of the developmental changes in
p75 and trkA mRNA levels in the trigeminal ganglion
because the results can be corrected for the known changes
in the total number of neurons in the ganglion during development (Davies and Lumsden, 1984; Davies, 1987b). Thus,
we have be able to calculate the mean levels of p75 and trkA
mRNAs in neurons at different stages during their early in
vivo development and relate these data to the changing
responsiveness of these neurons to neurotrophins (Buchman
and Davies, 1993).
We have shown that both p75 and trkA mRNAs are
expressed in the trigeminal ganglion as early as E9.5, the
stage when the first axons emerge from the ganglion and
start growing towards their peripheral targets. This suggests
that developing sensory neurons express NGF receptors
before their axons reach their targets. The mean neuronal
levels of p75 and trkA mRNAs are, however, low during this
period. After E12, however, the levels of both mRNAs, but
especially trkA mRNA, increase markedly. These increases
appear to be closely related to the time trigeminal neurons
become dependent on NGF for survival (Buchman and
Davies, 1993). Whether the apparent lack of NGF responsiveness earlier in development is due to a low level of trkA
receptor tyrosine kinase expression or to an inability of the
neurons to respond to a trkA-mediated signal is unclear.
Trigeminal neurons are presumably able to respond to a trkB
receptor tyrosine kinase-mediated signal during this early
stage of development because the survival of these neurons
is promoted by BDNF before they become NGF dependent
(Buchman and Davies, 1993). Furthermore, it is likely that
the molecular events immediately downstream of signal
transduction are common for trkA and trkB because microinjection of a trkA expression vector in the BDNF-dependent
trigeminal mesencephalic confers NGF responsiveness on
these neurons (Allsopp et al., 1993). The demonstration that
overexpression of trkA in PC12 cells accelerates NGFinduced differentiation (Hempstead et al., 1992), raises the
possibility that the level of receptor expression and tyrosine
kinase activity could be crucial in governing responsiveness.
644
A
S. Wyatt and A. M. Davies
B
C
Fig. 8. Expression of p75 mRNA and trkA mRNA in trigeminal neurons grown with 40 mM KCl. (A) Autoradiograph showing the results
of a series of RT-PCR reactions amplified using trkA-specific primers. All reactions contained 5 fg of trkA control RNA template and total
RNA from 50 E13 trigeminal neurons that had been cultured for 48 hours in the presence of: (1) 16 pg/ml NGF, (2) 2 ng/ml NGF, (3) 16
pg/ml NGF plus 40 mM KCl and (4) 2 ng/ml NGF plus 40 mM KCl. A control reaction (C) that contained 5 fg of control RNA template
plus total RNA from 50 E13 neurons but no reverse transcriptase resulted no reaction products. The 78 bp and 75 bp amplification
products of the trkA control RNA template trkA mRNA, respectively, are indicated. (B) Autoradiograph showing the results of a series of
RT/PCR reactions amplified using p75-specific primers. All reactions contained 5 fg of the p75 control template and the total RNA from
50 E11 trigeminal neurons that had been cultured in the presence of: (1) 3.2 pg/ml BDNF, (2) 2 ng/ml BDNF, (3) 3.2 pg/ml BDNF plus
40 mM KCl, (4) 2 ng/ml BDNF plus 40 mM KCl. A control reaction (C) that contained 5 fg of control RNA template plus total RNA
from 50 E11 neurons but no reverse transcriptase resulted in no reaction products. The 98 bp and 94 bp amplification products of the p75
control RNA template p75 mRNA, respectively, are indicated. (C) Bar charts of the levels of p75 mRNA and trkA mRNA in E12
trigeminal neurons grown with NGF at either 0.016 ng/ml or 2 ng/ml in the absence and presence of 40 mM KCl. The combined 24 and
48 hour data from 3 separate experiments are shown.
For this reason, it would be informative to ascertain if overexpressing trkA in early trigeminal neurons would confer
NGF dependence prematurely.
We have shown that the mean neuronal level of trkA
mRNA increases markedly from E12 to E15. Although we
do not know the relationship between trkA mRNA and
protein levels, it is likely that the number of trkA receptors
on trigeminal neurons also rises during this period. It is surprising, therefore, that the NGF dose-response curve shifts
by an order of magnitude to higher NGF concentrations
between E12 and E15 (Buchman and Davies, 1993). This
shift is not dependent on the expression of p75 because it
also occurs in the developing trigeminal neurons of embryos
homozygous for a null mutation of the p75 gene (Davies et
al., 1993). We do not know if this decrease in NGF sensitivity is due to a reduction in the efficiency of the NGF
signal transduction event mediated by trkA or to changes in
the cascade of events downstream of trkA. Several isoforms
of trkB and trkC have recently been identified that either
have truncations of the catalytic tyrosine kinase domain or
have insertions in this domain (Klein et al., 1990a;
Middlemas et al., 1991; Soppet et al., 1991; Parada et al.,
1993). Although the function of these isoforms is unknown,
it is possible that some may act as dominant-negative
receptors. Likewise, increasing expression of a trkA
dominant-negative receptor in developing trigeminal
neurons could account for the decreasing NGF sensitivity of
these neurons as they mature. Although only a single trkA
band was observed on northern blots, a trkA isoform with a
small insertion or deletion would not be detected by this
technique.
Influence of BDNF and NGF on the expression of
p75 mRNA
We have shown that BDNF and NGF have age-related and
concentration-dependent effects on the level of p75 mRNA
expression in developing trigeminal neurons. At E10 and
E11, when the survival of trigeminal neurons beyond 24
hours in vitro is dependent on the presence of BDNF, the
level of p75 mRNA was markedly affected by BDNF. In the
absence of BDNF, the level of p75 mRNA in E11 neurons
fell within the first 3 hours in vitro. This fall was not part of
a non-specific decrease in mRNA expression associated
with the reduced viability of these neurons because the level
of trkA mRNA was maintained for 24 hours in the absence
of BDNF. In the presence of the lowest concentration of
BDNF required to promote the survival of the neurons for
two or more days, the level of p75 mRNA was maintained
Regulation of p75 and trkA expression
close to its in vivo level. At higher concentrations of BDNF,
the level of p75 mRNA increased above its in vivo level by
as much as two-fold. These findings suggest that BDNF
maintains and is capable of up-regulating the expression of
p75 mRNA during the early stages of target field innervation. During this period of development, however, high
levels of NGF do not increase the level of p75 mRNA in
neurons surviving with low levels of BDNF.
At E12, when the survival of trigeminal neurons beyond
24 hours in vitro is dependent on the presence of NGF and
only a minority respond to BDNF, the level of p75 mRNA
was markedly affected by NGF. In the absence of NGF, the
level of p75 mRNA in E12 neurons decreased. This fall was
less pronounced in the presence of the lowest concentration
of NGF that promoted maximal survival, and at higher concentrations of NGF the level of p75 mRNA exceeded its in
vivo level. In contrast to E11 cultures, the level of p75
mRNA in E12 cultures fell in the presence of a high level
of BDNF. Although the level of p75 mRNA in the presence
of BDNF was significantly higher than in control cultures
during the first 24 hours in vitro, high levels of BDNF failed
to increase the level of p75 mRNA in cultures containing
suboptimal levels of NGF. These findings suggest that the
effects of BDNF and NGF on p75 mRNA expression in
developing sensory neurons are restricted to the respective
periods of development when the neurons depend on each
of these factors for survival.
In E13 and E14 cultures, the level of p75 mRNA was also
up-regulated by concentrations of NGF in excess of those
required for maximum neuronal survival. However, even in
the maximally effective concentrations of NGF, the level of
p75 mRNA was substantially lower than the corresponding
in vivo level. This suggests that neurotrophins alone are not
sufficient to maintain the appropriate level of p75 mRNA in
sensory neurons during this later stage of development.
Although this low level of p75 mRNA in vitro could be due
to the absence of a specific growth factor to which the
neurons are normally exposed in vivo, it is not part of a nonspecific decrease in mRNA levels in cultured neurons
because the level of trkA mRNA was maintained close to in
vivo levels in these neurons. Although previous work has
shown that NGF increases the level of p75 mRNA
expression in cultured adult DRG neurons (Lindsay et al.,
1990), because the relationship between in vitro and in vivo
levels of p75 mRNA was not determined, it is not known if
neurotrophins alone are sufficient to maintain the appropriate levels of p75 mRNA in viable adult sensory neurons.
There is evidence, however, that infusion of exogenous NGF
is able to up-regulate the in vivo level of p75 mRNA in adult
DRG neurons (Verge et al., 1992) and adult basal forebrain
cholinergic neurons (Cavicchioli et al., 1989; Gage et al.,
1989; Higgins et al., 1989).
Influence of BDNF and NGF on the expression of
trk-A mRNA
We have shown that BDNF and NGF have markedly
different effects on the expression of trkA and p75 mRNAs.
In contrast to the up-regulation of p75 mRNA by BDNF in
cultures of E10 and E11 trigeminal neurons, BDNF caused
a marked decrease in trkA mRNA levels in these neurons. It
has been argued that the purpose of the early survival
645
response of trigeminal neurons to BDNF may be to sustain
the survival of the neurons whose axons reach the target field
during the early stages of its innervation, and thereby delay
the onset of neuronal death in the trigeminal ganglion until
most of the neurons have started to innervate the target field
(Buchman and Davies, 1993). This would ensure that the
majority of neurons compete for a supply of NGF during the
same period of development and thereby maximise choice
in maintaining neurons on the basis of the appropriateness
of their axons terminations in the target field. BDNF mRNA
is expressed in the peripheral target field of the trigeminal
ganglion prior to the arrival of the earliest axons and its level
peaks during the earliest stages of target field innervation
and declines after E12 (Buchman and Davies, 1993).
Because the onset of NGF responsiveness in developing
trigeminal neurons appears to be associated with an increase
in the expression of trkA mRNA, it is possible that the
exposure of early trigeminal neurons to BDNF in vivo may
delay the onset of NGF dependence in these neurons until
they can compete with the later arriving neurons for the
limiting supply of NGF.
When virtually all trigeminal neurons have become
dependent on NGF for survival in E12 cultures, BDNF no
longer had discernible effect on the level of trkA mRNA.
Likewise, the level of trkA mRNA was also not greatly
affected by NGF at this stage. In contrast to p75 mRNA,
there was no significant difference in the level of trkA
mRNA in E12 neurons grown for 24 hours with and without
NGF in the culture medium. Interestingly, in marked
contrast to p75 mRNA, the level of trkA mRNA increased
in E12 control cultures during the first 18 hours. This clearly
demonstrates that NGF is not required to maintain the level
of trkA mRNA at this stage of development. Furthermore,
because the level of trkA mRNA increases markedly
between E12 and E13 in vivo, the autonomous increase in
trkA mRNA levels in neurotrophin-free cultures during this
same period in vitro raises the possibility that this increase
may be an intrinsically regulated feature of the neurons. In
this respect, it is perhaps significant that the time at which
different populations of placode-derived cranial sensory
neurons become dependent on neurotrophins for survival is
controlled, at least in part, by an intrinsic developmental
program in the neurons (Davies and Vogel, 1991; Vogel and
Davies, 1991, 1993). The apparent lack of effect of NGF on
the expression of trkA mRNA in E12 and older cultures is
also shown by the similar levels of trkA mRNA in cultures
containing the minimum level of NGF required for longterm survival and higher concentrations which cause a
marked elevation in the level of p75 mRNA.
Although it has been suggested that NGF increases trkA
mRNA expression in vivo, the reported effects are not only
small but are difficult to interpret because the data were
obtained from grain counting in autoradiograms which is
neither reliable nor accurate in quantifying specific mRNA
levels. Intraventricular infusion of NGF over a 2-week
period caused only a 1.7-fold increase in grain density over
forebrain cholinergic neurons in sections hybridised with a
trkA probe (Holtzman et al., 1992). In adult neurons,
intrathecal infusion of NGF increased the level of p75
mRNA but did not affect the level of trkA mRNA (Verge et
al., 1992). NGF infusion did, however, partially restore the
646
S. Wyatt and A. M. Davies
level of trkA mRNA in adult DRG neurons following
peripheral nerve section. Here again, however, the effect
was small, grain counts were 48% of normal after nerve
section alone and were elevated to only 61% of normal after
nerve section plus NGF infusion (Verge et al., 1992). It is
possible that this slightly higher level of trkA mRNA may
be due to the improved viability of lesioned sensory neurons
in the presence of exogenous NGF.
Influence of depolarising levels of KCl on the
expression of p75 and trk-A mRNAs
We have shown that depolarising levels of potassium
chloride do not affect the expression of either p75 mRNA
or trkA mRNA in developing trigeminal neurons. In
contrast, depolarisation induces the expression of trkA
mRNA in MAH cells, a retrovirally immortalised sympathoadrenal progenitor cell line (Birren et al., 1992).
Assuming this response is also a feature of normally developing sympathetic neuroblasts, the difference in the regulation of trkA mRNA expression in early sensory and sympathetic neurons could be related to the differences in the
acquisition of neurotrophin responsiveness in these two
classes of neurons. Whereas early sensory neurons acquire
neurotrophin responsiveness autonomously (Davies and
Lumsden, 1984; Ernsberger and Rohrer, 1988; Buchman
and Davies, 1993; Vogel and Davies, 1991), early sympathetic neurons that are cultured before acquiring NGF
responsiveness in vivo, die after several days in vitro even
if NGF is present in the culture medium (Leah and Kidson,
1983; Ernsberger et al., 1989). Early sympathetic neurons
do, however, acquire NGF dependence if retinoic acid is
included in the culture medium (Rodriguez-Tebar and
Rohrer, 1991). However, whereas retinoic acid induces the
expression of high-affinity NGF receptors in early sympathetic neurons (Rodriguez-Tebar and Rohrer, 1991), it does
not induce the expression of trkA mRNA in MAH cells
(Birren et al., 1992). The application of the highly sensitive
techniques that we have used for quantifying p75 and trkA
mRNA levels to low density cultures of normal sympathetic
neuroblasts may help clarify the factors that normally
regulate the expression of NGF receptor genes at successive
stages in their early development.
We are grateful to Luis Parada, Phillip Barker, Susan Meakin
and Eric Shooter for the rat and mouse trkA cDNA clones used in
this study. Our thanks also to Bill Mobley for the NGF and to John
Winslow and Gene Burton of Genentech for the purified recombinant BDNF. This work was supported by a grant from the Medical
Research Council. Some of the work in this paper was carried out
in St. George’s Hospital Medical School, London.
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(Accepted 23 August 1993)