Download Different neurotrophins are expressed and act in a developmental

Development 118, 989-1001 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
989
Different neurotrophins are expressed and act in a developmental
sequence to promote the survival of embryonic sensory neurons
Vladimir L. Buchman and Alun M. Davies
Department of Anatomy, St. George’s Hospital Medical School, Tooting, London SW17 0RE, UK
Present address: School of Biological and Medical Sciences, Bute Medical Buildings, St. Andrews University, St. Andrews, Fife KY16 9AJ, Scotland
SUMMARY
To investigate if different neurotrophins regulate the
survival of neurons at successive developmental stages,
we studied the effect of nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the survival of mouse trigeminal
neurons at closely staged intervals in development. We
show that during the earliest stages of target field innervation trigeminal neurons display a transitory survival
response to BDNF and NT-3. This response is lost as the
neurons become NGF-dependent shortly before
neuronal death begins in the trigeminal ganglion. BDNF
and NT-3 mRNAs are expressed in the peripheral
trigeminal target field before the arrival of the earliest
axons and the onset of NGF mRNA expression. The
levels of BDNF and NT-3 mRNAs peak during the early
stages of target field innervation and decline shortly after
the loss of neuronal responsiveness to BDNF and NT-3.
Our study provides the first clear evidence that different
target-derived neurotrophins can act sequentially to
promote the survival of developing neurons.
INTRODUCTION
synthesis of NGF begins in the peripheral target field with
the arrival of the earliest axons and the level of NGF mRNA
increases throughout the period that trigeminal axons reach
the peripheral target field (Davies et al., 1987). In the developing autonomic nervous system, NGF synthesis also begins
when sympathetic axons reach their targets (Korsching and
Thoenen, 1988).
The demonstration that NGF is a member of an homologous family of neurotrophins (Leibrock et al., 1989; Hohn
et al., 1990; Ernfors et al., 1990; Maisonpierre et al., 1990;
Rosenthal et al., 1990; Jones and Reichardt, 1990; Hallbook
et al., 1991; Berkemeier et al., 1991; Ip et al., 1992) has
raised the issue of whether similar correlations between
target field innervation, neurotrophin synthesis and neuronal
responsiveness exist for these more recently identified neurotrophins. This is especially pertinent as these neurotrophins, in contrast to NGF, have additional roles in
neuronal development before the onset of target field innervation. For example, there is some evidence that brainderived neurotrophin (BDNF) promotes neuronal differentiation in neural crest cell cultures (Sieber, 1991) and
neurotrophin-3 (NT-3) promotes the differentiation of
sensory neurons from their progenitors in early dorsal root
ganglion (DRG) cell cultures (Wright et al., 1992). BDNF
and NT-3 also enhance an early maturational change in
DRG neurons before they become dependent on neurotrophins for survival (Wright, et al., 1992).
For the above reasons, we undertook a detailed developmental study of embryonic mouse trigeminal system to
Work on nerve growth factor (NGF) supports the proposal
that neuronal target fields produce limiting quantities of neurotrophins that promote the survival of the appropriate
number of innervating neurons during development (Davies,
1988; Barde, 1989). NGF is synthesized in the target fields
of developing NGF-dependent neurons, which include sympathetic neurons and certain kinds of sensory neurons.
Experimental manipulation of the availability of NGF to
these neurons when they are innervating their targets influences the number of neurons that survive. Exogenous NGF
prevents naturally occurring cell death in populations of
NGF-dependent neurons, whereas NGF antibodies eliminate
these neurons.
Descriptive studies of the development of the embryonic
mouse trigeminal ganglion, a population of NGF-dependent
sensory neurons, and its innervation of the periphery
(Davies and Lumsden, 1984, 1986; Davies, 1987a; Davies
et al., 1987) have permitted the relationship between NGF
synthesis and NGF receptor expression to be ascertained.
When trigeminal axons are growing to their targets, the
neurons express low levels of the mRNAs encoding the lowaffinity NGF receptor (p75, Wyatt et al., 1990) and the
tyrosine kinase NGF receptor (trk, Wyatt and Davies,
unpublished data) and are unlabelled by iodinated NGF
(Davies et al., 1987). Near the time that the axons reach their
targets, the levels of p75 and trk mRNAs increase markedly
and the neurons become labelled by iodinated NGF. The
Key words: neurotrophins, sensory neurons, trigeminal system,
nerve growth factor, NGF, mouse nervous system
990
V. L. Buchman and A. M. Davies
relate the spatial and temporal patterns of BDNF and NT-3
expression in this system to the phases of neuronal development and neurotrophin responsiveness. We show that
trigeminal neurons exhibit a transient survival response to
BDNF and NT-3 during the earliest stages of target field
innervation that correlates with the early expression of
BDNF mRNA and NT-3 mRNA in the peripheral field of
these neurons.
MATERIALS AND METHODS
Dissection of embryonic tissues
Embryos were obtained from overnight matings of CD1 mice.
Pregnant females were killed by cervical dislocation and the 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.
Maxillary processes and mystacial whisker pads (the cutaneous
derivative of the maxillary process) were dissected from E10 to
E16 embryos. The region of cranial ectoderm and mesenchyme
from which the maxillary process develops was dissected from
E9.5 embryos. Mandibular and hyoid processes were dissected
from E11 embryos. Developing skin of the mandibular and ophthalmic territories was obtained at stages from E11 to E15 and the
hindbrain from stages E9.5 to E15. Dissected tissue was frozen in
siliconised Eppendorf tubes in liquid nitrogen and stored at −70°C.
Separation of cutaneous epithelium and mesenchyme
Developing skin from the maxillary, mandibular and ophthalmic
territories was separated into its component epithelium and mesenchyme by treatment with dispase (Boeringer-Manheim) for 30
minutes at 4°C followed by 5 minutes at 37°C. This preferentially
digests the basal lamina, allowing the sheet of epithelium to be
lifted off the mesenchyme after washing the tissue in Hanks
balanced salt solution (HBSS) containing 10% heat-inactivated
horse serum (HIHS). Tissues were frozen and stored as above.
Neuron cultures
Trigeminal ganglia were incubated for 5 minutes at 37°C with
0.05% trypsin (Worthington) in calcium- and magnesium-free
HBSS. After removal of the trypsin 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 250-500 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 µg/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-thyroxine, 38 ng/ml sodium selenite, 340 ng/ml triiodo-thyronine, 60 mg/ml penicillin and 100 mg/ml streptomycin.
Because morphological criteria are unreliable for identifying
neurons at the earliest stages of their development, the low
molecular weight neurofilament protein was localised by immunocytochemistry to identify positively and count the number of plated
neurons in each experiment. To do this, the cells in several dishes
were fixed with 4% paraformaldehyde 6 to 8 hours after plating.
After permeablizing with 0.05% Triton-X100 in phosphatebuffered saline (PBS) for 30 minutes, the cells were incubated for
3 hours at room temperature with 1:1 dilution of 2H3 monoclonal
antibody supernatant (gift of Tom Jessell). After washing, the
cultures were incubated for 30 minutes with a 1:100 dilution of
FITC-conjugated goat anti-mouse IgG secondary antibody and
were mounted in DABCO beneath coverslips. The number of fluorescent cells within a 12×12 mm square in the centre of each dish
was counted; the mean of these counts was taken as the initial
number of neurons the experiment.
After 24 or 48 hours incubation, neurons were clearly recognized
in early trigeminal cultures by their bipolar morphology under
phase-contrast optics. The percentage neuronal survival in the
absence or presence of different neurotrophins was estimated at
these times by counting the number of neurons in the same 12×12
mm area in each dish and expressing the results as a percentage of
the initial number of neurofilament-positive cells. In each experiment, triplicate cultures were set up for all conditions. The data
illustrated are compiled from the results of at least three separate
experiments.
Neurotrophins were added to the culture medium prior to plating
the neurons. Purified mouse submandibular salivary gland NGF
was a gift of Bill Mobley, UCSF. Purified recombinant full-length
BDNF and NT-3 were gifts of John Winslow and Gene Burton,
Genentech Inc.
To determine if the increased number of early trigeminal
neurons in the presence of BDNF or NT-3 was due to enhanced
neuronal survival, the fate of individual neurons was monitored in
culture (Vogel and Davies, 1991). This was done by recording 6
hours after plating the location of unambiguously identifiable
bipolar neurons within a 9×9 mm grid scored on the undersurface
of culture dishes and monitoring at 6 hourly intervals until 48 hours
whether each of these neurons was surviving or had degenerated.
All results are expressed as a percentage of the number of neurons
in the initial neuronal cohorts at 6 hours. To avoid any effects of
neurotrophins on the size of the initial cohorts, these factors were
added after the identification of cohorts at 6 hours.
Measurement of BDNF mRNA and NT-3 mRNA
Northern blotting was used to measure the level of BDNF mRNA
and NT-3 mRNA in dissected tissue. Total RNA was extracted
using guanidinium isothiocyanate (Chizgwin et al., 1979). After
electrophoresis in 1.2% agarose/formaldehyde gels, the RNA was
blotted to Hybond-N filters (Amersham) and was crosslinked to
these membranes by UV irradiation and baking at 80°C for 2 hours
under vacuum. The filters were hybridized with 32P-labelled cRNA
probes made by in vitro run-off transcription from either the rat
NT-3 cDNA (gift of Marc Tessier-Lavigne, UCSF) or the mouse
BDNF cDNA (gift of Patrick Enfors, Whitehead Institute)
subcloned into the pGEM riboprobe vector downstream of the SP6
promoter. Filters were pre-hybridized at 65°C for 5 hours in the
following solution: 50% formamide, 5× SSC, 50 mM sodium
phosphate pH 7.0, 5 mM EDTA, 0.5% SDS, 5× Denhardt’s
solution, 250 µg/ml salmon sperm DNA and 250 µg/ml E. coli
tRNA. Filters were hybridized at 65°C for 15 hours in the above
solution containing 107 cts/minute/ml of the 32P-labelled cRNA
probe. Filters were washed twice for 15 minutes in 1× SSC with
0.1% SDS at 65°C and twice for 30 minutes in 0.1× SSC with 0.1%
SDS at 65°C before exposure to Fuji X-ray film. On autoradiograms, two NT-3 mRNA transcripts of very similar size (approximately 1.4 kb) and two BDNF mRNA transcripts of 1.6 and 4 kb
were detected. Band intensities on autoradiograms were measured
using a Molecular Dynamics densitometer.
The levels of BDNF mRNA and NT-3 mRNA in tissues were
standardized to the level of β-actin mRNA. To do this, the filters
were boiled for 2× 5 minutes in 0.1× SSC with 0.1% SDS to
remove the BDNF cRNA or NT-3 cRNA probe and were subsequently hybridized with a 1 kb fragment of the mouse β-actin
cDNA labelled by nick-translation. The filters were prehybridized
at 42°C for 5 hours in a solution of the same composition as used
for BDNF and NT-3 cRNA hybridizations and were hybridized at
42°C for 48 hours in the same solution containing 107
cts/minute/ml of the 32P-labelled nick-translated β-actin probe. The
Changing neuronal response to neurotrophins
filters were washed as described above and were exposed to X-ray
film. Densitometry of the actin mRNA band intensities on the
resulting autoradiograms enabled the ratio between the actin
mRNA level and the previously determined BDNF or NT-3 mRNA
level to be calculated for each RNA sample on a filter. To compile
data on the relative BDNF or NT-3 mRNA levels in RNA samples
on different filters (as in the case of RNA samples from each age
series of maxillary or hindbrain tissue), several RNA samples from
each age series were run on the same gel and were northern blotted
onto a single filter. Subsequent hybridization of this filter with the
BDNF or NT-3 probe and the actin probe followed by autoradiography and densitometry permitted standardisation of the relative
BDNF or NT-3 mRNA levels in all age series. The data on the
developmental changes in the relative BDNF mRNA level are
compiled from three separate maxillary and hindbrain tissue age
series and the NT-3 mRNA data are compiled from five separate
maxillary tissue age series.
Because any developmental changes in the level of actin mRNA
expression in target field cells would cause corresponding shifts in
the relative BDNF and NT-3 mRNA levels determined by the
above method, several filters were reprobed with cDNA probes that
hybridize with two other ubiquitous constitutively expressed
RNAs. The L27 ribosomal protein mRNA was detected with a 0.3
kb fragment of the rat L27 cDNA that was labelled by nick-translation. This fragment of the rat L27 cDNA was isolated from rat
brain cDNA by PCR amplification using primers based on the
published L27 cDNA sequence (Tanaka et al., 1988). Filters were
hybridized with the 32P-labelled nick-translated L27 probe as
described for the β-actin probe. The 18S rRNA was detected with
a 32P-labelled cRNA probe that was made by run-off transcription
from the 18S rRNA cDNA (gift of Rolf Heuman) subcloned into
the pGEM vector. Filters were hybridized with this probe as
described previously (Harper and Davies, 1990). The developmental changes in BDNF or NT-3 mRNA levels were virtually
identical when expressed relative to actin mRNA, L27 mRNA or
18S rRNA.
To measure the absolute levels of BDNF mRNA and NT-3
mRNA in the maxillary process at the corresponding peaks of
expression during development, the BDNF or NT-3 mRNA band
intensities in northern blots of RNA samples from known numbers
of maxillary processes were quantified by reference to a series of
calibration standards. The calibration standards were known quantities of unlabelled, sense BDNF or NT-3 RNA transcripts that
were run on gels alongside the tissue RNA samples. These figures
were corrected for RNA losses during extraction, electrophoresis
and blotting by reference to the band intensities of known quantities of shorter unlabelled, sense BDNF or NT-3 recovery standards
that were added to tissue samples prior to RNA extraction, as
described for NGF and p75 mRNAs (Heumann et al., 1984; Wyatt,
1990). The mean levels of BDNF and NT-3 mRNA per maxillary
process were then related to the mean protein content of the
maxillary process determined by the method of Lowry (1951) at
the corresponding ages.
RESULTS
Early trigeminal neurons survive independently of
neurotrophins
When trigeminal neurons were cultured at low-density in
defined medium during the earliest stages of axonal
outgrowth in vivo (E10 and E11), more than 80% of the
neurons identified by neurofilament staining 6 hours after
plating were still alive after 24 hours incubation. In cultures
set up at later stages, the proportion of neurons surviving for
991
24 hours without neurotrophins decreased to less than 5%
in E14 cultures (Fig. 1). After 48 hours incubation, almost
all neurons were dead in cultures of E10 and older neurons
grown without neurotrophins (Fig. 1).
Trigeminal neurons exhibit a transitory survival
response to BDNF and NT-3 prior to naturally
occurring cell death
The survival effects of neurotrophins on cultured embryonic
trigeminal neurons are clearly observed after 48 hours incubation when virtually all neurons have died in control
cultures. Furthermore, since the serum-free medium used in
these experiments is not conducive for the growth of fibroblasts, the cultures remain virtually free of non-neuronal cells.
Thus, neuronal survival in this culture system is unlikely to
be influenced by neurotrophins produced by non-neuronal
cells.
In E10 cultures supplemented with either BDNF or NT3, the number of neurons after 48 hours incubation had
increased to between 120 and 150% of the number identified 6 hours after plating (Fig. 1). This effect of BDNF and
NT-3 on increasing or sustaining the number of neurons in
trigeminal cultures was only observed in cultures set up
during the earlier stages of trigeminal ganglion development. The response to BDNF was lost most rapidly, with
only 5% of the neurons surviving for 48 hours in E13
cultures containing 2 ng/ml of BDNF. The number of
neurons surviving for 48 hours in the presence of 2 ng/ml
of NT-3 had fallen to a similar level by E14 (Fig. 1).
Fig. 2 illustrates typical dose responses of E10 and E12
trigeminal neurons to BDNF. These resembled typical NGF
dose responses (see below) in that they were sigmoidal when
plotted on a semi-log graph. The concentration of BDNF
that promoted half-maximal survival was near the point of
inflexion and had a value in E11 cultures of 1.95±0.25 pg/ml
(mean±s.e.m., n=3 separate dose response experiments, each
set up in triplicate). Typical dose responses of E11, E12 and
E14 trigeminal neurons to NT-3 are also shown in Fig. 2. In
contrast to NGF and BDNF dose responses, these were not
markedly sigmoidal. Fig. 3A,B illustrates the bipolar morphology of early trigeminal neurons grown with BDNF and
NT-3.
Because early sensory neuron cultures contain neuron
progenitor cells that can differentiate in vitro (Rohrer et al.,
1985; Wright et al., 1992), it was necessary to exclude the
possibility that the increased number of early trigeminal
neurons surviving in the presence of BDNF or NT-3 was
due to enhanced proliferation or differentiation of these
progenitor cells. To do this, cohorts of neurons were identified by their bipolar morphology 6 hours after plating and
the fate of the individual neurons in each cohort was
monitored at intervals for the next 42 hours in vitro. BDNF
or NT-3 were added to these cultures after the identification
of neuronal cohorts at 6 hours incubation. Addition was
made at this time because any effect of these factors on
either cell attachment or neuronal differentiation would
influence cohort size and confound the results. In these
cohort experiments, 88%±2.8 (mean±s.e.m., n=6) of
neurons grown with BDNF and 51.8%±6.4 (mean±s.e.m.,
n=6) of neurons grown with NT-3 survived from 6 to 48
hours in culture. In control cultures, none of the neurons
992
V. L. Buchman and A. M. Davies
24hr
100
NGF
160
48hr
BDNF
140
NT-3
80
Percent survival
Percent survival
120
60
40
100
80
60
40
20
20
0
0
9
10
11
12
13
14
15
Embryonic age (days)
9
10
11
12
13
14
15
Embryonic Age (days)
Fig. 1. The survival of E10 to E15 trigeminal neurons in control cultures and neurotrophin supplemented cultures. The left-hand graph
shows the number of neurons surviving after 24 hours (open circles) and 48 hours (closed squares) in control cultures expressed as a
percentage of the number of neurons identified 6 hours after plating. The right-hand graph shows the number of neurons surviving after
48 hours incubation with 2 ng/ml NGF (open circles), 2 ng/ml BDNF (open squares) or 2 ng/ml NT-3 (closed triangles) expressed as a
percentage of the number of neurons identified 6 hours after plating. The mean±s.e.m. of triplicate cultures from three separate
experiments at each age are shown in both graphs.
Fig. 2. Dose-responses of trigeminal neurons to BDNF and NT-3. The left-hand graph shows the number of E11 neurons (filled circles)
and E12 neurons (open circles) surviving after 48 hours incubation with different concentrations of BDNF expressed as a percentage of
the number of neurons identified 6 hours after plating. The right-hand graph shows the number of E11 neurons (filled circles), E12
neurons (open circles) and E14 neurons (triangles) surviving after 48 hours incubation with different concentrations of NT-3 expressed as
a percentage of the number of neurons identified 6 hours after plating. The mean±s.e.m. of triplicate cultures from representative
experiments are shown in both graphs.
identified at 6 hours survived to 48 hours. This indicates that
BDNF and NT-3 have a direct survival-promoting effect on
early trigeminal neurons.
NGF promotes the survival of trigeminal neurons
during the period of naturally occurring cell death
In contrast to the marked survival response of E10 and E11
trigeminal neurons to BDNF and NT-3, NGF promoted the
survival of less than 10% of trigeminal neurons in E10
cultures and only 25% in E11 cultures. By E12, shortly
before the onset of cell death in the trigeminal ganglion,
NGF supported all the neurons. In cultures established
throughout the phase of naturally occurring cell death (E13
to E18), NGF continued to promote the survival of the great
Changing neuronal response to neurotrophins
A
B
C
D
E
F
majority of neurons (Fig. 1 and unpublished data). Fig. 3C
shows the typical bipolar morphology of E12 trigeminal
neurons grown in the presence of NGF. The small percentage of E14 or E15 neurons that survived in the presence of
either BDNF or NT-3 had distinctly larger cell bodies than
neurons of the same age grown in the presence of NGF (Fig.
3D-F).
Although NGF promoted the survival of the majority of
E12 and older trigeminal neurons, there was a marked shift
in the dose response of these neurons to NGF with increasing embryonic age (Fig. 4). The concentration of NGF that
elicits half maximal survival (estimated by interpolation)
increases by an order of magnitude from 4.7 pg/ml at E12
to 53.2 pg/ml at E15.
To investigate whether NGF, BDNF and NT-3 promote
the survival of the same or different subsets of neurons in
the early trigeminal ganglion, E11 and E12 neurons were
grown with each factor alone and with combinations of these
factors. After 48 hours incubation, there was no significant
additional neuronal survival in the presence of any two
factors (Fig. 5). This is clearly seen in E12 cultures in which
NGF promotes the survival of virtually all of the neurons
(98%) and there was no significant increase in the number
of neurons in the presence of either NGF + BDNF (105%)
or NGF + NT-3 (95%) even though BDNF alone and NT-3
alone promoted the survival of 27% and 65% of the neurons,
respectively. This indicates that the early trigeminal
993
Fig. 3. Phase-contrast photomicrographs of trigeminal
neurons. The typical bipolar morphology of E11
trigeminal ganglion neurons grown for 48 hours with 2
ng/ml BDNF (A) or 2 ng/ml NT-3 (B). E12 neurons
grown for 48 hours with 2 ng/ml NGF had a similar
morphology (C) as did E15 neurons grown with NGF
(D). The small proportion of E15 neurons surviving
with 2 ng/ml of either BDNF (E) or NT-3 (F) had larger
cell bodies than the majority of E15 neurons that
survive in the presence of NGF (D). Many E15 neurons
surviving with NGF, BDNF or NT-3 had a unipolar
morphology (illustrated by the two neurons shown in
micrograph E). Scale bar, 50 µm.
Fig. 4. Dose responses of trigeminal neurons to NGF. The number
of neurons at E12 (filled circles), E13 (open circles), E14 (filled
squares) and E15 (open squares) surviving after 48 hours
incubation with different concentrations of NGF is expressed as a
percentage of the number of neurons identified 6 hours after
plating. The mean±s.e.m. of triplicate cultures from typical
experiments are shown.
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V. L. Buchman and A. M. Davies
Fig. 5. The survival of trigeminal neurons grown
neurotrophins singularly and in combination. The percentage
survival of E11 (left-hand bar chart) and E12 (right-hand bar
chart) neurons after 48 hours incubation with neurotrophins
(each at 2 ng/ml) is expressed as a percentage of the number
of neurons identified 6 hours after plating. The mean±s.e.m. of
triplicate cultures from typical experiments are shown.
ganglion does not contain distinct subsets of neurons that
respond to different factors, but that the same neurons
respond to multiple factors.
Developmental changes in BDNF mRNA and NT-3
mRNA expression in the peripheral target field
Northern blotting revealed the presence of 1.6 kb and 4 kb
A
B
C
BDNF mRNA transcripts in the maxillary process (Fig. 6).
At E9.5 and E10, low levels of these transcripts were
present. From E10 to E12, the levels increased over ten-fold
(relative to actin mRNA). From the peak of expression at
E12, the levels fell to lower levels at E13 that remained
largely unchanged through later ages (Fig. 7). Densitometry
revealed that there was an approximate 1:1 ratio between the
1.6 and 4 kb transcripts throughout development (data not
shown). Quantitative northern blotting showed that the mean
level of BDNF mRNA per process (both transcripts
together) at the peak of expression at E12 was approximately 0.9 pg. Relative to the mean protein content of the
maxillary process at E12, the concentration of BDNF
mRNA per process was approximately 60 pg/mg protein.
Northern blotting revealed the presence of two NT-3
mRNA transcripts of very similar size that ran as a doublet
of approximately 1.4 kb (Fig. 6). Low levels of these transcripts were present at E9.5, E10 and E11. The levels then
increased four-fold (relative to actin mRNA) from E11 to
reach a peak of expression at E13. From E13 the level subsequently fell to reach the earlier low levels by E16 (Fig. 7).
Fig. 6. Developmental changes in the expression of BDNF mRNA
and NT-3 mRNA in the maxillary target field. Autoradiograms of
a northern blot of E9.5 to E16 maxillary total RNA hybridized
sequentially with the 32P-labelled BDNF riboprobe (A), the 32Plabelled NT-3 riboprobe (B) and the 32P-labelled nick-translated
actin DNA probe (C). The 1.6 and 4 kb BDNF mRNA transcripts
with maximal expression at E11 and E12 are indicated by small
arrows in A. A 0.95 kb BDNF RNA recovery standard is indicated
by the large arrow on the left-hand side of A. 25 pg of this
standard were added to each tissue same prior to RNA extraction.
Different amounts (2, 5, 10 and 20 pg) of this BDNF RNA
standard were run in separate lanes on the gel to calibrate the
levels of the BDNF recovery standard and BDNF mRNA in each
RNA sample lane. These calibration standards are indicated by a
large arrow on the right-hand side of A. These calibration
standards ran lower in the gel than the recovery standards because
they were not retarded by the presence of additional RNA. The 1.4
kb NT-3 mRNA doublet band with maximal expression between
E12 and E14 (relative to actin mRNA) is indicated by the double
arrows in B. The actin mRNA band is shown in C.
Changing neuronal response to neurotrophins
995
gave results that were virtually identical to those obtained
by standardising the changes to actin mRNA (data not
shown). Furthermore, quantitative northern blotting was
used to estimate the absolute amounts of BDNF mRNA and
NT-3 mRNA in the maxillary target field in two age series
(E10 to E15 in each case). When the results were standardised to the mean protein content of the maxillary target field
at each age, the respective developmental changes in the
levels of BDNF mRNA and NT-3 mRNA were very similar
to those obtained by standardising the changes to actin
mRNA (data not shown). Thus, data compiled from multiple
estimations of BDNF and NT-3 mRNAs levels (relative to
actin mRNA) in the maxillary target field from E9.5 and E16
(Fig. 7) accurately describe the changes in the overall concentrations of these neurotrophin mRNAs in the peripheral
target field prior to and throughout the phase of target field
innervation.
Fig. 7. Developmental changes in the levels of BDNF mRNA and
NT-3 mRNA in the peripheral and central target fields of the
trigeminal ganglion. The mean±s.e.m. of the BDNF mRNA level
(1.6 kb + 4 kb transcripts) relative to actin mRNA in the maxillary
target field (open circles, n=3) and hindbrain (open squares, n=3)
are shown from E9.5 to E16 and E9.5 to E15, respectively. The
mean±s.e.m. of the NT-3 mRNA level relative to actin mRNA in
the maxillary target field (closed circles, n=5) are shown from
E9.5 to E16.
Quantitative northern blotting showed that the mean level of
NT-3 mRNA per maxillary process was approximately 3.2
pg at the peak of expression at E13. Relative to the mean
protein content of the maxillary process at E13, the concentration of NT-3 mRNA per process was approximately 140
pg/mg protein which is just over twice the level of BDNF
mRNA at its peak of expression at E12.
Because BDNF and NT-3 mRNA levels in the developing peripheral target field were standardised to the level of
actin mRNA, any developmental changes in the level of
actin mRNA expression in target field cells would cause corresponding shifts in the relative BDNF and NT-3 mRNA
data. To exclude this possibility, several northern blots of
complete age series of maxillary tissue RNA were rehybridized with 32P-labelled probes that hybridize with two
other ubiquitous constitutively expressed RNAs: the L27
ribosomal protein mRNA and the 18S rRNA. Standardisation of the respective developmental changes in BDNF
mRNA and NT-3 mRNA to either L27 mRNA or 18S rRNA
Developmental changes in BDNF mRNA
expression in the hindbrain
Both the 1.6 and 4 kb BDNF mRNA transcripts were
detected in northern blots of total RNA extracted from the
developing hindbrain from E9.5 to E15 (the latest age
studied). In contrast to BDNF mRNA expression in the
peripheral target field, the levels were much lower in the
central target field and there was no peak in expression
between E10 and E12. Rather there was a gradual increase
in both transcripts (relative to actin mRNA) from E9.5 to
E15 (Figs 7 and 8). An approximate 1:1 ratio between the
levels of the 1.6 and 4 kb transcripts was maintained
throughout development (data not shown).
In contrast to the relatively high level of NT-3 mRNA
expression in the peripheral target field, NT-3 mRNA was
barely detectable in northern blots of E9.5 to E15 hindbrain
total RNA, yet when the same blots were re-screened with
the BDNF cRNA probe, two BDNF mRNA transcripts were
detected (data not shown).
Regional differences in BDNF and NT-3 mRNA
expression in the periphery
To ascertain the relative levels of BDNF mRNA and NT-3
mRNA in the epithelium and mesenchyme of the maxillary
target field, these tissue components were separated by
dispase treatment at ages ranging from E11 to E15 and were
assayed separately for BDNF mRNA and NT-3 mRNA by
northern blotting (Fig. 9). The levels of both BDNF mRNA
and NT-3 mRNA (relative to the levels of either actin
mRNA or L27 mRNA) were clearly higher in the mesenchyme compared with the epithelium during the earliest
stages of target field innervation at E11. By E13, however,
this pattern was reversed for BDNF mRNA with higher
levels in the epithelium compared with the mesenchyme
and, at E14 and E15, the overall levels of BDNF mRNA
were similar in both tissues. In the case of NT-3 mRNA, the
overall levels of this transcript were similar in epithelium
and mesenchyme from E13 to E15.
To determine if there are regional differences in the levels
of BDNF mRNA and NT-3 mRNA in cutaneous targets
during the earliest stages of their innervation, northern
blotting was used to estimate the levels of these mRNA tran-
996
V. L. Buchman and A. M. Davies
A
A
B
Fig. 8. Expression of BDNF mRNA in the developing hindbrain.
Autoradiograms of a northern blot of E9.5 to E15 hindbrain total
RNA (Hb) hybridized sequentially with the 32P-labelled BDNF
riboprobe (A) and the 32P-labelled nick-translated actin DNA
probe (B). The 1.6 and 4 kb BDNF mRNA transcripts are
indicated by small arrows in A. For comparison, similar quantities
of total RNA from E15 and E16 maxillary tissue (Mx) were run
on the gel alongside the hindbrain RNA samples.
scripts in the maxillary, mandibular and hyoid processes and
in the forelimb bud at E11. When standardised to the level
of actin mRNA, the levels of both BDNF mRNA transcripts
were almost 3-fold higher in the maxillary process compared
with the other cutaneous target tissues (Fig. 10). In contrast
to the expression of BDNF mRNA, there was no significant
difference in the level of NT-3 mRNA in the branchial arch
target tissues and the level of NT-3 mRNA in these tissues
was approximately two-fold higher than in the forelimb bud
(Fig. 10).
To determine if there are regional differences in the level
of NT-3 mRNA in cutaneous epithelia from sparsely innervated and densely innervated cutaneous territories during
the earliest stages of naturally occurring neuronal death, we
used northern blotting to estimate the levels of NT-3 mRNA
in the epithelia of the maxillary, mandibular and ophthalmic
territories of trigeminal ganglion at E13 and E14. At the
onset of neuronal death at E13, there were marked differences in the level of NT-3 mRNA (relative to actin mRNA)
in these epithelia. The epithelium of the sparsely innervated
ophthalmic territory possessed the lowest level of NT-3
mRNA, the epithelium of the densely innervated maxillary
territory possessed a 6-fold higher level and the epithelium
of the moderately innervated mandibular territory had the
highest level (Fig. 11). At E14, the levels of NT-3 mRNA
in these epithelia were correlated with final innervation
density. The ophthalmic epithelium possessed the lowest
level of NT-3 mRNA and maxillary epithelium possessed
the highest level.
B
C
D
Fig. 9. Developmental changes in the expression of BDNF mRNA
and NT-3 mRNA in maxillary epithelium and mesenchyme.
Autoradiograms of a northern blot of E11 to E15 total RNA
extracted from maxillary epithelium and mesenchyme hybridized
sequentially with the 32P-labelled BDNF riboprobe (A), the 32Plabelled NT-3 riboprobe (B), the 32P-labelled nick-translated actin
DNA probe (C) and the 32P-labelled nick-translated L27 DNA
probe (D). The 1.6 and 4 kb BDNF mRNA transcripts are
indicated by small arrows in A. Relative to actin mRNA (C) or
L27 mRNA (D) band intensities, the levels of both BDNF mRNA
transcripts are clearly higher in mesenchymal tissue than in
epidermal tissue at E11. The 1.4 kb NT-3 mRNA doublet band is
indicated by the double arrows in B. The relative level of NT-3
mRNA is also higher in mesenchymal tissue at E11. Comparison
of the band intensities in C and D indicate that the levels of actin
mRNA and L27 mRNA are correlated in these different tissues at
developmental stages from E11 to E15.
Changing neuronal response to neurotrophins
997
A
D
B
C
Fig. 10. Regional differences in the expression of BDNF mRNA and NT-3 mRNA in cutaneous tissues during the earliest stages of
sensory innervation. (A-C) Autoradiograms of a northern blot of E11 total RNA extracted from the forelimb bud (FLB) and the hyoid
(Hy), mandibular (Md) and maxillary (Mx) processes hybridized sequentially with the 32P-labelled BDNF riboprobe (A), the 32P-labelled
NT-3 riboprobe (B) and the 32P-labelled nick-translated actin DNA probe (C). The 1.6 and 4 kb BDNF mRNA transcripts with maximal
expression in the maxillary process are indicated by small arrows in A. The similar levels of the 1.4 kb NT-3 mRNA transcript in the
maxillary, mandibular and hyoid processes are seen in B. The actin mRNA band is shown in C. (D) Levels of BDNF mRNA (left-hand
bar chart) and NT-3 mRNA (right-hand bar chart) relative to actin mRNA in the E11 forelimb bud (FLB) and the hyoid (Hy), mandibular
(Md) and maxillary (Mx) processes.
DISCUSSION
Changing responses of developing trigeminal
neurons to neurotrophins
Earlier work on the mouse trigeminal system has shown that
neurite outgrowth from trigeminal ganglion explants occurs
in the absence of neurotrophins during the stage axons are
growing to their targets in vivo (Davies and Lumsden,
1984). Recent studies of avian cranial sensory neurons
grown in low-density dissociated cultures show that these
neurons survive independently of neurotrophins when
cultured before innervating their targets and that the duration
of neurotrophin independence correlates with the distance
and time that it takes their axons to grow to their targets in
vivo (Vogel and Davies, 1991). Here we show that trigeminal neurons cultured in low-density at E10 and E11, when
the earliest axons are growing to their peripheral targets,
survive for 24 hours without neurotrophins. A substantial
number of neurons also survive for 24 hours without neurotrophins in cultures set up at E12 and E13 (Fig. 1).
Because axons are recruited to the trigeminal nerve from
E9.5 to E13 in vivo (Davies and Lumsden, 1984), the
neurons that survive independently of neurotrophins in
culture are probably those whose axons are normally
growing to their targets at the time when the cultures were
set up. By E15, when virtually all trigeminal axons have
reached their targets in vivo, none of the neurons survive
independently of neurotrophins in vitro.
Before trigeminal neurons become responsive to NGF,
their survival is promoted by either BDNF or NT-3. In E10
cultures, virtually all neurons die by 48 hours unless the
medium is supplemented with BDNF or NT-3, but not NGF.
By following the fates of individual neurons from 6 to 48
hours in these cultures we demonstrated that BDNF and NT3 exert a direct survival-promoting effect on early trigeminal neurons.
The earliest trigeminal axons come into proximity with
the peripheral target epithelia of the mandibular process by
E10.5 and the maxillary process by E11 (Davies and
Lumsden, 1984). The finding that the early survival response
of trigeminal neurons to BDNF and NT-3 begins in E10
cultures between 24 and 48 hours in vitro, immediately after
the phase of neurotrophin independence, suggests that the
onset of this response in vivo is closely related to the arrival
of the earliest axons in the peripheral target field. Over the
next few days of development the proportions of neurons
responding to BDNF and NT-3 decrease whereas the proportion responding to NGF increases. The loss of the BDNF
and NT-3 survival responses could be due to the death of a
short-lived population of BDNF- and NT-3-responsive
neurons present in the early trigeminal ganglion. Alternatively, responsiveness to BDNF and NT-3 may be a stage
through which trigeminal neurons pass before becoming
dependent on NGF for survival. The former possibility is
unlikely because the BDNF survival response is over by the
time naturally occurring neuronal death begins in the
998
V. L. Buchman and A. M. Davies
A
B
C
Fig. 11. Regional differences in NT-3 mRNA levels in cutaneous
epithelia. (A,B) Autoradiograms of a northern blot of E13 and
E14 total RNA extracted from maxillary (Mx), mandibular (Md)
and ophthalmic (Op) epithelia hybridized sequentially with the
32P-labelled NT-3 riboprobe (A) and the 32P-labelled nicktranslated actin DNA probe (B). The 1.4 kb NT-3 mRNA doublet
band is indicated by the double arrows in A. High levels of NT-3
mRNA (relative to actin mRNA) are present in the mandibular
epithelium at E13 and in the maxillary epithelium at E14. The
ophthalmic epithelium has much lower levels of NT-3 mRNA at
both ages. The faint band below the main actin mRNA band
observed in B is due to a minor shorter actin mRNA transcript.
(C) Levels of NT-3 mRNA relative to actin mRNA in the
maxillary (Mx), mandibular (Md) and ophthalmic (Op) epithelia
at E13 and E14.
ganglion at E13 (Davies and Lumsden, 1984) and the NT-3
response is waning at this time. The demonstration that there
is no additional neuronal survival in cultures containing
NGF plus either BDNF or NT-3 in E11 and E12 cultures
indicates that the population of neurons that responds to
NGF overlaps with those that respond to BDNF or NT-3.
These findings suggest that there is indeed a switch in the
neurotrophin requirements of developing trigeminal neurons
from BDNF and NT-3 to NGF during the early stages of
target field innervation (Fig. 12).
Not all neurons become refractory to BDNF and NT-3.
Each of these factors promotes the survival of approximately
5% of trigeminal neurons at E14 and at stages throughout
the phase of naturally occurring neuronal death (data not
shown). The observation that these neurons are distinctly
larger than those surviving with NGF suggests that they are
distinct from those supported by NGF at this stage. Work
on the neurons of the embryonic chicken trigeminal
ganglion has clearly shown that during the phase of naturally
occurring neuronal death there are separate populations of
BDNF-responsive and NGF-responsive neurons. The ventrolateral part of the ganglion contains a small number of
large-diameter, placode-derived, BDNF-responsive neurons
(Davies et al., 1986b) and the dorsomedial part of the
ganglion is composed of numerous small-diameter, neuralcrest-derived, NGF-responsive neurons (Davies and
Lindsay, 1985; Davies et al., 1986b). The absence of a
similar anatomical segregation of placode- and neural-crestderived neurons in the embryonic rodent trigeminal
ganglion (Altman and Bayer, 1982; Davies and Lumsden,
1983) precludes similar definitive studies of the existence of
separate NGF- and BDNF-responsive neurons in the midembryonic mouse trigeminal ganglion.
The onset of the NGF survival response lags behind the
BDNF and NT-3 responses. In E10 cultures after 48 hours
incubation, NGF supports less than 5% of neurons that
survive in the presence of BDNF or NT-3. If neuronal development proceeds at the same rate in vitro, then only a very
minor proportion of the neurons in the E12 ganglion in vivo
are capable of responding to NGF. This is some 1.5 days
after the earliest trigeminal axons come into proximity with
target field epithelium in the mandibular process. There is
an increase in the proportion of NGF responsive neurons in
cultures set up at E10 to those set up at E11, but this only
amounts to 25% NGF-responsive neurons in E11 cultures
after 48 hours incubation. At the developmentally equivalent stage in vivo, E13, a prominent dermal plexus of nerve
fibres has formed and the innervation of developing whisker
follicles is well under way. Thus, contrary to the previous
assertion that NGF responsiveness commences in trigeminal neurons with the arrival of their axons in the peripheral
target field (Davies and Lumsden, 1984), our present
findings suggest that NGF responsiveness begins in these
neurons shortly after their axons reach the peripheral target
field.
A striking feature of the NGF dose response in embryonic
trigeminal neurons is that it undergoes a marked developmental shift (Fig. 4). In E12 cultures, when most if not all
neurons have started to respond to NGF, the concentration
of NGF that promotes half-maximal survival is 1.8×10-13 M.
In E15 cultures, however, the concentration of NGF that
Changing neuronal response to neurotrophins
999
Fig. 12. Summary of the developmental changes in neurotrophin responsiveness through which trigeminal ganglion neurons pass. Time in
the life history of a typical trigeminal ganglion neuron is represented from left to right in the diagram (not necessarily drawn to scale).
The onset of axon outgrowth and the approximate times when the peripheral axon reaches the vicinity of its target and the neuron is
susceptible to being eliminated during the phase of naturally occurring neuronal death are shown along the lower part of the diagram.
From the onset of axon outgrowth to the time the axon comes into proximity with its peripheral target, the neuron survives independently
of neurotrophins (upper box). As the axon reaches its target the neurons become responsive to both BDNF and NT-3. There is then a
transitional period during which the neurons respond to NGF as well as BDNF and NT-3. The BDNF response is lost first followed by the
loss of the NT-3 response, leaving the neuron dependent on NGF for survival during the time when it is competing with other neurons for
the limiting supply of NGF. By analogy with in vitro studies on sensory neurons from adult dorsal root ganglia (Lindsay, 1988), the
trigeminal neuron loses its survival dependence on NGF at some later stage of development.
promotes half-maximal survival has increased by over an
order of magnitude to 2×10-12 M. This shift in the NGF dose
response takes place during the phase of naturally occurring
neuronal death in the trigeminal ganglion, which extends
from E13 to E18 in vivo Thus, the number of neurons that
survive this phase of development in vivo is not only limited
by the supply of NGF but is limited by the increasing
requirement of individual trigeminal neurons for NGF.
In summary, our findings suggest that the neurotrophin
requirements of sensory neurons change during development (Fig. 12). Initially, when their axons are growing to
their targets, the neurons survive independently of neurotrophins. When their axons come into proximity with their
peripheral targets, the neurons become responsive to both
BDNF and NT-3. Shortly afterwards, the neurons additionally become responsive to NGF. With the onset of naturally
occurring neuronal death, the neurons lose their response
first to BDNF and then to NT-3. The NGF survival response
is maintained throughout the phase of naturally occurring
neuronal death but there is a marked shift in the NGF dose
response during this period.
Developmental significance of changing
neurotrophin requirements
It is unlikely that the early survival responses of developing
trigeminal neurons to BDNF and NT-3 play a direct role in
regulating the final number of neurons that survive in the
trigeminal ganglion because these responses are largely over
by the time naturally occurring neuronal death begins. It is
possible, however, that the function of these early survival
responses to BDNF and NT-3 may be to sustain the survival
of the neurons whose axons reach the target field during the
early stages of its innervation. This would delay the onset
of neuronal death in the trigeminal ganglion until most of
the neurons have started to innervate the target field, thereby
ensuring that the majority of neurons compete for a supply
of NGF during the same period of development. If, in the
absence of the early BDNF and NT-3 survival responses, the
supply of NGF were limiting from the earliest stages of
target field innervation, it is possible that too many neurons
would be eliminated before the capacity of the growing
target field has increased to support the required number of
neurons. It may also be advantageous for most of the
neurons that innervate a given target field to compete for
survival at the same time because this would maximise the
choice for selectively maintaining neurons on the basis of
the appropriateness of their axon terminations in the target
field.
Changing patterns of neurotrophin gene
expression during development
We have shown that BDNF mRNA and NT-3 mRNA are
expressed in presumptive maxillary tissue as early as E9.5.
This is approximately 1.5 days before the arrival of the
earliest trigeminal axons and the onset of NGF mRNA
expression in the maxillary process (Davies and Lumsden,
1984; Davies et al., 1987). Studies of neurite outgrowth from
trigeminal ganglia co-cultured with appropriate and inappropriate cutaneous target tissues have shown that the uninnervated maxillary process produces a diffusible substance
that promotes and attracts the growth of early trigeminal
neurites (Lumsden and Davies, 1983). It is unlikely,
however, that either BDNF or NT-3 is the trigeminal
chemotropic factor because BDNF and NT-3 are expressed
in both epithelium and mesenchyme whereas the
chemotropic factor is released by the epithelium alone
(Lumsden and Davies, 1986). Furthermore, the production
of the chemotropic factor is apparently restricted to the
1000 V. L. Buchman and A. M. Davies
trigeminal territory whereas BDNF mRNA and NT-3
mRNA are present in both the hyoid process and forelimb
bud during the earliest stages of target field innervation at
E11.
The level of BDNF mRNA remains low in the maxillary
target field at E9.5 and E10, then increases over ten-fold to
reach a peak at E12 followed by a fall to reach a lower level
by E13 that is maintained through later ages. NT-3 mRNA
also shows a peak of expression in the maxillary target field.
However, relative to BDNF mRNA, the increase begins a
day later (during E11), the peak occurs a day later than the
BDNF mRNA peak and the level of NT-3 mRNA does not
fall to comparable BDNF mRNA levels until E16. These
findings together with previous work on the time-course of
NGF mRNA expression in the developing maxillary target
field (Davies et al., 1987) indicate that developmental
changes in neurotrophin mRNA expression in the peripheral
trigeminal territory are correlated with the changing responsiveness of trigeminal neurons to neurotrophins. The early
expression of BDNF mRNA and NT-3 mRNA is related to
the early response of trigeminal neurons to these factors, and
the subsequent expression of NGF mRNA correlates with
the onset of NGF responsiveness. The early peak and decline
in BDNF mRNA relative to NT-3 mRNA accords with the
loss of BDNF responsiveness preceding the loss of NT-3
responsiveness. The expression of BDNF and NT-3 mRNAs
at lower levels after the majority of neurons have lost their
response to BDNF and NT-3 may be required to promote
the survival of the small proportion of trigeminal neurons
that remain dependent on these factors for survival.
In contrast to the early peaks of BDNF mRNA and NT3 mRNA expression in the peripheral trigeminal territory,
the level of BDNF mRNA is very low and NT-3 mRNA is
barely detectable in the hindbrain over the same period of
development. This suggests that early trigeminal neurons
obtain their supply of BDNF and NT-3 mainly, if not exclusively, from their peripheral targets. Although there is no
early peak of BDNF mRNA expression in the hindbrain, the
level increases slowly throughout the early period of
naturally occurring neuronal death in the trigeminal
ganglion. Thus, the hindbrain may be an important source
of BDNF for the small proportion of trigeminal neurons that
remains dependent on this factor for survival later in development. The hindbrain may also be a source of BDNF for
the BDNF-dependent trigeminal mesencephalic neurons
(Davies et al., 1986a, 1987) and placode-derived cranial
sensory neurons (Lindsay et al., 1985; Davies et al., 1986b)
whose central axons innervate various groups of neurons in
the hindbrain.
In addition to showing that each neurotrophin mRNA has
a distinct temporal pattern of expression in the developing
maxillary target field, we have shown that there are regional
differences in the expression of these mRNAs within and
among developing cutaneous territories. Throughout the
early stages of maxillary target field innervation up to the
onset of neuronal death, the level of NGF mRNA (relative
to tissue protein content) in the epithelium is 5-fold higher
than in the mesenchyme (Davies et al., 1987). During the
earliest stages of target field innervation, the levels of both
BDNF mRNA and NT-3 mRNA (relative to either actin
mRNA or L27 mRNA) are higher in mesenchyme than in
epithelium. At later stages, however, the overall levels of
each of these mRNAs are expressed at approximately
similar levels in both tissues. A recent in situ hybridization
study has also shown that NGF, BDNF and NT-3 mRNAs
are expressed in the epidermal and dermal tissues of the
embryonic mouse maxillary target field from E11.5 (the
earliest stage studied) to E17.5 (Schecterson and Bothwell,
1992). NGF mRNA was expressed mainly in the epithelium,
in agreement with previous studies (Davies et al., 1987),
whereas BDNF and NT-3 mRNAs were expressed primarily
in dermal mesenchyme. Although this latter observation
appears to conflict with our finding that the overall levels of
BDNF mRNA or NT-3 mRNA are similar in these tissues
in the later stages of innervation, northern blotting and in
situ hybridization provide complementary information on
mRNA expression. Whereas northern blotting provides
valuable quantitative data on the mean level of neurotrophin
mRNA expression in different tissues, this technique does
not provide information about any regional differences in
mRNA expression within tissus. Thus, both epithelium and
mesenchyme may express similar mean levels of NT-3
mRNA by northern blotting, yet within the mesenchyme
NT-3 mRNA could be preferentially expressed in the presumptive dermal region and give a stronger signal in this
part of the mesenchyme compared with the overlying epithelium in in situ hybridization.
The significance of the different regional patterns of neurotrophin mRNA expression within developing cutaneous
tissues is unclear. It is likely, however, that expression of
BDNF and NT-3 mRNAs at relatively high levels in the
early target field mesenchyme results in BDNF and NT-3
being made available to growing axons from the earliest
stages of reaching the target field. The predominant
expression NGF mRNA with the epithelium and subjacent
mesenchyme indicates that NGF is synthesized in the region
where the great majority of sensory axons terminate and as
such is more likely to play a major role in selectively supporting the survival of neurons that make appropriate axon
terminations in the target field.
We have demonstrated that there are differences in neurotrophin mRNA levels in the epithelia of different
cutaneous territories. Previous work has shown that during
the earliest stages of naturally occurring neuronal death in
the trigeminal ganglion at E13 and E14, there are marked
differences in the level of NGF mRNA in the epithelia of
the maxillary, mandibular and ophthalmic innervation territories; the level is highest in the epithelium of the densely
innervated maxillary territory and lowest in the epithelium
of the sparsely innervated ophthalmic territory (Harper and
Davies, 1990). This suggests that regional differences in
NGF production govern the number of neurons that survive
to innervate each territory. Likewise, in the present study,
we have shown that the levels of NT-3 mRNA in the
epithelia of the maxillary, mandibular and ophthalmic territories during the early stages of naturally occurring neuronal
death at E14 are also correlated with final innervation
density. Thus, regional differences in NT-3 production may
also govern regional differences in innervation density by
the small proportion of trigeminal neurons that retain dependence on NT-3 throughout the phase of naturally occurring
neuronal death.
Changing neuronal response to neurotrophins 1001
In summary, we have demonstrated a sequence of neurotrophin gene expression in developing cutaneous targets
and corresponding changes in the response of the innervating sensory neurons to neurotrophins. How these temporal
patterns of gene expression and neuronal responsivenss are
controlled are important questions that need to be resolved
in future studies in order to provide a more complete understanding of the trophic interactions between neurons and
their targets that govern the formation and maintenance of
appropriate neural connections.
We thank John Winslow and Gene Burton of Genentech for the
purified recombinant BDNF and NT-3, Bill Mobley for the NGF,
Marc Tessier-Lavigne for the rat NT-3 cDNA, Patrick Ernfors for
the mouse BDNF cDNA, Rolf Heuman for the mouse 18S rRNA
cDNA and Tom Jessell for the 2H3 monoclonal antibody. We
thank Sarah Harper for carrying out some preliminary studies of
NT-3 mRNA expression in the trigeminal system. This work was
supported by grants from the Wellcome Trust, Action Research,
Medical Research Council and The Royal Society.
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(Accepted 30 March 1993)