1 Unit of Molecular Neurobiology, MBB, Karolinska Institutet, 171 77 Stockholm,
Sweden
2 Inserm U432, CNRS, Université de Montpellier 2, Montpellier,
France
3 Department of Physiology and Pharmacology, Karolinska Institutet, 171 77
Stockholm, Sweden
4 Laboratory of Oral Neurobiology, Department Biologic and Materials Sciences,
School of Dentistry, University of Michigan, Ann Arbor, MI, USA
* Author for correspondence (e-mail: patrik.ernfors{at}mbb.ki.se)
Accepted 7 January 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: BDNF, NT3, Sensory neurons, Ccochlea, Neurotrophin, Taste, Mouse
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The analysis of knockout mice for the neurotrophins and their receptors
conveyed important information on the requirement of neurotrophins for
selective neuronal subpopulations in the peripheral nervous system
(Bibel and Barde, 2000;
Snider and Wright, 1996
).
These knockout mice show losses of different subpopulations of sensory neurons
in the dorsal root ganglia. NGF/ and
TrkA/ mice show a similar phenotype with a
loss predominantly in the unmyelinated and the small myelinated dorsal root
ganglia neurons conveying nociceptive information, while the NT3 and TrkC
deficient mice lose the large myelinated neurons that convey limb
proprioception. The selective neuronal loss of these specific dorsal root
ganglia subpopulations is correlated with the expression of one particular Trk
receptor in each of these subclasses of neurons. Furthermore, NT3 is expressed
in the intrafusal muscle fibres and in golgi tendon organs, the target tissues
of the large myelinated neurons that convey limb proprioception
(Copray and Brouwer, 1997
;
Oakley et al., 1995
;
Oakley et al., 1997
) and NGF
in the skin, which is a major target of the nociceptive neurons
(Davies et al., 1987
;
Schornig et al., 1993
). Thus,
specificity of these subclasses of dorsal root ganglia neurons is attained by
a selective expression of Trk receptors and by a spatially regulated
expression of their ligands in the respective target tissues.
Unexpectedly, the mechanism for achieving specificity of dorsal root
ganglia neuron subpopulations to neurotrophins is not generic. In most cranial
sensory ganglia, Trk receptors are more widely expressed than in the dorsal
root ganglia. For instance, in both the vestibular and spiral ganglia, TrkB
and TrkC are ubiquitously expressed and BDNF and NT3 are present in both
vestibular and auditory sensory epithelia. Yet, ligand and receptor null
mutant mice display distinct and specific deficits. BDNF is of major
importance for the development of the vestibular system. In
BDNF/ mice there is an 80% loss of
vestibular ganglion neurons and a complete loss of innervation of the target
sensory epithelia at birth, while in NT3/
mice only 20% of the vestibular neurons are lost
(Bianchi et al., 1996;
Ernfors et al., 1995a
;
Ernfors et al., 1994a
;
Ernfors et al., 1995b
). In
contrast to the vestibular neurons, the majority of spiral ganglion neurons
depend on NT3 for survival (90% loss in NT3/
mice) while BDNF supports only about 10% at birth
(Ernfors et al., 1994a
;
Ernfors et al., 1995b
;
Farinas et al., 1994
;
Fritzsch et al., 1997a
). Most
spiral ganglion neurons lost in NT3/ mice
are type I neurons that constitute around 90% of the ganglion and that
innervate inner hair cells (IHC) while nearly all type II neurons are lost in
BDNF/ mice. These amount to about 10% of the
ganglion and innervate outer hair cells (OHC)
(Ernfors et al., 1995b
). In
addition to this specific action of selective neurotrophins upon distinct
spiral neuron subclasses, BDNF and NT3 might also act in a spatial gradient.
Neuronal loss in NT3/ mice is almost
complete in the basal part of the cochlea with the remaining few percent are
localised apically. In contrast, BDNF/ mice
an almost complete loss of type II neurons and OHC innervation. However, in
these mice a minor residual OHC innervation is more pronounced in the base
than in the apex (Farinas et al.,
2001
; Fritzsch et al.,
1997a
). A similar degree of specificity has been observed in
TrkB/ and
TrkC/ mice
(Fritzsch et al., 1998
). These
results suggest that BDNF and NT3 are not functionally redundant in the inner
ear even though TrkB and TrkC have overlapping expression in individual
neurons of both the vestibular and spiral ganglia
(Ernfors et al., 1992
;
Ylikoski et al., 1993
).
TrkB and TrkC but not TrkA expression has been demonstrated in the
geniculate ganglion, which largely provides innervation for the tongue
(Ernfors et al., 1992). The
expression of TrkB in the geniculate ganglion agrees with results showing
expression of BDNF selectively in taste buds of gustatory papillae.
Consistently, there is also a distinct difference in the lingual phenotypes in
the BDNF/ and
NT3/ mice.
BDNF/ mice display a specific set of
deficits in their peripheral gustatory system, including general loss of taste
buds, innervation deficits in the remaining taste buds, and fewer and
malformed tongue papillae. Instead, NT3/
animals lose their lingual somatosensory innervation
(Nosrat et al., 1997
;
Zhang et al., 1997
).
We have generated mice in which the coding exon of the BDNF gene has been replaced with NT3. Thus, the normal temporal and spatial expression of BDNF during development has been replaced by NT3. In the auditory system, we find that replacement with NT3 largely restores neuronal survival, target innervation, and hearing, indicating that the specificity of BDNF in the inner ear is attained by the temporal and spatial ligand availability. In the vestibular system we find a rescue from neuronal death but a loss of innervation and function (and as a consequence an additional delayed loss of neurons). Loss of geniculate taste neurons and inappropriate target innervation in the gustatory system of BDNFNT3/NT3 mice were similar to those of BDNF/ mice, indicating that a specific BDNF-TrkB activation is required for proper development of the peripheral gustatory system.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR
DNA was extracted from tails and used for genotyping with the following
primers:
A mixture of BL1, BL2 and PGK was used to determine the BDNFNT3/NT3 and wild-type alleles. NT3-04 and BL2 as well as Neo-3' and Neo-5' were used to distinguish between loxed, BDNFNT3lox/NT3lox, and not loxed, BDNFNT3/NT3, animals.
In situ hybridisation
For in situ hybridisation fresh frozen brains from postnatal day 14 (P14)
mice were used. 35S-labeled riboprobe in situ hybridisation was
performed as previously described (Trupp
et al., 1997). Riboprobes complementary to rat BDNF and
mouse NT3 coding sequence were used. The probes were generated from
linearised plasmids using T3 and T7 RNA polymerase and 35S-labelled
-UTP. Slides were exposed to ß-max X-ray film (Amersham) for 4
days.
BDNF and NT3 ELISA
Brains from P14 mice were removed and the hippocampus and part of the
cortex were dissected out, frozen fresh and kept at 70°C until
further processing. The tissue was homogenised using syringes with decreasing
diameter down to 0.7 mm. BDNF and NT3 ELISA were then performed according to
the manufacturer's protocol (Promega).
Auditory brainstem response (ABR)
ABR was recorded in anaesthetised mice (0.05 mg ketalar and 0.003 mg rompun
per gram body weight i.p.) as previously described
(Duan et al., 2000). The
stimuli consisted of full cycle sine waves at 6.3, 8, 12.5 and 20 kHz
synthesised digitally by SigGen software [Tucker Davis Technologies (TDT),
Florida, USA]. The potentials were amplified by 100,000, averaged for 1000
sweeps, and then processed with BioSig software (TDT, Florida, USA). Threshold
was defined as the lowest intensity at which a visible ABR wave was seen in
two averaged runs.
DiI tracing
Wild type (n=14), BDNFNT3/NT3 (n=11),
and BDNF/ (n=7), were sacrificed by
cervical dislocation at P5. The heads were removed and the brain lifted out.
The intact fifth and seventh nerve were identified and cut to reveal the
eighth cranial nerve. The eighth nerve was cut and DiI crystals (1,
1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate; Molecular Probes) were applied directly to the end of the eighth
nerve. The tissue was incubated in 4% PFA at 37°C for 2 days. The cochleae
were dissected into 3 pieces each being approximately 2 mm long and analysed
using fluorescence microscopy. Confocal images were acquired and the number of
OHCs in row one was estimated in each dissected part. The number of nerve
fibres projecting to the first, second and third row of OHCs was then
determined and results presented as percentage of the number of OHCs per
dissected part (apex, middle or base).
Semithin sections
Semithin 1 µm plastic sections were prepared as previously described
(Ernfors et al., 1995b).
Tissue preparation
Embryos were obtained from overnight mating and the morning of the
appearance of the vaginal plug was considered as embryonic day 0 (E0). Tissues
were immersion fixed in 4% PFA overnight, equilibrated in 10% PFA, followed by
20% sucrose, frozen and sectioned in a cryostat at a thickness of 14
µm.
Immunohistochemistry
Immunohistochemistry was performed on 14 µm sections or on whole mounts
of the inner ear and on primary cell cultures using primary antibodies against
the p75 receptor (rabbit 9651; 1:200) or monoclonal acetylated tubulin 6-11B1
(1:500; Sigma) together with FITC-conjugated phalloidin (1:80; Molecular
Probes) or calretinin (1:250; Swant Antibodies) and NF200 N52 (1:400; Sigma)
as previously described (Ernfors et al.,
1995b). Immunohistochemistry on tongue with antiserum against
protein gene product 9.5 (PGP 9.5; Chemicon) was performed as previously
described (Nosrat et al.,
1997
). Photomicrographs were obtained on a confocal microscope
(Zeiss LSM510 or BioRad Radiance 2100). Selected levels or single projections
of stacked images were used.
Quantification
14 µm cryostat serial sections were stained with Cresyl Violet. Neuronal
numbers were established by counting neurons with a clear nucleus and nucleoli
in every third section in spiral and vestibular ganglia (in the DRG and
geniculate ganglia all sections were counted, in the trigeminal ganglion every
12th section and in the nodose ganglion every sixth). The number of neurons
counted was multiplied by section separation to give a total estimated number
of profiles (n). This number was multiplied by section thickness (T), divided
by T plus the average diameter of the nuclei (D) to give the neuronal number
(N); N=nxT/(T+D) (Abercrombie,
1946; Coggeshall,
1992
).
Cell culture
Isolated vestibular ganglion cells were obtained from 3-day old wild-type
Balb/C mice via enzymatic (trypsin and collagenase, 1 mg/ml each, for 5
minutes at 37°C) and mechanical dissociation. The cells were plated at a
density of 500 cells/mm2 on laminin (10 µg/ml) coated coverslips
in serum-free MEM Earle's (GIBCO BRL) supplemented with defined additives N2
(2%), glucose (4.5 g/l), glutamine (0.3 g/l), pyruvate (0.1 g/l) and
penicillin (100 U/ml). All additives were purchased from Life Technologies,
penicillin and enzymes from Sigma. Vestibular sensory epithelial cells from
wild type, BDNF/, or from
BDNFNT3/NT3 mice were seeded on wild-type vestibular
ganglion cell cultures after dissociation with DNAse (0.5 mg/ml; Roche
Diagnostics), protease type X and collagenase (both at 1 mg/ml; Sigma) in MEM
Earl's (GIBCO). Co-cultures were fixed after 6 to 9 days of development in
vitro and stained using immunohistochemistry for neurons and hair cells.
Scanning electron microscopy (SEM)
Tongues were fixed in 4% PFA, rinsed in PBS and kept at 4°C. Upon use,
tongues were dehydrated in a graded serie of ethanol that was exchanged during
three subsequent washes in hexamethyldisilazane (HMDS). The HMDS was allowed
to evaporate in a fume hood overnight. The tongues were then mounted on stubs,
lightly sputter-coated with gold/palladium, and studied in a scanning electron
microscope.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
Neuronal survival but not innervation and function is rescued in the
vestibular system
Both NT3 and BDNF mRNA are strongly expressed in the sensory epithelia of
the saccular and utricular maculae during development
(Ernfors et al., 1992;
Pirvola et al., 1992
). The
levels of NT3 decrease with maturation and at P9 the expression is almost gone
(Fig. 5A). BDNF mRNA, but not
NT3 mRNA is also seen in the ampullar cristae
(Pirvola et al., 1992
). As in
the spiral ganglion, both TrkB and TrkC are expressed in the embryonic,
postnatal as well as adult vestibular ganglion with an evenly distributed
pattern throughout most or all neurons
(Ylikoski et al., 1993
). If
the mechanisms establishing specificity of the neurotrophins and their
receptors were the same in the vestibular system as in the cochlea, the
balance defects of BDNF/ mice should be
rescued in BDNFNT3/NT3 mice. However,
BDNFNT3/NT3 mice displayed a similar defect in balance as
BDNF/ mice.
|
BDNF/TrkB but not NT3/TrkC elicit terminal innervation and maturation
of functional sensory nerve endings
The failure to innervate the saccular and utricular maculae despite the
survival of many vestibular ganglion neurons suggested that BDNF and NT3 play
different roles in the vestibular ganglion neurons. We therefore first
determined whether the reduced innervation is caused by a failure to maintain
the nerves or whether the sensory nerve fibres never innervate the saccular
and utricular maculae. Already at E16, at which time the fibres should have
reached the epithelium but not yet formed calyces,
BDNFNT3/NT3 mice showed less innervation
(Fig. 6C) compared with
wild-type mice (Fig. 6A).
Similar results were obtained in neonatal mice
(Fig. 6D-F). At all these
stages, nerve fibres were seen in the subepithelial layer with sparse
innervation of the sensory epithelia, suggesting that BDNF is only required
for terminal innervation and formation of functional nerve endings, and not
for nerve fibres to project to the utricular and saccular compartments. The
ampullar cristae completely lacked innervation in both
BDNF/ and BDNFNT3/NT3
mice as shown by p75 immunohistochemistry
(Fig. 6G-I).
|
|
The NT3/NT3 alleles supports neither survival of taste
neurons, nor proper innervation of the peripheral gustatory system
In the rodent tongue, BDNF and NT3 show distinct non-overlapping expression
patterns. BDNF mRNA is expressed in the epithelium of the developing gustatory
papillae prior to the formation of taste buds and continues to be expressed
throughout adulthood (Nosrat et al.,
1996; Nosrat and Olson,
1995
). NT3 mRNA is located in those areas were BDNF is not
expressed, such as the superior surface and the lateral epithelium of the
fungiform papillae (Nosrat et al.,
1996
) (Fig.
8A).
|
In order to assess whether these BDNF/-like deficits in BDNFNT3/NT3 mice were caused by a loss of gustatory neurons, neurons in the geniculate ganglion were counted, which in part consists of neurons projecting to fungiform taste buds. The geniculate ganglion in BDNFNT3/NT3 mice showed the same extent of neuronal loss as in BDNF/ mice, compared to wild-type mice (Fig. 8H). We conclude that NT3 can neither preserve the BDNF-dependent gustatory innervation of the taste buds, nor rescue the neurons from death in the peripheral gustatory system, indicating that NT3 does not play a physiological role as a ligand for TrkB in vivo in this system.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A selective expression of receptors in separate subpopulations of dorsal
root ganglia has been shown to occur as a mechanism of creating specificity of
neurotrophins. BDNF/,
NT3/ and
TrkB/ mice
(Fritzsch et al., 1997b;
Nosrat, 1998
;
Nosrat et al., 1997
), as well
as transgenic mice overexpressing truncated TrkC receptors
(Palko et al., 1999
) show
distinct differences in the role of BDNF and NT3 in the complex innervation of
the tongue. In mice in which NT3 or its receptor has been manipulated,
gustatory neurons and innervation are not affected, while manipulating the
function of BDNF or TrkB leads to defects in gustatory innervation and loss of
geniculate ganglion neurons. By placing NT3 in the BDNF
locus we have challenged this sensory system by expressing NT3 at the level,
time and location where BDNF is normally present. Expression of NT3 in the
place of BDNF did not rescue any of the observed phenotypes of
BDNF/ mice in the gustatory system. Thus,
specificity of BDNF is not alone elicited by the distinct expression patterns
of the ligands. In situ hybridisation studies have shown that TrkB mRNA is
expressed in most cells in the geniculate ganglion at E13 to E18 in the rat
while TrkC is expressed only at E13, with very few cells showing expression at
later stages during embryonic development
(Ernfors et al., 1992
). NT3
mRNA is also expressed in the embryonic geniculate ganglion
(Ernfors et al., 1992
). The
early expression of TrkC could therefore correlate with a local role for NT3
during neurogenesis, as previously shown for NT3 in the early forming dorsal
root ganglia (ElShamy and Ernfors,
1996
; Farinas et al.,
1996
). In agreement with this, the nestin-driven NT3
overexpressing transgenic mice, in which NT3 is expressed in neuronal
precursor cells, appear to have a normal tongue surface morphology and
innervation, while other sensory systems where NT3 is physiologically provided
from the target show severe malformations
(Ringstedt et al., 1997
).
Combined with the in situ hybridisation study for TrkB, our results suggest
that the failure of rescue of the gustatory system in
BDNFNT3/NT3 mice is, just as in the dorsal root ganglia
during target innervation, caused by a temporally selective expression of TrkB
in the gustatory neurons that prevent these neurons from responding to NT3 and
as a consequence, the neurons fail to survive and innervate the target tissue.
The failure of NT3-dependent perigemmal somatosensory nerve fibres to invade
the intragemmal territory, which in these mice express NT3, indicates that in
addition to neurotrophins stimulating innervation of these territories, other
mechanisms participate in preventing innervation of a tissue by a particular
class of sensory neurons (Rochlin and
Farbman, 1998
; Rochlin et al.,
2000
).
In BDNF/ and
NT3/ mice, the loss of spiral ganglion
neurons at birth coincide with the number of type II (8-10%) and type I
neurons (around 90%) in the spiral ganglion, respectively. In both
BDNF/ and
TrkB/ mice there is a highly selective loss
of OHC innervation and death of type II spiral ganglion neurons
(Ernfors et al., 1995b;
Minichiello et al., 1995
;
Schimmang et al., 1995
). Based
on the preferential loss of type II neurons in the BDNF mutant mice, the loss
of 90% of spiral ganglion neurons in NT3/
mice and the complete loss of all neurons in double mutant mice
(Ernfors et al., 1995b
), we
hypothesised that NT3 must be acting primarily on type I neurons innervating
IHCs (Ernfors et al., 1995b
).
Recent expression analysis using lacZ knock-in mice in the
NT3 and BDNF loci, suggest an apical-basal difference in the
levels of BDNF and NT3 expression, with NT3 being preferentially expressed in
the base and BDNF in the apex (Farinas et
al., 2001
). In contrast to the above studies, the most pronounced
deficit in the BDNF/ mice reported by
Farinas et al. is that of a marked selective loss of radial fibres in the
apex, indicating a general loss of both IHC and OHC innervation and type I and
type II neurons in the apex, but not in the middle and basal turns of the
cochlea (Bianchi et al., 1996
;
Farinas et al., 2001
). Our
results from a large number of DiI tracings (n=32) reveal that there
is no loss of radial fibres or IHC innervation in either
BDNF/ or BDNFNT3/NT3
mice. This finding is supported by an independent analysis of the presence of
radial fibres using immunohistochemical staining. It has been shown that the
number of radial fibres per IHC in the apex is only one third of the number in
the middle and base of the cochlea
(Liberman et al., 1990
). It is
also well known that the apex is not as well organised as the middle and basal
regions of the cochlea; stereocilia can often be immature and the hair cell
rows in the apex are sometimes disorganised with extra outer or inner hair
cells added (Borg and Viberg,
1995
; Bredberg,
1968
; Roth and Bruns,
1992
). It is possible that the variation of radial fibres in the
apex reported in some previous studies
(Bianchi et al., 1996
;
Farinas et al., 2001
) is due
to biological variation rather than a direct consequence of the BDNF null
allele.
Another apical-basal difference reported in
BDNF/ and
TrkB/ mice is that some OHC innervation is
retained in the basal portion of the cochlea
(Bianchi et al., 1996;
Fritzsch et al., 1997c
).
However, none of these studies have been quantitative and the extent of the
retained innervation in BDNF/ mice has thus
been unknown. Our semi-quantitative measurements showed a very limited
residual OHC innervation present at all turns of the cochlea, but those that
were present showed an apical-basal difference in the amount of fibres. In
addition, we found a gradient along the three rows of OHCs where the third row
of OHCs did not receive innervation at any turn of the cochlea in the
BDNF/ mice. Taken together, the results
reported here are consistent with our earlier results (Ernfors, 1995b), and
demonstrate unequivocally that the main function of BDNF is to specifically
support spiral type II neurons and OHC innervation.
Neuronal survival, innervation of the cochlea and hearing was rescued in
BDNFNT3/NT3 mice. These results imply that NT3/TrkC
promotes spiral ganglion type II neuron survival and innervation similarly
(but not equally) to BDNF/TrkB, provided that the ligand is expressed in the
right place and at the right time. Thus, we conclude that the specificity of
BDNF in the cochlea is dependent on a controlled spatial and temporal
regulation of ligand expression. This finding raises another issue. Why does
NT3 expressed by the IHC not attract innervation of the type II spiral
ganglion neurons, which we show in this study responds to NT3 similarly to
BDNF? The parsimonious explanation might be that type I and type II spiral
ganglion neuron phenotypes are induced by the target they innervate, as are
certain properties of both motor neurons and sensory dorsal root ganglia
neurons determined by their targets (Arber
et al., 2000). One major difference between the OHC and IHC
innervation is that while many type I neurons converge onto each IHC, each
type II neuron innervates at least 10 OHC through spirally running fibres.
Consistent with results in the BDNFNT3/NT3 mice, showing
reduced branching and a reduction of spiral nerve fibres, mice in which NT3
has been replaced by BDNF display a massive hypertrophy of nerve fibres with
spirally running fascicles at the level of the OHCs. These fibres appear to
fail to innervate the hair cells. There also seems to be a recruitment of
neurons projecting to the OHC as seen by thick fascicles of radial fibres
projecting to the OHCs (Coppola et al.,
2001
).
Because there are both TrkB and TrkC mRNAs in the vestibular ganglion
neurons (Pirvola et al.,
1994), the simplest explanation for any specificity in the
vestibular system would be, just as in the cochlea, a temporal and spatially
restricted expression of the different ligands. In
BDNF/ mice 85% of the neurons are lost at
birth and the remaining are atrophic, and at 2 weeks after birth all neurons
are gone (Bianchi et al., 1996
;
Ernfors et al., 1994a
;
Ernfors et al., 1995b
) while
only 20-30% die in the NT3/ mice
(Ernfors et al., 1994b
;
Ernfors et al., 1995b
;
Farinas et al., 1996
). We
found that the NT3/NT3 alleles rescued many of the vestibular
ganglion neurons from death and some innervation of the utricular and saccular
maculae but not the crista. We have however, with NF staining, observed one
calyx formation in the crista of one BDNFNT3/NT3 mouse
(data not shown). It is conceivable that the progressive postnatal loss of
neurons in the BDNFNT3/NT3 mice is caused by reduced
levels of NT3 reaching the vestibular axons as a consequence of the failure of
the neurons to innervate the sensory epithelium.
It is intriguing that most or all vestibular afferents reach the
subepithelial layer of the sensory organs but do not properly invade the
epithelia in the BDNFNT3/NT3 mice. We found occasional
nerve fibres that did invade the epithelia, but these fibres shot through the
hair cell layer and ended up on the surface of the epithelium and failed to
form calyces. These results suggest that NT3 activation of TrkC is not
equivalent to BDNF activation of TrkB and indicates that TrkB is required for
two developmental processes that cannot be replaced by TrkC; (i) invasion of
vestibular afferents into the sensory epithelium and (ii) inducing hair cell
contact and formation of the highly specialised sensory nerve endings in the
vestibular system (calyces). Since these functions of BDNF have not previously
been addressed we set up an in vitro model that would allow us to directly
dissect the physiological role of BDNF produced in the target cells. These
experiments confirmed a role for BDNF in terminal arborisation and maturation
of calyces. There are several previous lines of evidence that has indicated
the importance of BDNF activation of TrkB in target innervation. TrkB and TrkC
have been proposed to elicit survival in vivo through similar intracellular
signalling pathways and this pathway does not involve the Shc adaptor binding
sites of the receptors (Postigo et al.,
2002). In contrast, mice carrying a signalling mutation of the Shc
adaptor-binding site showed a loss of target innervation in the case of the
TrkB receptor whereas in the TrkC, the surviving neurons maintained target
innervation. Consistently, although both BDNF and NT3 have been shown to cause
growth cone turning in an in vitro assay, they do it by different
intracellular signalling pathways (Song et
al., 1997
). Thus, activation of TrkB and TrkC is not equivalent
and we show in this study that BDNF activation of TrkB, but not NT3 activation
of TrkC, is required for target innervation and synaptic formation in
vivo.
We show that specificity of neurotrophins is achieved by multiple
mechanisms during development of the nervous system. These include selective
receptor expression in neuronal populations, selective ligand expression in
different targets and different signalling between Trk receptors. Conceivably,
many other mechanisms can lead to distinct read-outs between different
neurotrophins in selective neuronal populations, such as differences in levels
of expression of both full-length receptors as well as differences in the
proportion of endogenous truncated dominant negative and scavenging TrkB and
TrkC receptors to full-length receptors
(Biffo et al., 1995;
Eide et al., 1996
;
Ninkina et al., 1996
;
Palko et al., 1999
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abercrombie, M. (1946). Estimation of nuclear population from microtome sections. Anat. Rec. 94,239 -247.
Arber, S., Ladle, D. R., Lin, J. H., Frank, E. and Jessell, T. M. (2000). ETS gene Er81 controls the formation of functional connections between group Ia sensory afferents and motor neurons. Cell 101,485 -498.[Medline]
Barbacid, M. (1994). The Trk family of neurotrophin receptors. J. Neurobiol. 25,1386 -1403.[Medline]
Bianchi, L. M., Conover, J. C., Fritzsch, B., DeChiara, T.,
Lindsay, R. M. and Yancopoulos, G. D. (1996). Degeneration of
vestibular neurons in late embryogenesis of both heterozygous and homozygous
BDNF null mutant mice. Development
122,1965
-1973.
Bibel, M. and Barde, Y. A. (2000).
Neurotrophins: key regulators of cell fate and cell shape in the vertebrate
nervous system. Genes. Dev.
14,2919
-2937.
Biffo, S., Offenhauser, N., Carter, B. D. and Barde, Y. A.
(1995). Selective binding and internalisation by truncated
receptors restrict the availability of BDNF during development.
Development 121,2461
-2470.
Borg, E. and Viberg, A. (1995). Extra inner hair cells: prevalence and noise susceptibility. Hear. Res. 83,175 -182.[CrossRef][Medline]
Bredberg, G. (1968). Cellular pattern and nerve supply of the human organ of Corti. Acta Otolaryngol. 236, 1-135.
Coggeshall, R. E. (1992). A consideration of neural counting methods. Trends Neurosci. 15, 9-13.[CrossRef][Medline]
Coppola, V., Kucera, J., Palko, M. E., Martinez-De Velasco, J.,
Lyons, W. E., Fritzsch, B. and Tessarollo, L. (2001).
Dissection of NT3 functions in vivo by gene replacement strategy.
Development 128,4315
-4327.
Copray, J. C. and Brouwer, N. (1997). Neurotrophin-3 mRNA expression in rat intrafusal muscle fibres after denervation and reinnervation. Neurosci. Lett. 236, 41-44.[CrossRef][Medline]
Davies, A. M., Bandtlow, C., Heumann, R., Korsching, S., Rohrer, H. and Thoenen, H. (1987). Timing and site of nerve growth factor synthesis in developing skin in relation to innervation and expression of the receptor. Nature 326,353 -358.[CrossRef][Medline]
Davies, A. M., Minichiello, L. and Klein, R. (1995). Developmental changes in NT3 signalling via TrkA and TrkB in embryonic neurons. EMBO J. 14,4482 -4489.[Abstract]
Duan, M., Agerman, K., Ernfors, P. and Canlon, B.
(2000). Complementary roles of neurotrophin 3 and a
N-methyl-D-aspartate antagonist in the protection of noise and
aminoglycoside-induced ototoxicity. Proc. Natl. Acad. Sci.
USA 97,7597
-7602.
Eide, F. F., Vining, E. R., Eide, B. L., Zang, K., Wang, X. Y.
and Reichardt, L. F. (1996). Naturally occurring truncated
trkB receptors have dominant inhibitory effects on brain-derived neurotrophic
factor signaling. J. Neurosci.
16,3123
-3129.
ElShamy, W. M. and Ernfors, P. (1996). A local action of neurotrophin-3 prevents the death of proliferating sensory neuron precursor cells. Neuron 16,963 -972.[Medline]
Ernfors, P., Kucera, J., Lee, K. F., Loring, J. and Jaenisch, R. (1995a). Studies on the physiological role of brain-derived neurotrophic factor and neurotrophin-3 in knockout mice. Int. J. Dev. Biol. 39,799 -807.[Medline]
Ernfors, P., Lee, K. F. and Jaenisch, R. (1994a). Mice lacking brain-derived neurotrophic factor develop with sensory deficits. Nature 368,147 -150.[CrossRef][Medline]
Ernfors, P., Lee, K. F., Kucera, J. and Jaenisch, R. (1994b). Lack of neurotrophin-3 leads to deficiencies in the peripheral nervous system and loss of limb proprioceptive afferents. Cell 77,503 -512.[Medline]
Ernfors, P., Merlio, J.-P. and Persson, H. (1992). Cells expressing mRNA for neurotrophins and their receptors during embryonic rat development. Eur. J. Neuroscience 4,1140 -1158.[Medline]
Ernfors, P., Van De Water, T., Loring, J. and Jaenisch, R. (1995b). Complementary roles of BDNF and NT-3 in vestibular and auditory development. Neuron 14,1153 -1164.[Medline]
Ernfors, P., Wetmore, C., Olson, L. and Persson, H. (1990). Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family. Neuron 5,511 -526.[Medline]
Farinas, I., Jones, K. R., Backus, C., Wang, X. Y. and Reichardt, L. F. (1994). Severe sensory and sympathetic deficits in mice lacking neurotrophin-3. Nature 369,658 -661.[CrossRef][Medline]
Farinas, I., Jones, K. R., Tessarollo, L., Vigers, A. J., Huang,
E., Kirstein, M., de Caprona, D. C., Coppola, V., Backus, C., Reichardt, L. F.
et al. (2001). Spatial shaping of cochlear innervation by
temporally regulated neurotrophin expression. J.
Neurosci. 21,6170
-6180.
Farinas, I., Wilkinson, G. A., Backus, C., Reichardt, L. F. and Patapoutian, A. (1998). Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo. Neuron 21,325 -334.[Medline]
Farinas, I., Yoshida, C. K., Backus, C. and Reichardt, L. F. (1996). Lack of neurotrophin-3 results in death of spinal sensory neurons and premature differentiation of their precursors. Neuron 17,1065 -1078.[Medline]
Fritzsch, B., Barbacid, M. and Silos-Santiago, I. (1998). The combined effects of trkB and trkC mutations on the innervation of the inner ear. Int. J. Dev. Neurosci. 16,493 -505.[CrossRef][Medline]
Fritzsch, B., Farinas, I. and Reichardt, L. F.
(1997a). Lack of neurotrophin 3 causes losses of both classes of
spiral ganglion neurons in the cochlea in a region-specific fashion.
J. Neurosci. 17,6213
-6225.
Fritzsch, B., Sarai, P. A., Barbacid, M. and Silos-Santiago, I. (1997b). Mice with a targeted disruption of the neurotrophin receptor trkB lose their gustatory ganglion cells early but do develop taste buds. Int. J. Dev. Neurosci. 15,563 -576.[CrossRef][Medline]
Fritzsch, B., Silos-Santiago, I., Bianchi, L. M. and Farinas, I. (1997c). Effects of neurotrophin and neurotrophin receptor disruption on the afferent inner ear innervation. Semin. Cell. Dev. Biol. 8,277 -284.[CrossRef][Medline]
Jones, K. R., Farinas, I., Backus, C. and Reichardt, L. F. (1994). Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development. Cell 76,989 -999.[Medline]
Liberman, M. C., Dodds, L. W. and Pierce, S. (1990). Afferent and efferent innervation of the cat cochlea: quantitative analysis with light and electron microscopy. J. Comp. Neurol. 301,443 -460.[Medline]
Minichiello, L., Piehl, F., Vazquez, E., Schimmang, T., Hokfelt,
T., Represa, J. and Klein, R. (1995). Differential effects of
combined trk receptor mutations on dorsal root ganglion and inner ear sensory
neurons. Development
121,4067
-4075.
Ninkina, N., Adu, J., Fischer, A., Pinon, L. G., Buchman, V. L. and Davies, A. M. (1996). Expression and function of TrkB variants in developing sensory neurons. EMBO J. 15,6385 -6393.[Abstract]
Nosrat, C. A. (1998). Neurotrophic factors in
the tongue: expression patterns, biological activity, relation to innervation
and studies of neurotrophin knockout mice. Ann. NY Acad.
Sci. 855,28
-49.
Nosrat, C. A., Blomlof, J., ElShamy, W. M., Ernfors, P. and
Olson, L. (1997). Lingual deficits in BDNF and NT3 mutant
mice leading to gustatory and somatosensory disturbances, respectively.
Development 124,1333
-1342.
Nosrat, C. A., Ebendal, T. and Olson, L. (1996). Differential expression of brain-derived neurotrophic factor and neurotrophin 3 mRNA in lingual papillae and taste buds indicates roles in gustatory and somatosensory innervation. J. Comp. Neurol. 376,587 -602.[CrossRef][Medline]
Nosrat, C. A. and Olson, L. (1995). Brain-derived neurotrophic factor mRNA is expressed in the developing taste bud-bearing tongue papillae of rat. J. Comp. Neurol. 360,698 -704.[Medline]
Oakley, R. A., Garner, A. S., Large, T. H. and Frank, E.
(1995). Muscle sensory neurons require neurotrophin-3 from
peripheral tissues during the period of normal cell death.
Development 121,1341
-1350.
Oakley, R. A., Lefcort, F. B., Clary, D. O., Reichardt, L. F.,
Prevette, D., Oppenheim, R. W. and Frank, E. (1997).
Neurotrophin-3 promotes the differentiation of muscle spindle afferents in the
absence of peripheral targets. J. Neurosci.
17,4262
-4274.
Palko, M. E., Coppola, V. and Tessarollo, L.
(1999). Evidence for a role of truncated trkC receptor isoforms
in mouse development. J. Neurosci.
19,775
-782.
Phillips, H. S., Hains, J. M., Laramee, G. R., Rosenthal, A. and Winslow, J. W. (1990). Widespread expression of BDNF but not NT3 by target areas of basal forebrain cholinergic neurons. Science 250,290 -294.[Medline]
Pirvola, U., Arumae, U., Moshnyakov, M., Palgi, J., Saarma, M. and Ylikoski, J. (1994). Coordinated expression and function of neurotrophins and their receptors in the rat inner ear during target innervation. Hear. Res. 75,131 -144.[CrossRef][Medline]
Pirvola, U., Ylikoski, J., Palgi, J., Lehtonen, E., Arumae, U. and Saarma, M. (1992). Brain-derived neurotrophic factor and neurotrophin 3 mRNAs in the peripheral target fields of developing inner ear ganglia. Proc. Natl. Acad. Sci. USA 89,9915 -9919.[Abstract]
Postigo, A., Calella, A. M., Fritzsch, B., Knipper, M., Katz,
D., Eilers, A., Schimmang, T., Lewin, G. R., Klein, R. and Minichiello, L.
(2002). Distinct requirements for TrkB and TrkC signaling in
target innervation by sensory neurons. Genes Dev.
16,633
-645.
Ringstedt, T., Kucera, J., Lendahl, U., Ernfors, P. and Ibanez,
C. F. (1997). Limb proprioceptive deficits without neuronal
loss in transgenic mice overexpressing neurotrophin-3 in the developing
nervous system. Development
124,2603
-2613.
Rochlin, M. W. and Farbman, A. I. (1998).
Trigeminal ganglion axons are repelled by their presumptive targets.
J. Neurosci. 18,6840
-6852.
Rochlin, M. W., O'Connor, R., Giger, R. J., Verhaagen, J. and Farbman, A. I. (2000). Comparison of neurotrophin and repellent sensitivities of early embryonic geniculate and trigeminal axons. J. Comp. Neurol. 422,579 -593.[CrossRef][Medline]
Roth, B. and Bruns, V. (1992). Postnatal development of the rat organ of Corti. I. General morphology, basilar membrane, tectorial membrane and border cells. Anat. Embryol. 185,559 -569.[Medline]
Schimmang, T., Minichiello, L., Vazquez, E., San Jose, I.,
Giraldez, F., Klein, R. and Represa, J. (1995). Developing
inner ear sensory neurons require TrkB and TrkC receptors for innervation of
their peripheral targets. Development
121,3381
-3391.
Schornig, M., Heumann, R. and Rohrer, H. (1993). Synthesis of nerve growth factor mRNA in cultures of developing mouse whisker pad, a peripheral target tissue of sensory trigeminal neurons. J. Cell Biol. 120,1471 -1479.[Abstract]
Schwenk, F., Baron, U. and Rajewsky, K. (1995). A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res. 23,5080 -5081.[Medline]
Snider, W. D. and Wright, D. E. (1996). Neurotrophins cause a new sensation. Neuron 16,229 -232.[Medline]
Song, H. J., Ming, G. L. and Poo, M. M. (1997). cAMP-induced switching in turning direction of nerve growth cones. Nature 388,275 -279.[CrossRef][Medline]
Trupp, M., Belluardo, N., Funakoshi, H. and Ibanez, C. F.
(1997). Complementary and overlapping expression of glial cell
line-derived neurotrophic factor (GDNF), c-ret proto-oncogene, and GDNF
receptor-alpha indicates multiple mechanisms of trophic actions in the adult
rat CNS. J. Neurosci.
17,3554
-3567.
Ylikoski, J., Pirvola, U., Moshnyakov, M., Palgi, J., Arumae, U. and Saarma, M. (1993). Expression patterns of neurotrophin and their receptor mRNAs in the rat inner ear. Hear. Res. 65,69 -78.[CrossRef][Medline]
Zhang, C., Brandemihl, A., Lau, D., Lawton, A. and Oakley, B. (1997). BDNF is required for the normal development of taste neurons in vivo. Neuroreport 8,1013 -1017.[Medline]