1 Neural Development Group, Mouse Cancer Genetics Program, NCI, Frederick, MD 21701, USA
2 Department of Neurology, Boston University School of Medicine, Boston, MA 02118, USA
3 Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA
*Author for correspondence (e-mail: tessarol{at}ncifcrf.gov)
Accepted July 27, 2001
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SUMMARY |
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Key words: NT3, BDNF, TrkA, TrkB, TrkC, DRG, Cochlea, Sensory neurons, Mouse
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INTRODUCTION |
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The availability of genetically engineered mouse models obtained by targeted deletion of neurotrophins or their receptors has led to important information about the neurotrophin requirement of specific cell populations of the PNS and the specificity of ligand/receptors interactions in vivo (Snider, 1994; Tessarollo, 1998). For example, both Ngf/ and TrkA/ (Ntrk1/ Mouse Genome Informatics) mice have similar phenotypes. Ablation of either gene results in the virtual absence of the superior cervical ganglia (SCG) neurons and insensitivity to pain, owing to the loss of small-diameter nociceptive sensory neurons. By contrast, the phenotype of BDNF and TrkB knockout mice is not identical because of the presence of another physiologically relevant ligand, NT4/5. Interestingly, BDNF and NT4/5 double mutations cause PNS losses comparable with the ones caused by the TrkB null mutation, confirming the relevance of the same receptor to both neurotrophins in vivo. For example, the geniculate ganglion is lost completely in TrkB null mutants or in BDNF-NT4/5 double mutants but shows only 50% reduction in either BDNF- or NT4/5-deficient mice (Tessarollo, 1998). However, the abnormal rescue of the BDNF null mice phenotype by NT4/5 knock-in in the BDNF locus suggests that these neurotrophins may differentially activate TrkB and its downstream signals in vivo (Fan et al., 2000).
NT3 and TrkC knockout mice display remarkable differences. Even though both exhibit the same deficiencies in movement and posture due to proprioception defects, spinal and cephalic sensory ganglia display greater loss of neurons in Nt3/ than in TrkC/ (Ntrk3/ Mouse Genome Informatics) mice (Tessarollo, 1998). Moreover, while the TrkC null mutant mice have no deficiencies in the SCG, Nt3/ mice exhibit severe neuronal losses, indicating that also in the sympathetic system NT3 activates other receptors (Tessarollo et al., 1997).
Thus, these mouse models have provided essential information on the neurotrophin requirements of peripheral neurons in vivo. One major obstacle in understanding neurotrophin actions other than survival has been the early demise of PNS neurons before target interactions (ElShamy and Ernfors, 1996; Fariñas et al., 1996; Liebl et al., 1997; Tessarollo et al., 1994). An interesting approach has recently been used to circumvent this problem. Snider and colleagues (Patel et al., 2000) have generated mice that lack not only NGF or TrkA but also the proapoptotic BAX protein. This strategy blocks cell death in the PNS and suggested that TrkA/NGF signaling is not required for innervation of central targets, as dorsal root ganglia (DRG) axons extended centrally into the dorsal roots and established collaterals in the superficial laminae of the spinal cord. This study also showed that NGF is still necessary for biochemical differentiation of DRG neurons (Patel et al., 2000).
Analysis of NT3 signaling is complicated by its potential interactions with TrkA and TrkB, and suggests that a similar rescue strategy would not differentiate between the role of different receptors. For example, NT3 mutant mice lose about 70% of DRG sensory neurons, whereas only 20% (Klein et al., 1994) to 35% are lost in the TrkC-deficient mice (Tessarollo et al., 1997). Two hypotheses have been put forward to explain the severe sensory losses of NT3 null mice. Initial studies suggested that TrkC-expressing proliferating precursors in the DRG undergo apoptosis when NT3 is absent, resulting in a depletion of neuron precursors (ElShamy and Ernfors, 1996). However, a subsequent study of TrkC- and NT3-deficient DRG suggested that failure of NT3 to activate TrkB during the proliferation stage of neurogenesis is responsible for these neuron losses (Fariñas et al., 1998; Fariñas et al., 1996). Equally conflicting results have been obtained from the analysis of the inner ear of NT3 and TrkC null mice. NT3 mutant mice lose 85% of the cochlear sensory neurons (Fariñas et al., 1994; Tessarollo et al., 1997), whereas 55% (Schimmang et al., 1995) to 70% losses (Tessarollo et al., 1997) have been reported for TrkC-deficient mice. In addition, initial studies have proposed differential functions for BDNF and NT3 suggesting that BDNF supports outer hair cell innervation whereas NT3 supports inner hair cell innervation (Ernfors et al., 1995). By contrast, subsequent analysis has shown that NT3 null mutants lose spiral neurons in a topologically restricted fashion and that the outer hair cell innervation is more reduced than the inner hair cell innervation in either BDNF or NT3 null mutants, suggesting that spatial-temporal gradients of neurotrophin expression control inner ear innervation (Fritzsch et al., 1997). Such gradients were recently visualized using NT3 and BDNF lacZ fusion reporter systems (Fariñas et al., 2001).
To study the temporal activation of Trk receptors and to dissect the dynamics of NT3 requirement by sensory neurons in development, we have replaced the Nt3 gene with Bdnf (B/N allele). This strategy was aimed at eliminating activation of TrkA and TrkC, while preserving only TrkB-specific signaling by NT3. For the DRG, we found that BDNF in place of NT3 can rescue a substantial number of neurons during neurogenesis suggesting a major role for TrkB activation at this stage. However, during the period of target tissue innervation when the majority of DRG sensory neurons express TrkA, we observed a substantial loss of neurons in the B/N DRG, indicating that at this stage TrkA activation by NT3 becomes crucial for promoting sensory neuron survival. In the inner ear, we found that the B/N allele could rescue almost completely the sensory losses caused by NT3 deficiency in agreement with the reported expression of both TrkB and TrkC by spiral neurons (Fariñas et al., 2001; Pirvola et al., 1994). This result indicates that either NT3 or BDNF can support spiral neurons and that gradients of neurotrophin expression control inner ear innervation (Fariñas et al., 2001). In the spinal cord of the B/N mice, we have detected central sensory fibers, which suggests a chemoattractant function of BDNF on subpopulations of TrkB-expressing neurons (Song and Poo, 1999; Wang and Tessier-Lavigne, 1999). By contrast, thermo- and nociceptive neurons do not require NGF for sending collaterals into the dorsal laminae (Patel et al., 2000). Thus, our data indicate that not all spinal sensory neurons behave in a similar fashion and some subpopulations that express TrkB may require neurotrophin for central projections of collaterals to appropriate regions of the spinal cord (Song and Poo, 1999; Wang and Tessier-Lavigne, 1999).
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MATERIALS AND METHODS |
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Animals, histological techniques and in situ hybridization
Timed embryos were obtained by overnight mating of heterozygotes. The morning when the vaginal plug was observed was considered embryonic (E) day 0.5. Gravid uteri were removed from timed pregnant females at different stages of gestation (E11.5, 12.5, 13.5, 14.5, 15.5), and embryos dissected and fixed overnight in either 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.2) for in situ hybridization or Carnoys fixative (60% ethanol, 30% chloroform, 10% acetic acid) for immunostaining and cell counts. Anesthetized neonate animals were perfused with 4% PFA for retrograde labeling experiments or decapitated and fixed in Bouins fixative for neuronal counts, as described elsewhere (Tessarollo et al., 1997). Tissues from one mutant and one wild-type littermate were embedded in the same block, sectioned at 5 µm thickness and mounted on sialinated slides. In situ hybridization protocols employing the full-length antisense coding sequence of the NT3 and BDNF genes, and the previously described TrkB- and TrkC-specific probes (Tessarollo et al., 1993) were performed as previously reported (Tessarollo and Parada, 1995). All analyses were carried out on age-matched mice processed in parallel. All animals employed in this study were genotyped by DNA blot analysis as described (Tessarollo et al., 1997; Tessarollo et al., 1994).
Neuronal cell counts and immunocytochemistry
One mutant and one wild-type embryo fixed in Carnoys solution were embedded in the same paraffin block and serially sectioned at 5 µm. For neuronal counts, the sections were immunostained with antibodies to the 150 kDa neurofilament subunit as described (Fariñas et al., 1998). Individual ganglia were identified using the ribs as landmarks. Cells were counted in every fourth section to calculate the total number in the ganglion, and numbers were multiplied by four and not corrected. For neonatal neuronal counts, the heads (for C1 and T1 counts) or spinal cords (for L1 counts) of one mutant and one wild-type littermate were embedded in the same block, sectioned at 5 µm sagittally, for C1 and T1 analysis, or tranversally, for L1 analysis, and Nissl-stained with 0.1% Cresyl Violet. Neurons with a clear nucleus and nucleolus were counted in every sixth section, and the sum of counts multiplied by 6. To assure unbiased analysis, blind neuronal counts were made.
Primary antibodies included rabbit polyclonal antibodies to the neurofilament subunit of relative molecular mass 150,000 (Chemicon, Temecula, CA; dilution 1:2,000) and the monoclonal antibody against parvalbumin (Swant Swiss antibodies; Bellinzona, Switzerland; dilution 1:5000). For bright field immunocytochemistry, sections were incubated with the appropriate biotinylated secondary antibody followed by avidin-biotin-peroxidase complex (Vector Labs, Burlingame, CA), according to the instructions of the manufacturer. Sections were developed in 0.05% 3-3'-diaminobenzidine tetrahydrochloride and 0.003% hydrogen peroxide in 0.1 M Tris-HCl (pH 7.5), dehydrated and mounted with DPX.
Using one-way analysis of variance (ANOVA), mean neuronal counts from each ganglion (C1, T1, L1) for TrkC/, B/N/ and Nt3/ mice were compared at E12.5, E13.5, E14.5 and at P0. The significance of any differences between strains at each timepoint was determined by Tukey post-hoc testing.
DiI tracing, spindle and nerve fiber counts
E15.5 and newborn mutant and normal littermates were decapitated and perfused with phosphate-buffered saline and 4% PFA, eviscerated, and incubated overnight at 4°C in 4% PFA. The skin was removed and crystals of DiI (Molecular Probes) were inserted into axial muscles or DRG. After 5-11 days of incubation in 10% formalin (37°C), the spinal cords were removed, embedded in 3.5% agar/sucrose and sectioned transversely at 100 µm on a vibratome. Sections were viewed on a Zeiss Axiophot microscope using the rhodamine filter set to visualize DiI-labeled afferents. For muscle spindle counts, tissues from newborn mutant and wild-type littermates were prepared as previously described (Kucera et al., 1995). Muscle spindles were identified and counted in the soleus (SOL), medial gastrocnemius (MG) and plantaris (PL). Myelinated axons of the nerve to the SOL muscles were also counted in neonate, as described (Kucera et al., 1995). The total number of nerve fibers (both myelinated and unmyelinated) of mutant and wild-type newborns was determined from electron micrographs taken at 5000x magnification. Differences between mutant and wild-type values were analyzed using Students t-test.
Analysis of inner ear innervation
We have analyzed a total of 15 mutant ears (three ears from P6, four ears each from P3, P1 and E 14.5 null mutants) and compared them with P1 NT3 null mutants and P6 and P1 wild-type control animals. Analysis consisted of labeling the afferent and efferent fibers to the ear from the brainstem using DiI. After insertion of the DiI-soaked filter strips, the fixed heads were incubated for four days at 36°C until the dye had diffused into the fine axonal terminals. The inner ears were then dissected and mounted flat in glycerol. Images were captured using a cooled CCD camera and were photographed. The P6 ears were analyzed by confocal microscopy (BioRad Radiance 200). Image stacks of flat mounted cochlea were taken and collapsed in the z-axis to reveal the entire pattern of innervation in the control mice. In the B/N mice, two sets of fibers were collapsed: one set below and another one on top of the basilar membrane. Cochlea were subsequently osmicated, embedded in epoxy resin and thick and ultra-thin sections were cut to reveal the pattern of innervation and synapses on various cells. One mutant and one wild-type cochlea were reacted for acetylated tubulin using an Alexa 568-conjugated secondary antibody, frozen and sectioned at 20 µm thickness, and viewed in a confocal microscope using the residual autofluorescence to visualize cellular details.
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RESULTS |
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Expression of the B/N allele parallels that of the wild-type Nt3 gene
The objective of our approach depended on the ability to reproduce the precise developmental expression pattern of NT3 with the exogenous Bdnf gene under the control of the NT3 promoter. Therefore, we analyzed the expression of the B/N allele in comparison with NT3 and BDNF. The B/N allele produced an mRNA that was similar in size to the endogenous NT3 transcripts (Fig. 2C). Northern blot analysis of tissues obtained from B/N mutant mice and wild-type control littermates revealed that indeed the exogenous Bdnf gene was expressed in a pattern overlapping that of the wild-type Nt3 gene (Fig. 2). For example, in the B/N mutant mouse a BDNF-specific band (B/N band) is present in cortex, intestine, heart and kidney at levels that are comparable with NT3 in the control. In the mutant mouse, we did not observe significant changes in expression pattern of the endogenous Bdnf gene, compared with the wild type animal, indicating that the exogenous BDNF allele did not alter the expression level of the endogenous BDNF locus (Fig. 2). Occasionally, in the B/N mouse we have observed small differences in the levels of the endogenous BDNF and the B/N transcripts compared with BDNF and NT3 levels in wild-type controls. However, these differences were not consistent between different animals and we attributed them to variations in tissue dissections (data not shown). One notable exception is the cerebellum, where the relative level of the B/N transcripts was consistently significantly higher than that of NT3 in the wild-type animal (see Discussion).
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Chemotropic effects of BDNF on central afferent projections
The B/N mutant mice display the same behavioral phenotype, including abnormal movements and postures attributed to defects in proprioception that are characteristic of Nt3- and TrkC-deficient mice (not shown) (Ernfors et al., 1994; Fariñas et al., 1994; Tessarollo et al., 1994; Klein et al., 1994; Tessarollo et al., 1997). Proprioception deficit in NT3 null mice is characterized by loss of Ia peripheral afferent projections and of muscle spindles in the limbs (Kucera et al., 1995). In addition, spinal cord collateral branches of Ia proprioceptive neurons are completely missing at any developmental stage (Ernfors et al., 1994; Tessarollo et al., 1994) (Fig. 3B). In the B/N mutant mouse, by contrast, by retrograde labeling of B/N sensory and motor neurons innervating lumbar and thoracic axial muscles we found central neuronal fibers projecting ventrally in the spinal cord at day 15.5 of embryonic development (Fig. 3C). As TrkC-expressing neurons are missing in the DRG of B/N mice at E12.5 and E13.5, while TrkB expression is not altered (Fig. 4), these data suggest that expression of BDNF instead of NT3 in the motoneuron region of the spinal cord has chemotropic effects on central projections that express TrkB. To assure that the observed fibers were central sensory projections, we selectively retrograde labeled dorsal root at E15.5. As shown in Fig. 3F, a substantial number of fibers projected ventrally in the B/N spinal cord, in contrast to NT3 deficient spinal cord where these fibers are completely absent (Fig. 3E). However, these projections appear abnormal when compared with those in control mice, indicating that other factors in addition to neurotrophins are involved in the precise routing of central sensory fibers. Despite this apparent embryonic rescue, by P0 only few residual projections towards motoneurons persisted, consistent with the mouse behavior and indicating proprioception deficits (Fig. 3G,H). To determine whether these residual fibers belong to proprioceptive neurons, we analyzed mutant spinal cord for expression of parvalbumin (PV), a proprioceptive neuronal marker (Ernfors et al., 1994). Similar to what was observed in the NT3 (Fig. 3J) or TrkC/ (data not shown) mice, no PV-positive fibers were detected in the thoracic or lumbar B/N mutant spinal cord, suggesting that most projecting fibers observed during embryogenesis fail to differentiate and degenerate by birth (Fig. 3K).
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Differential rescue of DRG sensory neurons at neurogenesis and target tissue innervation by B/N
Comparison of neonatal mice deficient in TrkC or Nt3 has shown that there is a much greater deficiency in the number of spinal sensory neurons in the ligand mutant. This suggests that NT3 acts through other receptors in addition to TrkC presumably supporting the survival of non-proprioceptive neurons. If so, replacement of NT3 with BDNF, a preferred TrkB ligand, should rescue at least in part the neuronal losses caused by NT3 deficiency. During embryogenesis, sensory neurons undergo dynamic changes in neurotrophin receptor expression, raising the possibility that sequential activation of different Trk receptors by NT3 may be required to support neuronal survival (Davies 1994; Fariñas et al., 1998). As the effect of NT3 on sensory neurons may vary between ganglia at different sites of the spinal cord, we investigated the dynamics of neuronal losses in cervical 1 (C1), thoracic 1 (T1) and lumbar 1 (L1) DRG during embryonic development in B/N mice compared with NT3 and TrkC null mutant animals (Table 1, Fig. 5). At E13.5, immediately after most DRG neurons are born (Lawson and Biscoe, 1979) all three ganglia of B/N mice contained significantly more neurons than the NT3 nulls. In T1 and L1 DRG, neuronal losses in B/N mice were similar to that observed in TrkC/ mice. A similar trend was also observed in C1. Collectively, these results suggest that BDNF expressed in the NT3 locus can rescue neurons during the major period of neurogenesis, and support the notion that NT3 can function through TrkB during neurogenesis (Fariñas et al., 1998). We next investigated the nature of this rescue by studying the dynamics of Trk-expressing neurons in wild-type and B/N DRG between days 11.5 and 13.5 of development (Fig. 4). In situ hybridization analysis showed that, as observed in the NT3 knockout mouse (Tessarollo et al., 1994; Fariñas et al., 1998), neurons that express TrkC are already reduced at E11.5 and are completely absent by E12.5 (Fig. 4P-R). In NT3 null mice, the DRG TrkB neuronal population is 40-50% reduced at E13.5 (Fariñas et al., 1998). However, quantitative analysis of TrkB expression demonstrated a normal sized population in the B/N DRG at E13.5 (Fig. 4G,L; Table 2). This result is important because it demonstrates that the TrkB neuronal populations in the B/N mouse are restored to a physiological level and are not abnormally expanded by the ectopic expression of BDNF. Thus, expression of BDNF activity from the NT3 locus rescues TrkB-expressing neurons which would otherwise be lost in the NT3 deficient mouse. These data demonstrate that activation of TrkB by NT3 is a crucial function during neurogenesis.
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The B/N allele supports the development of inner ear spiral neurons
Spiral ganglion sensory neurons innervate the cochlea from the apex to the basal turn. These cells give rise to innervation of both the inner hair cells and outer hair cells via numerous radial fibers (Fig. 6B). In NT3 null mutants, both the basal turn spiral neurons and the radial fibers emanating from them are absent (Fig. 6A,D) (Fritzsch et al., 1997). By contrast, at P0 the B/N mouse shows the presence of spiral neurons that extend as far to the basal turn as in wild-type animals (Fig. 6B,C). As in wild type animals, numerous fibers extend radially from these sensory neurons to the basal turn to innervate densely the inner and outer hair cells. Contrary to the NT3 null mutants, where the density of outer hair cell innervation in the basal turn is particularly reduced or even absent, the B/N mice show dense innervation, in particular, of outer hair cells (Fig. 6D,E). This apparently complete innervation rescue is paralleled by an almost complete rescue of neurons in the spiral ganglion of B/N mice (85% of wild type; 8646±936 in B/N (n=4) versus 10740±894 in wild type (n=4)) compared with NT3- (15% of wild type) and TrkC- (30% of wild type) deficient mice (Tessarollo et al., 1997). These data are in agreement with the overlapping expression of TrkB and TrkC in spiral neurons, and suggest that if both receptors are expressed in the same neuron, substituting BDNF for NT3 will not affect neuronal survival (Fariñas et al., 2001; Pirvola et al., 1994).
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DISCUSSION |
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In addition to their specific Trk tyrosine kinase receptors, neurotrophins also bind the so-called p75 low-affinity neurotrophin receptor (Kaplan and Miller, 1997; Bibel and Barde, 2000). As p75 is highly expressed in the sensory neuronal populations analyzed in this study, it is possible that a neurotrophin replacement may impact on neurotrophin effects mediated by this receptor. However, it has been shown that BDNF and NT3 elicit similar biological responses when binding to p75. For example, while all neurotrophins stimulate p75-mediated sphingomyelin hydrolysis with similar kinetics in mouse fibroblasts, BDNF and NT3 show a more potent effect than NGF (Dobrowsky et al., 1995). However, only NGF, but not BDNF or NT3, induces p75-mediated activation of NF-B (Kaplan and Miller, 1997; Bibel and Barde 2000). Similarly, only NGF causes association of p75 with the intracellular NADE protein (Mukai et al., 2000) or p75-mediated regulation of the subcellular localization of the zinc-finger protein SC-1 (Chittka and Chao, 1999). Thus, the specific replacement of NT3 with BDNF is not expected to alter activation of p75 significantly as NT3 and BDNF have similar effects on p75.
BDNF expression in the lateral motor column of the spinal cord supports the development of central afferent projections
The B/N mutant mouse completely lacks proprioceptive functions, as shown by the behavioral deficit and absence of muscle spindles. As only muscle-specific expression of NT3 can rescue these deficits (Wright et al., 1997), our data confirm that the proprioceptive neurons are entirely dependent on TrkC activity. Nevertheless, we have detected the presence of central afferent projections at embryonic day E15.5 in the spinal cord of mice expressing the B/N allele (Fig. 3). By contrast, central fibers are completely missing in mice that lack NT3 or its receptor TrkC (Klein et al., 1994; Tessarollo et al., 1994). The B/N-BDNF is expressed by the lateral motor column in a region that normally expresses NT3 (Fig. 2) (Schecterson and Bothwell, 1992). Thus, these fibers may be derived from misrouted TrkB-expressing neurons that are sensitive to BDNF gradients in the spinal cord. Despite mounting experimental evidence that neurotrophic factors may serve as axon guidance molecules during neuronal development in vitro (Ming et al., 1997; Song and Poo, 1999) such a notion is still controversial in vivo (Chen and Frank, 1999; Oakley et al., 1995; Patel et al., 2000). The recent finding that TrkA/ sensory neurons rescued by deletion of the pro-apoptotic Bax gene can extend collaterals into the superficial lamina of the spinal cord suggests that NGF/TrkA signaling does not have chemoattractive roles in vivo (Patel et al., 2000). This idea is also supported by the fact that NGF is not present at significant levels in the spinal cord (Shelton and Reichardt, 1986) and that it has a late onset of expression in the peripheral targets (Davies et al., 1987). However, both BDNF and NT3 are expressed much earlier during development (Maisonpierre et al., 1990), making them potential candidates for a role in axonal growth and guidance. Furthermore, BDNF and NT3 exert much stronger effects than NGF as in vitro growth cone guidance molecules and in modulating the response of growth cones to axon guidance molecules (Paves and Saarma, 1997; Song and Poo, 1999; Tuttle and OLeary, 1998). However, it is not known which specific group(s), if any, of sensory neurons are sensitive to BDNF and/or NT3 as guidance cues. In chick, injections of a NT3 neutralizing antibody in the spinal cord does not reduce the number of Ia afferent projections or the extent of their ventral extension, suggesting that NT3 does not have chemotropic effects on Ia fibers (Oakley et al., 1995), which are known to belong to TrkC-expressing proprioceptive neurons (Klein et al., 1994). Thus, it appears that only TrkB-expressing fibers may be sensitive to BDNF or possibly even NT3 gradients. Interestingly, in a transgenic mouse expressing NT3 under the control of the nestin promoter a few central fibers develop but fail to contact the motoneurons in the lateral column. Instead, they project to the midline of the spinal cord, an area coinciding with highest levels of NT3 expression (Ringstedt et al., 1997). As the identity of those fibers is not known, including whether they express TrkB or TrkC, it is conceivable that TrkB-expressing central sensory fibers may be the ones that responded to the ectopic NT3. Therefore, while TrkA-expressing nociceptive and TrkC-expressing proprioceptive axons may not require NGF and NT3, respectively, for reaching their central target, TrkB-expressing axons may sense and react to BDNF or even NT3 gradients.
Different neurotrophins can have opposite effects on growth cone behavior in vitro, depending on the context in which they function (Song and Poo, 1999). Thus, the lack of a unified view of their role as chemoattractant molecules on sensory neurons in vivo may be symptomatic of the heterogeneity among sensory neuron populations (Paves and Saarma, 1997; Scott, 1992; Tuttle and OLeary, 1998).
Temporal activation of TrkB and TrkA receptors by NT3 during DRG development
Expression of Trk receptors in the DRG is dynamically regulated during development (Fig. 8) (Fariñas et al., 1998; Ma et al., 1999; Tessarollo et al., 1993; Tessarollo et al., 1994). The transcription factors neurogenin 1 and 2 are required to generate all classes of Trk-expressing DRG neurons. However, the mechanisms that determine the expression of specific Trk receptors is still unknown (Anderson, 1999; Ma et al., 1999). TrkC is the main receptor expressed by progenitor cells (Tessarollo et al., 1993). During neurogenesis, cells expressing TrkA, TrkB and TrkC are all well represented in the DRG (Fariñas et al., 1998). However, after all neurons are born, at the beginning of the period of target tissue innervation, most neurons express TrkA. This dynamic pattern of neurotrophin receptor expression may reflect the changes in neurotrophin dependency of DRG neurons during development (Davies, 1994). However, it is still not clear what roles specific neurotrophins play in DRG neuronal survival during embryogenesis. For example, NT3 plays a major role in DRG development, as indicated by the severe losses in the sensory ganglia of Nt3/ mice (Ernfors et al., 1994; Fariñas et al., 1994; Tessarollo et al., 1997). However, mice that lack all isoforms of the high-affinity receptor TrkC do not lose as many neurons, suggesting that some TrkA- or TrkB-expressing neurons also depend on NT3 (Tessarollo et al., 1997). Two different mechanisms have been proposed to explain the severe neuronal losses caused by NT3 deficiency. As these losses appear early in development before the target tissue innervation period (ElShamy and Ernfors, 1996; Fariñas et al., 1996; Liebl et al., 1997; Tessarollo et al., 1994) and seem to coincide with an increased level of apoptosis of precursors, it has been first suggested that proliferating precursors undergo apoptosis when NT3 is absent, resulting in a depletion of pro-neuronal progenitors (ElShamy and Ernfors, 1996). However, analysis of the rate of proliferation and differentiation of precursor cells in NT3-deficient mice has lead to the proposal that a decrease in neurogenesis and not apoptosis is responsible for the severe sensory losses (Fariñas et al., 1996). Furthermore, the pattern of expression of Trk receptors in proliferating neuroblasts suggests that NT3 is required to maintain neurons that express TrkB but not neurons expressing TrkA (Fariñas et al., 1998). Our data support a role for NT3 on TrkB-expressing neurons at neurogenesis, as this neuronal population was rescued at this stage. This specific effect was true for all DRG analyzed, although the rescue was less prominent in the C1 ganglion. This result may reflect unique characteristics of this particular DRG. For example, the role of NT3 activation of different Trk receptors may vary among DRG at this early developmental stage or NT3 may act on the cell cycle control of sensory precursors cells (ElShamy et al., 1998).
While the B/N allele could rescue some neurons during neurogenesis, a large number is lost soon after most neurons switch to TrkA expression at the start of target tissue innervation (Table 1; Fig. 5). The dynamic of this loss is similar in the different ganglia, but in agreement with the reported rostrocaudal gradient of maturation of DRG neurons it occurs more rapidly at the C1 level (Lawson and Biscoe, 1979). The rapid loss of neurons immediately after neurogenesis was unexpected in light of the reports suggesting that only NGF is required at this stage (Davies, 1994; White et al., 1996). During the earliest stages of target tissue innervation, immediately after sensory neurons switch to TrkA expression they survive only briefly without neurotrophins in culture (Buchman and Davies, 1993). Because of this sensitivity to neurotrophin deprivation our data is consistent with a role for NT3 in supporting neurons during this delicate transition to a complete dependence on NGF (Fig. 8) (Davies et al., 1987; White et al., 1996). Expression data have shown that the peripheral axons of sensory neurons that undergo a switch in neurotrophin dependence are exposed to NT3 en route to their peripheral tissues where NGF is produced. Thus, these neurons may see only NT3 during the earliest stage of target tissue innervation (Fariñas et al., 1998). Alternatively, NGF and NT3 may both be required for sensory neuron survival at this specific stage as demonstrated for neurons of the sympathetic lineage for which simultaneous requirement of both NGF and NT3 has been reported (Tafreshi et al., 1998).
The B/N allele supports sensory innervation of the inner ear
We found that replacement of NT3 with BDNF rescues almost completely the severe inner ear neuronal and innervation losses caused by NT3 deletion. The implication of this rescue is significant in light of the current controversy about the role of NT3 and BDNF in inner ear development. Initial analysis of NT3- and BDNF-deficient mice suggested that these neurotrophins act on separate subsets of spiral ganglion neurons, with BDNF and NT3 supporting outer and inner hair cell innervation, respectively (Ernfors et al., 1995). This hypothesis was supported by the apparently restricted loss of neurons that innervate outer hair cells of the cochlea in BDNF and TrkB mutants, and the complete loss of inner hair cell innervation in the NT3 and TrkC mutant mice (Ernfors et al., 1995; Schimmang et al., 1995). In this case, the B/N allele should have had almost no effect on restoring the innervation losses caused by NT3 ablation.
More recent studies of the expression of neurotrophins and their receptors (Fariñas et al., 2001) and of inner ear innervation losses in mutant mice (Fritzsch et al., 1999) have lead to the hypothesis that region-specific expression of BDNF and NT3 may control the spatial shaping of cochlear innervation (Fariñas et al., 2001). Analysis of Nt3 mutants indicates an almost complete loss of innervation in the basal turn and a more severe reduction of the outer hair cell than the inner hair cell innervation (Fritzsch et al., 1997). Similar spatial effects on cochlear innervation are also present in TrkC receptor mutants (Fritzsch et al., 1998). As in situ hybridization studies showed that spiral sensory neurons express both the TrkB and TrkC receptors (Fariñas et al., 2001; Pirvola et al., 1994) it appeared that region-specific expression of the neurotrophins might account for the differential sparing of sensory neurons along the cochlea. The limited loss of spiral sensory neurons in the B/N mice indicates that only some signal through both TrkC and TrkB receptor is needed for survival of all neurons.
Indeed, recent work described a developmental countercurrent of neurotrophin expression in the cochlea, with BDNF expanding from the apex toward the base and Nt3 expanding from the base toward the apex as development progresses (Fariñas et al., 2001). In agreement with this scenario, replacement of NT3 with BDNF causes an almost complete rescue of the topological loss of basal turn spiral neurons caused by lack of NT3 (Fig. 6). Most importantly the effect of the exogenous BDNF on the basal turn innervation cannot be explained by a simple additive effect of endogenous and exogenous BDNF. In fact, the innervation rescue in the B/N mouse occurs before expression of the endogenous BDNF in the basal turn (data not shown). Beyond the simple rescue of spiral sensory neurons in the basal turn we have also observed an even increased density of outer hair cells innervation in the basal turn at birth. Closer examination at P7 showed that many of the fibers that appear to reach the outer hair cells are actually never entering the organ of Corti. Instead, they remain between the tympanic border cells where they run for various distances longitudinally underneath the organ of Corti (Fig. 7). These fibers, contrary to the rescued afferent and efferent fibers contacting the hair cells do not form recognizable synapses with border cells. The endogenous expression of NT3, which is predominately in supporting cells in the embryonic basal turn and the inner hair cells in the adult basal turn (Fariñas et al., 2001; Pirvola et al., 1994) does not correlate well with the innervation of hair cells in normal cochlea. Thus, it appears that BDNF, which is expressed exclusively in hair cells in the basal turn, may be more important in the cochlea for short range navigation of ingrowing afferents to reach preferentially the hair cells. Consistent with this assumption is our finding of misdirected growth of some fibers which spiral underneath the organ of Corti in the B/N mice. We assume that the synthesis of BDNF under NT3 promoter control in the cochlear supporting cells will lead to a diffusion away from those cells. Such a diffusion halo appears to allow afferent fibers to extend into the foreign territory of tympanic border cells and to survive there at least until P7. Later stages, which are known to display exclusive expression of NT3 in inner hair cells, need to be examined to reveal the ultimate fate of these fibers.
In summary, we have shown that replacement of NT3 with BDNF can support some neuronal systems that are impaired by NT3 deficiency. This rescue revealed for the first time physiological spatial-temporal interactions of NT3 with the non-preferred TrkA and TrkB receptors. Further analysis of this as well as of similar mouse models may allow the dissection of some of the most complex traits of neurotrophin functions in vivo.
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