Abstract
Slitrks are type I transmembrane proteins that share conserved leucine-rich repeat domains similar to those in the secreted axonal guidance molecule Slit. They also show similarities to Ntrk neurotrophin receptors in their carboxy-termini, sharing a conserved tyrosine residue. Among 6 Slitrk family genes in mammals, Slitrk6 has a unique expression pattern, with strong expression in the sensory epithelia of the inner ear. We generated Slitrk6-knockout mice and investigated the development of their auditory and vestibular sensory organs. Slitrk6-deficient mice showed pronounced reduction in the cochlear innervation. In the vestibule, the innervation to the posterior crista was often lost, reduced, or sometimes misguided. These defects were accompanied by the loss of neurons in the spiral and vestibular ganglia. Cochlear sensory epithelia from Slitrk6-knockout mice have reduced ability in promoting neurite outgrowth of spiral ganglion neurons. Indeed the Slitrk6-deficient inner ear showed a mild but significant decrease in the expression of Bdnf and Ntf3, both of which are essential for the innervation and survival of sensory neurons. In addition, the expression of Ntrk receptors, including their phosphorylated forms was decreased in Slitrk6-knockout cochlea. These results suggest that Slitrk6 promotes innervation and survival of inner ear sensory neurons by regulating the expression of trophic and/or tropic factors including neurotrophins from sensory epithelia.
Introduction
The Slitrk family consists of neuronal transmembrane proteins that control neurite outgrowth [1], [2]. Structurally, Slitrks share leucine-rich repeat (LRR) domains located amino-terminal to the transmembrane domain. LRR domains are present in many proteins and mediate protein–protein interactions [3]. LRR domains in the Slitrk family proteins are similar to those in all Slit family proteins, which control axon guidance and branching [4]. Tyrosine residues in the carboxy-terminus are another structural feature of the Slitrk family; these tyrosines are flanked by amino acid sequences similar to those in the carboxy-terminal domain of the Ntrk neurotrophin receptor [5].
The mouse Slitrk family contains 6 genes (Slitrk1-6) [1]. Slitrk1 is required for higher brain functions [6]. However, little is known about the physiological roles of the other family members. Although Slitrk1 through Slitrk5 are expressed broadly throughout the brain, the expression of Slitrk6 is highly restricted to thalamic nuclei [1]. Furthermore, a comprehensive expression analysis of Slitrk6 revealed a strong expression in the auditory and vestibular sensory epithelia of the ear [7]. This unique expression pattern led us to investigate the role of Slitrk6 in inner ear development. Inner ear sensory epithelia contain mechanosensory hair cells that recognize sound as well as linear and angular acceleration for balance [8]. During development, sensory epithelia also play an important role in the development of sensory neurons of the inner ear by releasing diffusible factors that promote survival and outgrowth of sensory neurons [9].
In the present study, we generated Slitrk6-knockout mice and investigated the development of their auditory and vestibular sensory organs. Histological examination and marker labeling studies revealed a reduction in the innervation density in the cochlea of Slitrk6-deficient mice. In addition, the innervation to the posterior crista was lost, reduced, or sometimes misguided in the vestibule. Many sensory neurons within the spiral and vestibular ganglia were lost during development. In the explant co-culture experiment, cochlear sensory epithelia from Slitrk6-knockout mice had less activity in promoting neurite outgrowth of the spiral ganglion neurons. These results indicate that Slitrk6 plays an important role in the sensory neural development of the inner ear by regulating the expression of molecules that promote survival and neurite outgrowth of sensory neurons.
Results
Slitrk6 Expression during Inner Ear Development
First, we carried out in situ hybridization analysis (Fig. 1) to know the Slitrk6 transcripts distribution in the course of inner ear development. Slitrk6 transcripts are first detected at embryonic day (E)8.5 in the otic placode, which invaginates to form the otic vesicle [7]. In the E10.5 otic vesicle, Slitrk6 transcripts were strongly expressed in the ventromedial and laterodorsal regions (Fig. 1A), which give rise to the cochlear and vestibular sensory epithelia [8], [10], [11]. At E15.5, Slitrk6 expression marked the presumptive organ of Corti (Fig. 1C). In addition, we detected relatively weak expression of Slitrk6 in a narrow region of the spiral ganglion near the developing cochlear sensory epithelium from E12.5 to E15.5 (Fig. 1C). In the postnatal day (P)1 cochlea, Slitrk6 transcripts were detected strongly in supporting cells and weakly in both inner and outer hair cells (Fig. 1D, D'). Furthermore, Slitrk6-positive signals were detected in vestibular sensory epithelia, including ampullary cristae, saccular and utricular maculae (Fig. 1B, E, F, and data not shown). Slitrk6 transcripts were densely located at the lumenal surface of the sensory epithelium, where hair cells localize (Fig. 1F'). The hybridization signal was also detected weakly in supporting cells located at the basal cell layer of the vestibular sensory epithelium (Fig. 1F'). We also found weak expression of Slitrk6 in vestibular ganglion neurons near the sensory epithelium from E11.5 to E14.5, comparable to what was observed in spiral ganglion neurons (Fig. 1B).
Next, we examined the localization of the Slitrk6 protein using an anti-Slitrk6 antibody. Sltrk6–immunopositive signals were detected early in the otic vesicle (Fig. 2A, B), and later in cochlear and vestibular sensory epithelia where Slitrk6 transcripts were distributed (Fig. 2C–L, Fig. S1, S2). In the presumptive organ of Corti, Slitrk6–immunopositive signals were strongly detected by E14.5 (Fig. 2C, D), and then confined to the supporting cell types at later stages of embryonic development and at the newborn (Fig. 2E, F, Fig. S2A–F). In the vestibular sensory epithelia, the signal was strong at the lumenal surface (Fig. 2G–L). However, double labeling with a hair cell marker calretinin indicated that the strong signals were in the non-hair cell region, presumably in the lumenal processes of the supporting cells (Fig. S2G–I). Transient expression of Slitrk6 in the spiral and vestibular ganglion neurons was also confirmed at the protein level, and positive signals were partly overlapped with neurofilament immunostaining (Fig. 2C, D, G, H). The cell type preferences of the Slitrk6 expression in the cochlear and vestibular organs are comparable to each other.
Generation of Slitrk6-Null Mutant Mice
To analyze the role of Slitrk6 in inner-ear development, we generated Slitrk6-deficient mouse lines. A targeting vector was designed to replace the open reading frame (ORF) of Slitrk6 with a phosphoglycerol kinase (PGK)-neo expression cassette flanked by a loxP sequence (Fig. 3A). We isolated 5 independent embryonic stem (ES) clones with homologous recombination, and 2 ES clones yielded chimeric mice capable of transmitting the disrupted allele (+neo) through the germline. Subsequently, the PGK-neo cassette was removed by crossing the heterozygous mice with mice transgenic for the Cre recombinase gene under the control of the cytomegalovirus immediate-early enhancer/chicken β-actin hybrid (CAG) promoter [12], which express Cre recombinase in their zygotes (Δneo, Fig. 3B). Ablation of Slitrk6 mRNA was confirmed by RT-PCR (Fig. 3C). Heterozygous mating produced Slitrk6 +/+, Slitrk6 +/−, and Slitrk6 −/− pups at the expected Mendelian frequencies. Both male and female mice grew without showing any external abnormalities and were fertile. The inner ear phenotypes described in the present study were observed in two mouse lines derived from two independent ES clones. Thus, the phenotypes are considered to correctly reflect the effects of the inserted mutation.
Cochlear Innervation Defects in the Absence of Slitrk6
The gross morphology of the Slitrk6-deficient inner ear was normal. In addition, light microscopic analysis of hematoxylin and eosin (H&E)-stained sections showed that the general organization of the organ of Corti and the size of cochlear sensory epithelium in the Slitrk6-deficient mice was not obviously different from those of wild-type littermates (Fig. S3A, B, E, F).
To initially characterize the innervation patterns of the inner ear in wild-type and mutant mice, we selectively labeled the afferent innervation of the cochleae at P0 by applying DiI to the alar plate of the brainstem, where the fibers terminate centrally. In contrast to the dense, evenly spaced innervation by radial fibers of the wild-type cochlea (Fig. 4A, C), the radial fibers in the Slitrk6-knockout mice were fasciculated with large spaces between the bundles (Fig. 4B, D). In Slitrk6-knockout mice, the innervation defects were distributed evenly throughout the cochlea, and all the cochlear turns were affected to the same degree (Fig. 4A–D).
To label the efferent fibers, we then placed DiI crystals in the olivo-cochlear efferent bundle near the floor plate [13], [14]. Selective DiI tracing of the olivo-cochlear efferents in the cochleae revealed that the efferent fibers were also abnormally fasciculated into fewer radial bundles, showing innervation defects similar to afferent fibers (Fig. 4E, F). Efferent fibers use afferent fibers as a scaffold during pathfinding, and the track of the efferents closely follows the afferent innervation pattern [15], [16]. Therefore, the observed cochlear efferent innervation defects are likely to be secondary to the afferent innervation defects.
We next examined the developmental changes of the cochlea innervation defects between E16.5 and P7, using whole-mount surface preparations of the cochlea labeled with anti-neurofilament antibody to visualize the innervation pattern (Fig. 5A–F). The radial fiber projection to the organ of Corti was strongly reduced in the mutant mice, leaving larger gaps between radial bundles than in the wild-type mice (Fig. 5A–F). At P7, a higher magnification view revealed that the type II spiral fibers that extend through the tunnel of Corti to the three rows of outer hair cells were reduced in number in Slitrk6-knockout mice (Fig. 5E', F'). Myelin staining by osmium tetroxide revealed that the defects in the innervation pattern were still prominent in Slitrk6-deficient mice at P28 (Fig. 5G, H).
Vestibular Innervation Defects in the Absence of Slitrk6
We next examined the abnormalities in the vestibular region of Slitrk6-deficient mice. Light microscopic examination revealed that the sensory hair cells in all of the vestibular regions (ampullary cristae and saccular and utricular maculae) appeared normal with regard to their number, shape, and tissue architecture (Fig. S3C, D). However, the sensory epithelia in saccular macula and posterior crista were slightly smaller than those of wild-type littermates at E16.5, but not at E13.5 (Fig. S3E–G).
DiI labeling of vestibular afferent fibers revealed that, from E13.5 to E15.5, innervation to the posterior crista was frequently absent in the Slitrk6-deficient inner ear (78.3% at E13.5 [n = 83] and 85.7% at E15.5 [n = 28]), and they sometimes showed reduced and abnormal trajectories (19.3% at E13.5 and 3.6% at E15.5) (Fig. 6A–F). At P28, myelin staining with osmium tetroxide confirmed that innervation to the posterior crista was still frequently absent in Slitrk6-knockout mice (Fig. 6G, H). After P14, 90.3% of posterior cristae of Slitrk6-deficient inner ear had no innervation (n = 62).
Loss of Neurons in Spiral and Vestibular Ganglia of the Slitrk6-Deficient Mice
To examine whether the number of neurons in the spiral and vestibular ganglia is affected by the deletion of Slitrk6, we calculated the number of neurons in each ganglion and its volume. In the spiral ganglion, neither the number of neurons nor ganglion volume was affected at E13.5; however, both were significantly reduced at E16.5, and at P0, they decreased to about a half of those of wild-type mice (Fig. 7A, B). Cell death in the spiral ganglion was significantly higher in Slitrk6-deficient mice at E16.5 than in the wild-type (Fig. 7C, G, K, Fig. S4A, B). Although the incidence of cell death was markedly reduced at P0 relative to the incidence at E16.5, it was still significantly higher in Slitrk6-deficient than in wild-type mice. In contrast to the change in the spiral ganglion, in the vestibular ganglion both the number of neurons and the volume began to decrease from E13.5, and their values were about 75% of those of wild-type mice from E16.5 to P0 (Fig. 7D, E). The incidence of cell death in the vestibular ganglion was significantly higher in Slitrk6-knockout mice at E13.5 and E16.5 than in wild-type mice (Fig. 7F, I, M, Fig. S4C, D). At P0, H&E-stained sections showed no apparent differences between wild-type and knockout mice in cell size and organization in either the spiral or vestibular ganglion (Fig. 7H, J, L, N). Reduction in both the number of neurons and volume in vestibular ganglion was prominent in inferior vestibular ganglion that gives rise to posterior crista innervation (Fig. S5) [17], [18]. This result suggests that the neuronal loss within the vestibular ganglion is largely due to the loss of neurons that innervate the posterior crista.
Decrease in the Neurite Outgrowth Activity in Cochlear Sensory Epithelia of Slitrk6-Deficient Mice
Inner ear sensory epithelia are known to release diffusible factors that promote survival and outgrowth of statoacoustic ganglion neurons [9]. Thus we examined whether Slitrk6-deficient sensory epithelia promote the survival and outgrowth of ganglion neurons by using culture experiments. In the explant culture, spiral ganglion neurons of both wild-type and Slitrk6-knockout mice could extend neurites toward wild-type sensory epithelia (Fig. 8A, B, E). However, sensory epithelia of knockout mice weakly attracted the neurites of both wild-type and knockout spiral ganglia (Fig. 8C–E). These results suggest that there is a reduction in the expression of trophic and/or tropic factors in Slitrk6-deficient sensory epithelia.
Decreased Expression of Neurotrophins and Their Receptors in the Slitrk6-Deficient Inner Ear
Among the molecules that promote survival and outgrowth of inner ear sensory neurons, neurotrophins are best characterized by multiple experiments. Brain-derived neurotrophic factor (Bdnf) and Neurotrophin-3 (Ntf3) are two neurotrophins that are produced in the developing sensory epithelia, and gene knockout studies revealed that they promote the innervation and support the survival of sensory neurons [19], [20]. Receptors for Bdnf (Ntrk2) and Ntf3 (Ntrk3) are expressed in sensory neurons, and mice deficient in these receptors also show reduced innervation and the loss of sensory neurons that mirror the ligand defects [21]. Therefore, we analyzed the expression of these molecules in E14.5 inner ear (which included the cochlear and vestibular sensory epithelia and the spiral and vestibular ganglia) by using real-time PCR. The expression of Bdnf and Ntf3 mRNA was significantly decreased in Slitrk6-deficient inner ear, whereas the expression of Ntrk2 and Ntrk3 was not affected (Fig. 9A). In situ hybridization of Bdnf and Ntf3 did not show any differences in the distribution of these mRNAs between wild-type (data not shown). We further examined the expression of Ntrk receptors using protein samples extracted from E14.5 cochleae (which included the cochlear sensory epithelium and spiral ganglion). We used cochleae at this developmental stage because neuronal loss in the spiral ganglion might affect the results at later stages. The expression level of both Ntrk2 and Ntrk3 proteins was significantly decreased in Slitrk6-deficient cochleae (Fig. 9B, C). In addition, the amount of phosphorylated form of Ntrk receptor was decreased in knockout mice (Fig. 9B, C). However, their spatial localization was not altered in the knockout mice (Fig. 9D–U). This suggests that Slitrk6 deletion interferes with the level of neurotrophin mRNA but affects Ntrks predominantly at the post-transcriptional level.
Discussion
In the present study, we developed Slitrk6-knockout mice and analyzed the development of their auditory and vestibular sensory organs. The overall innervation density of the cochlea was markedly reduced in the Slitrk6-deficient mice, and the innervation to the posterior crista in the vestibule was reduced, disoriented, or lost. Many sensory neurons in both the spiral and vestibular ganglia died during late embryonic development.
Slitrk6 is predominantly expressed in the sensory epithelia, which release trophic and/or tropic factors for sensory neurons. Especially, supporting cells of the organ of Corti in which Slitrk6 is strongly expressed, have been shown to play an important role in the survival of cochlear sensory neurons [22], [23]. The present results suggest that Slitrk6 participates in the transmembrane signal transduction processes that enhance the expression of such factors in sensory epithelia. Among these factors, neurotrophins are best understood by numerous studies [19], [20], [21]. In the developing inner ear, Ntf3 is expressed predominantly in the supporting cell and Bdnf in the hair cell lineages, and both Ntrk2 and Ntrk3 are expressed in the sensory neurons [22], [24], [25]. Mice deficient in one of these neurotrophins or Ntrk receptors show reduced innervation and loss of sensory neurons (Fig. S6) [16], [26]–[29]. Slitrk6-deficient inner ear showed mild but significant decrease in the expression level of both Bdnf and Ntf3 (Fig. 9A). In addition, we found the decrease in the expression of both Ntrk2 and Ntrk3 at protein level, and the amount of phosphorylated form of Ntrk receptors also decreased in Slitrk6-deficient cochlea (Fig. 9B, C). These results suggest a disturbance in the neurotrophin-Ntrk signaling in Slitrk6-deficient inner ear. Indeed, there are some similarities in the inner ear phenotypes of Slitrk6- and neurotrophin/Ntrk-deficient mice (Fig. S6, Table S1). Although the phenotypes of the inner ear with a partial reduction of both Bdnf-Ntrk2 and Ntf3-Ntrk3 signalings are still unknown, Bdnf heterozygous mutant mice display an intermediate decrease in size and neuronal number of the vestibular ganglion [28]. Thus, it is likely that quantitative changes in neurotrophin signaling contribute to the development of the inner ear phenotypes of Slitrk6-knockout mice.
Together with the co-culture experiment (Fig. 8), the reduction of the neurotrophins from cochlear sensory epithelia may be primarily responsible for the spiral ganglion innervation defects. This idea is also supported by a result that isolated spiral ganglia of Slitrk6-knockout mice can extend their neurites almost the same as those of wild-type mice in the presence of Ntf3 (Fig. S7). On the other hand, the sizes of the sensory epithelia in saccular macula and posterior crista were slightly lower in Slitrk6-deficient mice compared to those of wild-type littermates in late embryonic stages (Fig. S3E, F). This result indicates that Slitrk6 play some unidentified roles in the development of sensory epithelia per se. However, we believe that the innervation defects are not due to the sensory epithelial size reduction, because the innervation defect to the posterior crista was observed as early as E13.5 when the size reduction in the epithelium was not apparent.
The results in the present study indicate the involvement of neurotrophin–Ntrk signaling in the inner ear defects of Slitrk6-knockout mice. However, the decrease in the expression of neurotrophins in Slitrk6-knockout mice was relatively small compared to their dramatic phenotype. Culture experiments identified several candidate molecules (TGF-β, LIF, MCP-1, erythropoietin and Fgf2) that affect the survival and outgrowth of inner ear sensory neurons [30]–[33]. Slitrk6 may regulate the expression of some of those or other unidentified trophic and/or tropic factors that promote neuronal survival and neurite extension in addition to neurotrophins. Decrease in the expression of such factors may also affect the dynamics of Ntrk neurotrophin receptors.
Several genes are known to be critical for sensory neural development in the mouse inner ear, including NeuroD1, Brn3a (also called as Pou4f1), and ErbB [34]–[38]. Mice lacking NeuroD1 or Brn3a display defects in posterior crista innervation similar to those in Slitrk6-knockout mice, as well as loss of the inner ear sensory neurons [34]–[36]. The expression of Ntrk neurotrophin receptors is decreased in NeuroD1- and Brn3a-knockout mice, suggesting that neurotrophin-Ntrk receptor signaling is involved in the innervation defects and neuronal loss in these mutant mice [34]–[36]. Mice lacking ErbB function also show a marked decrease in the number of spiral ganglion neurons and disorganized inner ear innervation [37], [38]. ErbB2 and ErbB3 are highly expressed in the supporting cells of the organ of Corti and regulate the expression of neurotrophins, especially Ntf3. However, we could not detect any significant alteration in the expression of NeuroD1, Brn3a, ErbB2, or ErbB3 in the E14.5 cochleae (Fig. S8), in contrast to the reduction of neurotrophin receptors (Fig. 9B, C). Therefore, these genes may not function downstream of Slitrk6 during inner ear development.
Besides the strong expression in the sensory epithelia, Slitrk6 is also expressed in ganglion neurons weakly and transiently. However, isolated spiral ganglia of Slitrk6-knockout mice can extend their neurites almost the same as those of wild-type mice in a tissue culture experiment (Fig. 8A, B, E, Fig. S7). Although we cannot exclude the possibility that Slitrk6 expressed in ganglion neurons play some roles in target recognition or axon pathfinding, we consider that the observed abnormalities of Slitrk6-knockout mice are mainly attributed to the loss of Slitrk6 in sensory epithelia. Interestingly, previous studies have also reported the transient expression of neurotrophins in the inner ear ganglia, similar to the expression of Slitrk6 [22], [25]. The neurons that transiently express neurotrophins are cells delaminating from the sensory epithelia [39], and are postulated to have a role in guiding neurites from sensory neurons back to the area they delaminated from [40]. On the basis of its spatial distribution, we believe that Slitrk6 is localized in the delaminating cells, where it could participate in the neuronal organization of the inner ear and provides guidance as shown in the derailed pathfinding of posterior crista projections.
In conclusion, our results indicate that Slitrk6 is involved in the survival and innervation of sensory neurons in the inner ear, at least in part by modulating neurotrophin–Ntrk signaling. The molecular mechanisms involved in auditory and vestibular axon pathfinding are beginning to be unraveled [41], [42]. The results presented here provide new insight into the mechanisms of innervation in the developing inner ear. Further investigation of the molecular mechanisms of Slitrk6 function will increase our knowledge of sensory neural development of the inner ear.
Materials and Methods
Animals
Animal experiments were approved by the Animal Experiment Committee of the RIKEN Brain Science Institute. The mice were maintained by the Laboratory Animal Facility, Research Resource Center, RIKEN Brain Science Institute.
Generation of Slitrk6-Null Mutant Mice
Slitrk6-null mutant mice were generated as described previously [6], [43]. Briefly, to construct the Slitrk6 targeting vector, overlapping Slitrk6 genomic clones were isolated from a phage library made from mice of the 129SV strain (Stratagene, La Jolla, CA). The targeting construct contained the 1.8-kb 5′ and 5.3-kb 3′ homology regions, and the 3.3-kb fragment containing the ORF of Slitrk6 was replaced with a floxed PGK-neo expression cassette (Fig. 3A). E14 ES cells were transformed with the targeting construct by electroporation and selected with G418. Drug-resistant clones were screened by Southern blot analysis using PstI- and SpeI-digested genomic DNA hybridized, respectively, with a 0.7-kb 5′ genomic fragment corresponding to a genomic sequence outside of the sequence used in the targeting vector and a 0.6-kb PstI PGK-neo probe (Fig. 3A). Chimeric mice were generated by the injection of targeted ES cells into C57BL/6J blastocysts. To excise the PGK-neo cassette, mice with germline transmission of the transgene were first mated with CAG-Cre mouse. The correct excision of the PGK-neo cassette was confirmed by Southern blot analysis (Fig. 3B). The mice carrying the mutated Slitrk6 allele were backcrossed to C57BL/6J for more than 4 generations before analysis. Genotyping of progeny was performed by Southern blot or PCR analysis of genomic DNA extracted from tails. The PCR primers used were Slitrk6S (5′-CAGGAAGCATTCTCTTCTCTGTGA-3′), Slitrk6WTAS (5′-CATTGGTGACTGGGACTGTAGA-3′), and Slitrk6KOAS (5′-GGAAGCTCAGAACACTTACCTG-3′).
Generation of an Anti-Slitrk6 Antibody
A polyclonal anti-Slitrk6 antibody was raised in a rabbit against peptides corresponding to the carboxy-terminal region of mouse Slitrk6 (KNEYFELKANLHAEPDYLEVLEQQT). Peptides were synthesized and conjugated to keyhole limpet hemocyanin through a cysteine added to the N-terminus of the peptide. After immunization by conventional methods, antiserum was obtained and the antibody was purified by affinity chromatography with the immunized peptide. The antibody specificity was confirmed by the absence of Slitrk6 immunopositive signals in the Slitrk6-deficient inner ear (Fig. S1, Fig. 9B).
Morphological Analyses
In situ hybridization analysis for Slitrk6 was performed on paraformaldehyde-fixed paraffin sections obtained from wild-type mice as described previously [1], [7].
Immunohistochemistry was performed on paraffin or frozen sections and whole mounts. Rabbit polyclonal anti-Slitrk6 and anti–phospho-Ntrk (phosphorylated Tyr490 in NTRK1 and its corresponding residues in Ntrk2 and Ntrk3, Cell Signaling Technology, Beverly, MA), goat polyclonal anti-Ntrk2 (R&D Systems, Minneapolis, MN) and anti-Ntrk3 (R&D Systems), and mouse monoclonal anti-neurofilament (NF, Sigma, St. Louis, MO) and anti-βIII tubulin (Promega, Madison, WI) antibodies were used as primary antibodies. Alexa488 or 594-conjugated anti-rabbit, -goat, and -mouse IgG (Molecular Probes, Eugene, OR) were used as the secondary antibodies. To visualize inner and outer hair cells, whole mounts were stained with phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) (Sigma). For the visualization of myelinated nerve fibers, relatively mature inner ears were impregnated with osmium tetroxide.
To reveal the general pattern of innervation, nerve fibers were visualized with a lipophilic tracer. DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; Molecular Probes) crystals were placed into the brainstem alar plate to label eighth nerve afferent fibers to the inner ear, or into the olivo-cochlear efferent bundle near the floor plate to label the efferent fibers [13], [14]. After an appropriate diffusion time of 3 to 5 days at 37°C, whole inner ears or cochleae were then dissected out and viewed as whole-mount surface preparations. All the specimens mentioned above were examined with an Axioscop 2 plus (Carl Zeiss, Göttingen, Germany), MZ 16 FA (Leica Microsystems, Wetzlar, Germany), or FV1000 confocal laser microscope (Olympus, Tokyo, Japan).
For morphometrical analyses, inner ears from wild-type and Slitrk6-deficient mice were fixed in paraformaldehyde, embedded in paraffin, and serially sectioned at 6 µm. Every fourth section was stained with H&E. Comparisons of neuronal numbers in the spiral and vestibular ganglia were made by counting the number of neurons with clear nucleoli. No correction was made for split nucleoli. Dying neuronal cells that could be identified by pyknotic nuclei were counted in the same way. The volume of each ganglion was calculated by measuring the area occupied by neurons in each section using AxioVision software (Carl Zeiss).
Explant Culture of Cochlear Sensory Epithelium and Spiral Ganglion
Sensory epithelium and spiral ganglion were dissected from E14.5 cochlea. A cochlear sensory epithelium and a spiral ganglion were randomly chosen and placed in three-dimensional collagen gel (Cell Matrix; Nitta Gelatin, Osaka, Japan) about 500 µm apart each other. Then they were cultured in neurobasal medium (Invitrogen, Carlsbad, CA) containing 2 mM L-glutamine, B27 supplement (Invitrogen) for 48 hours. Neurites were visualized by the immunostaining for neurofilament (Sigma). Neurite outgrowth was analyzed with ImageJ (http://rsb.info.nih.gov/ij/). Area occupied by the neurofilament-positive neurites were measured and relative to the area of the ganglion body (%) is presented.
RNA Extraction and Real-Time RT-PCR Analyses
Inner ears from 5 to 6 E14.5 wild-type and Slitrk6-deficient mice were pooled, and total RNA was extracted with an RNeasy Protect Kit (QIAGEN, Hilden, Germany). Reverse transcription was performed with Superscript II reverse transcriptase (Invitrogen). Real-time PCR was performed with SYBR Green Real-time PCR Master Mix (Applied Biosystems, Foster City, CA) on an ABI Prism 7900HT (Applied Biosystems). The primers used for detection of Bdnf and Ntf3 were designed by Stankovic and Corfas [44], and those for Ntrk2 and Ntrk3 were as follows: Ntrk2, sense: 5′-AGCCTTCTCCAGGCATCGT-3′, antisense: 5′-TCCGGGTCAACGCTGTTAG-3′, and Ntrk3, sense: 5′-TGTAGTTTCTGGCGGATTTTCTT-3′, antisense: 5′-AGCACGGAGCCCACATAGTC-3′. The intensity relative to G3PDH was calculated, and the fold change relative to the relative intensity in wild-type mice is presented.
Protein Extraction and Western Blot Analysis
Cochleae from 12 to 13 E14.5 wild-type and Slitrk6-deficient mice were dissected and homogenized in RIPA buffer (50 mM Tris-HCl pH 7.8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 2 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail [Roche Diagnostics, Mannheim, Germany]). Approximately 20 µg of extract was loaded onto a SDS-PAGE gel, electrophoresed, and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Rabbit polyclonal anti-Slitrk6, anti–phospho-Ntrk (Cell Signaling Technology), anti–Myosin VIIa (Abcam, Cambridge, UK), anti–β-actin (Sigma), goat polyclonal anti-Ntrk2 (R&D Systems), and anti-Ntrk3 (R&D Systems), and mouse monoclonal anti-βIII tubulin (Promega) antibodies were used as primary antibodies. After incubation with appropriate secondary antibodies conjugated to horseradish peroxidase, the signals were detected with an ECL Plus kit (GE Healthcare, Buckinghamshire, UK). Signals in the Western blots were analyzed with ImageJ. The relative intensity against β-actin was calculated, and fold change relative to the relative intensity in wild-type mice is presented.
Supporting Information
Acknowledgments
We are grateful to Dr. Doris K. Wu (NIH) and Dr. Lisa V. Goodrich (Harvard Medical School) for their helpful comments on the manuscript. We thank following RIKEN BSI Research Resource Center staff for their technical assistance: Dr. Mika Tanaka and Ms. Chieko Nishioka for generating Slitrk6-knockout mice, Mr. Masaki Kumai for generating the anti-Slitrk6 antibody, Mr. Keisuke Fukumoto for real-time PCR analysis, and Mr. Hiromasa Morishita for the affinity purification of the antibody.
Footnotes
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was funded by RIKEN BSI and supported by the Japan Society for the Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
- 1.Aruga J, Mikoshiba K. Identification and characterization of Slitrk, a novel neuronal transmembrane protein family controlling neurite outgrowth. Mol Cell Neurosci. 2003;24:117–129. doi: 10.1016/s1044-7431(03)00129-5. [DOI] [PubMed] [Google Scholar]
- 2.Aruga J, Yokota N, Mikoshiba K. Human SLITRK family genes: genomic organization and expression profiling in normal brain and brain tumor tissue. Gene. 2003;315:87–94. doi: 10.1016/s0378-1119(03)00715-7. [DOI] [PubMed] [Google Scholar]
- 3.Kobe B, Kajava AV. The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol. 2001;11:725–732. doi: 10.1016/s0959-440x(01)00266-4. [DOI] [PubMed] [Google Scholar]
- 4.Brose K, Tessier-Lavigne M. Slit proteins: key regulators of axon guidance, axonal branching, and cell migration. Curr Opin Neurobiol. 2000;10:95–102. doi: 10.1016/s0959-4388(99)00066-5. [DOI] [PubMed] [Google Scholar]
- 5.Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem. 2003;72:609–642. doi: 10.1146/annurev.biochem.72.121801.161629. [DOI] [PubMed] [Google Scholar]
- 6.Katayama K, Yamada K, Ornthanalai VG, Inoue T, Ota M, et al. Slitrk1-deficient mice display elevated anxiety-like behavior and noradrenergic abnormalities. Mol Psychiatry in press. 2009 doi: 10.1038/mp.2008.97. [DOI] [PubMed] [Google Scholar]
- 7.Aruga J. Slitrk6 expression profile in the mouse embryo and its relationship to that of Nlrr3. Gene Expr Patterns. 2003;3:727–733. doi: 10.1016/s1567-133x(03)00141-8. [DOI] [PubMed] [Google Scholar]
- 8.Kelley MW. Regulation of cell fate in the sensory epithelia of the inner ear. Nat Rev Neurosci. 2006;7:837–849. doi: 10.1038/nrn1987. [DOI] [PubMed] [Google Scholar]
- 9.Bianchi LM, Cohan CS. Effects of the neurotrophins and CNTF on developing statoacoustic neurons: comparison with an otocyst-derived factor. Dev Biol. 1993;159:353–365. doi: 10.1006/dbio.1993.1247. [DOI] [PubMed] [Google Scholar]
- 10.Fekete DM. Cell fate specification in the inner ear. Curr Opin Neurobiol. 1996;6:533–541. doi: 10.1016/s0959-4388(96)80061-4. [DOI] [PubMed] [Google Scholar]
- 11.Giraldez F, Fritzsch B. The molecular biology of ear development - “Twenty years are nothing”. Int J Dev Biol. 2007;51:429–438. doi: 10.1387/ijdb.072390fg. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Sakai K, Miyazaki J. A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. Biochem Biophys Res Commun. 1997;237:318–324. doi: 10.1006/bbrc.1997.7111. [DOI] [PubMed] [Google Scholar]
- 13.Cole KS, Robertson D. Early efferent innervation of the developing rat cochlea studied with a carbocyanine dye. Brain Res. 1992;575:223–230. doi: 10.1016/0006-8993(92)90083-l. [DOI] [PubMed] [Google Scholar]
- 14.Fritzsch B, Nichols DH. DiI reveals a prenatal arrival of efferents at the differentiating otocyst of mice. Hear Res. 1993;65:51–60. doi: 10.1016/0378-5955(93)90200-k. [DOI] [PubMed] [Google Scholar]
- 15.Fritzsch B, Silos-Santiago I, Bianchi LM, Farinas I. Effects of neurotrophin and neurotrophin receptor disruption on the afferent inner ear innervation. Semin Cell Dev Biol. 1997;8:277–284. [PubMed] [Google Scholar]
- 16.Fritzsch B, Barbacid M, Silos-Santiago I. The combined effects of trkB and trkC mutations on the innervation of the inner ear. Int J Dev Neurosci. 1998;16:493–505. doi: 10.1016/s0736-5748(98)00043-4. [DOI] [PubMed] [Google Scholar]
- 17.Maklad A, Fritzsch B. Development of vestibular afferent projections into the hindbrain and their central targets. Brain Res Bull. 2003;60:497–510. doi: 10.1016/s0361-9230(03)00054-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Maklad A, Fritzsch B. Partial segregation of posterior crista and saccular fibers to the nodulus and uvula of the cerebellum in mice, and its development. Brain Res Dev Brain Res. 2003;140:223–236. doi: 10.1016/s0165-3806(02)00609-0. [DOI] [PubMed] [Google Scholar]
- 19.Rubel EW, Fritzsch B. Auditory system development: primary auditory neurons and their targets. Annu Rev Neurosci. 2002;25:51–101. doi: 10.1146/annurev.neuro.25.112701.142849. [DOI] [PubMed] [Google Scholar]
- 20.Pirvola U, Ylikoski J. Neurotrophic factors during inner ear development. Curr Top Dev Biol. 2003;57:207–223. doi: 10.1016/s0070-2153(03)57007-7. [DOI] [PubMed] [Google Scholar]
- 21.Fritzsch B, Tessarollo L, Coppola E, Reichardt LF. Neurotrophins in the ear: their roles in sensory neuron survival and fiber guidance. Prog Brain Res. 2004;146:265–278. doi: 10.1016/S0079-6123(03)46017-2. [DOI] [PubMed] [Google Scholar]
- 22.Farinas I, Jones KR, Tessarollo L, Vigers AJ, Huang E, et al. Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression. J Neurosci. 2001;21:6170–6180. doi: 10.1523/JNEUROSCI.21-16-06170.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Sugawara M, Corfas G, Liberman MC. Influence of supporting cells on neuronal degeneration after hair cell loss. J Assoc Res Otolaryngol. 2005;6:136–147. doi: 10.1007/s10162-004-5050-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Pirvola U, Ylikoski J, Palgi J, Lehtonen E, Arumae U, et al. Brain-derived neurotrophic factor and neurotrophin 3 mRNAs in the peripheral target fields of developing inner ear ganglia. Proc Natl Acad Sci U S A. 1992;89:9915–9919. doi: 10.1073/pnas.89.20.9915. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Schecterson LC, Bothwell M. Neurotrophin and neurotrophin receptor mRNA expression in developing inner ear. Hear Res. 1994;73:92–100. doi: 10.1016/0378-5955(94)90286-0. [DOI] [PubMed] [Google Scholar]
- 26.Ernfors P, Van De Water T, Loring J, Jaenisch R. Complementary roles of BDNF and NT-3 in vestibular and auditory development. Neuron. 1995;14:1153–1164. doi: 10.1016/0896-6273(95)90263-5. [DOI] [PubMed] [Google Scholar]
- 27.Schimmang T, Minichiello L, Vazquez E, San Jose I, Giraldez F, et al. Developing inner ear sensory neurons require TrkB and TrkC receptors for innervation of their peripheral targets. Development. 1995;121:3381–3391. doi: 10.1242/dev.121.10.3381. [DOI] [PubMed] [Google Scholar]
- 28.Bianchi LM, Conover JC, Fritzsch B, DeChiara T, Lindsay RM, et al. Degeneration of vestibular neurons in late embryogenesis of both heterozygous and homozygous BDNF null mutant mice. Development. 1996;122:1965–1973. doi: 10.1242/dev.122.6.1965. [DOI] [PubMed] [Google Scholar]
- 29.Fritzsch B, Farinas I, Reichardt LF. Lack of neurotrophin 3 causes losses of both classes of spiral ganglion neurons in the cochlea in a region-specific fashion. J Neurosci. 1997;17:6213–6225. doi: 10.1523/JNEUROSCI.17-16-06213.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Marzella PL, Gillespie LN, Clark GM, Bartlett PF, Kilpatrick TJ. The neurotrophins act synergistically with LIF and members of the TGF-beta superfamily to promote the survival of spiral ganglia neurons in vitro. Hear Res. 1999;138:73–80. doi: 10.1016/s0378-5955(99)00152-5. [DOI] [PubMed] [Google Scholar]
- 31.Bianchi LM, Daruwalla Z, Roth TM, Attia NP, Lukacs NW, et al. Immortalized mouse inner ear cell lines demonstrate a role for chemokines in promoting the growth of developing statoacoustic ganglion neurons. J Assoc Res Otolaryngol. 2005;6:355–367. doi: 10.1007/s10162-005-0013-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Berkingali N, Warnecke A, Gomes P, Paasche G, Tack J, et al. Neurite outgrowth on cultured spiral ganglion neurons induced by erythropoietin. Hear Res. 2008;243:121–126. doi: 10.1016/j.heares.2008.07.003. [DOI] [PubMed] [Google Scholar]
- 33.Hossain WA, D'Sa C, Morest DK. Interactive roles of fibroblast growth factor 2 and neurotrophin 3 in the sequence of migration, process outgrowth, and axonal differentiation of mouse cochlear ganglion cells. J Neurosci Res. 2008;86:2376–2391. doi: 10.1002/jnr.21685. [DOI] [PubMed] [Google Scholar]
- 34.Liu M, Pereira FA, Price SD, Chu MJ, Shope C, et al. Essential role of BETA2/NeuroD1 in development of the vestibular and auditory systems. Genes Dev. 2000;14:2839–2854. doi: 10.1101/gad.840500. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Huang EJ, Liu W, Fritzsch B, Bianchi LM, Reichardt LF, et al. Brn3a is a transcriptional regulator of soma size, target field innervation and axon pathfinding of inner ear sensory neurons. Development. 2001;128:2421–2432. doi: 10.1242/dev.128.13.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Kim WY, Fritzsch B, Serls A, Bakel LA, Huang EJ, et al. NeuroD-null mice are deaf due to a severe loss of the inner ear sensory neurons during development. Development. 2001;128:417–426. doi: 10.1242/dev.128.3.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Stankovic K, Rio C, Xia A, Sugawara M, Adams JC, et al. Survival of adult spiral ganglion neurons requires erbB receptor signaling in the inner ear. J Neurosci. 2004;24:8651–8661. doi: 10.1523/JNEUROSCI.0733-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Morris JK, Maklad A, Hansen LA, Feng F, Sorensen C, et al. A disorganized innervation of the inner ear persists in the absence of ErbB2. Brain Res. 2006;1091:186–199. doi: 10.1016/j.brainres.2006.02.090. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Fritzsch B. Development of inner ear afferent connections: forming primary neurons and connecting them to the developing sensory epithelia. Brain Res Bull. 2003;60:423–433. doi: 10.1016/s0361-9230(03)00048-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Fritzsch B, Beisel KW, Jones K, Farinas I, Maklad A, et al. Development and evolution of inner ear sensory epithelia and their innervation. J Neurobiol. 2002;53:143–156. doi: 10.1002/neu.10098. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Webber A, Raz Y. Axon guidance cues in auditory development. Anat Rec A Discov Mol Cell Evol Biol. 2006;288:390–396. doi: 10.1002/ar.a.20299. [DOI] [PubMed] [Google Scholar]
- 42.Fekete DM, Campero AM. Axon guidance in the inner ear. Int J Dev Biol. 2007;51:549–556. doi: 10.1387/ijdb.072341df. [DOI] [PubMed] [Google Scholar]
- 43.Inoue T, Hatayama M, Tohmonda T, Itohara S, Aruga J, et al. Mouse Zic5 deficiency results in neural tube defects and hypoplasia of cephalic neural crest derivatives. Dev Biol. 2004;270:146–162. doi: 10.1016/j.ydbio.2004.02.017. [DOI] [PubMed] [Google Scholar]
- 44.Stankovic KM, Corfas G. Real-time quantitative RT-PCR for low-abundance transcripts in the inner ear: analysis of neurotrophic factor expression. Hear Res. 2003;185:97–108. doi: 10.1016/s0378-5955(03)00298-3. [DOI] [PubMed] [Google Scholar]
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