Survival of inner ear sensory neurons in trk mutant mice

ELSEVIER

Mechanisms of Development 64 (1997) 77-85

Survival of inner ear sensory neurons in trk mutant mice Thomas Schimmang a, 1,,, Gonzalo Alvarez-Bolado a,1, Liliana Minichiello b,1, Esther Vazquez a, Fernando Giraldez a, Rtidiger Klein b, Juan Represa a alnstituto de Biologfa y Gen#tica Molecular, Universidad de Valladolid y Consejo Superior de Investigaciones Cientificas, Facultad de Medicina, 47005 Valladolid, Spain bCell Regulation Programme, European Molecular Biology Laborato~., 69117 Heidelberg, Germany

Received 4 February 1997; revised version received 27 March 1997; accepted 27 March 1997

Abstract Analysis of trkB-/-; trkC -/- double mutant mice revealed that peripheral and central inner ear sensory neurons are affected in these mice. However, a substantial amount of cochlear and vestibular neurons survive, possibly due to maintenance or upregulation of TrkA expression. To clarify the function of the TrkA receptor during development of the cochlear and vestibular ganglion we analysed trkA -Imice and the expression of this receptor in inner ear sensory neurons of trkB-/-; trkC -/- animals. TrkA homozygous mutant mice showed normal numbers of neurons and no TrkA expression was detected in neurons of trkB-/-; trkC-/- double mutant mice. We conclude that TrkA is not essential for inner ear development and that in the absence of any of the known catalytic Trk receptors peripheral inner ear sensory neurons are prone to undergo cell death or must use a different signaling mechanism to survive. © 1997 Elsevier Science Ireland Ltd. Keywords: Neurotrophin receptor; Mouse mutant; Inner ear; Sensory neurons; Central targets

1. Introduction Proper development of the peripheral sensory nervous system depends on the action of neurotrophic factors. Four of these neurotrophic factors, nerve growth factor (NGF), brain-derived factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT-4) form the family of neurotrophins in mammals (Glass and Yancopoulos, 1993). The biological effects of neurotrophins are mediated by the trk genes, which encode a family of protein tyrosine kinase receptors composed of TrkA, TrkB and TrkC (Barbacid, 1994). TrkB and TrkC also encode for non-catalytic neurotrophin receptor isoforms of an as yet unknown function (Klein et al., 1990; Middlemas et al., 1991; Tsoulfas et al., 1993; Valenzuela et al., 1993). Finally, all neurotrophins bind to the low-affinity NGF receptor, p75, whose role may be to modulate Trk signaling (Glass and Yancopoulos,

* Corresponding author. Tel.: +34 83 423609; fax: +34 83 423588; e-mail: [email protected] These authors contributed equally to this work.

1993; Davies et al., 1994; Hantzopoulos et al., 1994; Verdi et al., 1994). The cochlear and vestibular ganglia contain neurons which innervate the inner ear and connect the sensory epithelia of the cochlea and the vestibular apparatus via afferent nerve fibres with their respective central nuclei in the brainstem (Spoendlin, 1988). trkB, trkC and p 7 5 are expressed in the cochlear and vestibular ganglia (Ernfors et al., 1992; Tessarollo et al., 1993; Ylikoski et al., 1993; Lamballe et al., 1994; Schecterson and Bothwell, 1994; Vazquez et al., 1994) and their central nuclei (Merlio et al., 1992; Zhou et al., 1993; Gibbs and Pfaff, 1994; Hafidi et al., 1996). Additionally, the truncated forms of TrkB and TrkC are detected in non-neuronal structures of the inner ear (Pirvola et al., 1994; Biffo et al., 1995). BDNF and NT-3, which bind to their high-affinity receptors TrkB and TrkC, respectively, are found in the sensory epithelia (Pirvola et al., 1992, 1994; Schecterson and Bothwell, 1994). Bioassays of whole ganglia or dissociated neuron cultures show the effectiveness of BDNF and NT-3 to promote neurite outgrowth and neuronal survival (Pirvola et al., 1994; Vazquez et al., 1994). The recent analysis of

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knockout mice in either neurotrophins or Trk receptors underlines their essential role for neuronal survival and target innervation of the inner ear in vivo. Mutant mice for either BDNF, NT-3, TrkB or TrkC show reduced numbers of inner ear sensory neurons (Farifias et al., 1994; Jones et al., 1994; Ernfors et al., 1995; Fritsch et al., 1995; Minichiello et al., 1995; Schimmang et al., 1995). b d n f -/- and trkB -/- mice lose the majority of vestibular neurons and type 2 cochlear neurons, whereas nt-3 - / - and trkC - t mice lack specifically type 1 cochlear neurons and a subset of vestibular neurons. The role of N G F and its high-affinity receptor TrkA for inner ear development is controversial. TrkA and N G F are expressed in the cochleovestibular ganglion during early embryonic development (day 1 1 - 1 4 p.c.) and both are also detected in the central targets of these neurons during adult life (Gibbs and Pfaff, 1994; Nishio et al., 1994; Pirvola et al., 1994; Schecterson and Bothwell, 1994; Staecker et al., 1996). At high concentrations (up to 200 ng/ml) N G F promotes neuronal survival and neuritogenesis at day 11 and 12 p.c., respectively (Lefebvre et al., 1990, 1991). On the other hand, ganglion explants of the identical developmental stages do not evoke neurites upon treatment with the same dose of N G F (Pirvola et al., 1992). Only a very weak survival-promoting effect is measured in dissociated neurons of day 13 p.c. at lower concentrations of N G F (100 pg/ ml and 10 ng/ml; Pirvola et al., 1994) and no effect is seen on postnatal cochlear and vestibular neurons at high doses of N G F (50 ng/ml; Zheng et al., 1995a,b). Downregulation of N G F in explants of otocysts and cochleovestibular ganglia of day 10.5 p.c. using antisense oligonucleotides causes an inhibition of neuritogenesis and a decrease of neuronal survival (Staecker et al., 1996). Finally, whereas b d n f - / - ; n t - 3 - / - double mutant mice lack 99% of their vestibular and all of their cochlear neurons at day 18 p.c., a substantial amount (about 40%) of cochlear and vestibular neurons survive in t r k B - / - ; trkC - / - animals at birth (Ernfors et al., 1995; Minichiello et al., 1995). The latter results may be explained by expression of TrkA in cochlear and vestibular

neurons of t r k B - / - ; trkC - / - animals which would allow neuronal survival via N G F supplied from a local source or the central targets. To investigate this possibility and to clarify the function of TrkA and N G F for the development of the inner ear we analysed trkA - / - and t r k B - / - ; trkC - t double mutant mice. Central auditory and vestibular nuclei are present in t r k B - / - ; trkC - / - mice, but reveal defects due to increased cell death compared to wild-type animals. t r k A - / - animals show normal neuronal numbers and target innervation, indicating that TrkA is usually not required for the development of the vestibular and auditory system. Moreover, the surviving neurons in t r k B - / - ; trkC -/- mice do not express the high-affinity receptor TrkA. In absence of any of the known catalytic Trk receptors the cochlear and vestibular neurons in t r k B - t - ; t r k C - / - mutant animals may eventually die during later development or depend on other signaling pathways distinct from the ones exerted by the catalytic forms of Trk receptors. The potential roles of truncated Trk receptors and the low-affinity receptor p75 are discussed.

2. Results 2.1. Neuronal survival in the central targets o f inner ear sensory neurons is also affected in TrkB and TrkC double mutant mice

To determine the roles of Trk receptors for neuronal survival in the auditory and vestibular system we analysed t r k B - / - ; trkC -/- double mutant animals. Morphometric measurements of the cochlear and vestibular ganglia reveal a volume reduction of about 70% which is likely a consequence of the recently observed neuronal loss of approximately 60% in these mice compared to wild-type animals (Fig. 1, Table 1) (Minichiello et al., 1995). Figs. 1 and 2A,C show representative examples of cresyl violet-stained sections through a cochlear ganglion of a neonatal wildtype and a t r k B - / - ; trkC - / - mouse. At the cellular level

Fig. 1. Cochlear ganglion of a neonatal wild-type and a trkB-/-; trkC -/- double mutant mouse. Cresyl violet-stained sections of the cochlear ganglion (Gc VIII) of a wild-type (A) and a trkB-/-; trkC-/- animal (B). The circumference of the ganglion is indicated by arrowheads. Note the reduced size and staining of the ganglion in the mutant animal. Scale bar in (B) corresponds to 100/~m in (A,B). Fig. 2. Cochlear phenotypes of trk mutant animals. Cresyl violet-stained sections of the cochlear ganglia of a wild-type (A), a trkA -/- (B) and a trkB-/-; trkC / mouse (C). Note the wild-type phenotype observed in a trkA /- mouse. The trkB-/-; trkC -/- animal contains many weakly stained neurons and some pycnotic figures (arrows). (D,E) Innervation of the cochlear sensory epithelia (se) in a neonatal trkA -/- and a wild-type mouse. An antibody raised against/~-tubulindetects neurites (indicated by arrows) which reach and enter the sensory epithelium. Scale bar in (A) corresponds to 50 t~m in (A-C). Scale bar in (D) corresponds to 100/zm in (D,E). Fig. 3. Central cochlear and vestibular targets of wild-type and trkB-/-; trkC -/- animals. Cresyl violet-stained sections of the ventral cochlear nucleus (A-D) and the lateral vestibular nucleus (E,F) of wild-type (A,C,E) and trkB / ; trkC -/- mice (B,D,F). Note the smaller size of the ventral cochlear nucleus (VCO; arrowheads delimit the dorsal and medial boundaries) in the mutant mouse (B) compared to the wild-type (A). Abbreviations: CBX, cerebellar cortex primordium; chp, choroid plexus; Lme, membranous labyrinth; VCO, ventral cochlear nucleus; wm, white matter (includes part of the vestibular nerve and inferior cerebellar peduncle); asterisk, bony labyrintk. (C,D) High magnificationsof the ventral cochlear nucleus reveal numerous pycnotic nuclei (arrows) in trkB-/-: trkC -/ animals(D). (E,F) High magnificationdetail of the lateral vestibular nucleus. The arrow in (E) points to a large neuron characteristic for this area. Note the presence of pycnotic nuclei (arrows) in the mutant animal (F). Scale bar in (A) correspondsto 250 t~m in (A,B). Scale bar in (C) corresponds to 25/~m in (C-F).

T. Schimmang et al. / Mechanisms of Development 64 (1997) 77-85

sections from mutant animals reveal neurons which stain less and often contain pycnotic figures, indicative of cell death.

The sensory epithelia of the inner ear which represent the peripheral targets of inner ear sensory neurons have been shown to be affected in t r k B - / - ; trkC - I - mutant animals

3

2

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T. Schimmang et al. / Mechanisms of Development 64 (1997) 77-85

Table 1 Volumes of cochlear and vestibular ganglia and cochlear central nuclei in trkB-/-; trkC /- mutant mice Wild-type Cochlear ganglion Vestibular ganglion Cochlear nuclei

trkB-/-; trkC -/- % reduction

25.8 ± 0.6 (7) 17.0 + 0.5 (7)

9.1 +_0.6 (3) 5.2 + 0.3 (3)

155.2 + 4.4 (4)

78.7 + 17.3 (4)

65** 69* 49*

The volumes in 106/zm3 of ganglia and nuclei were calculated from newborn mice (P1) as described in Schimmang et al., 1995. Mean number of and volumes ( ± SEM) are listed and the sampling number is shown in parentheses. Differences were tested using Student's t-test. (Minichiello et al., 1995). The central targets of these neurons are composed of the cochlear and vestibular nuclei which express trkB and trkC (Merlio et al., 1992; Zhou et al., 1993) and therefore may also be affected in t r k B - / - ; trkC - / - mice. Microscopic examination of cresyl violetstained sections shows that neonatal t r k B - / - ; trkC - / - double mutant mice develop central cochlear and vestibular nuclei (Fig. 3). However, at high magnification numerous pycnotic nuclei are detected in mutant animals (Fig. 3D,F), pointing out a defect in cell survival. For morphometric measurements we chose to analyse the cochlear nuclei because their boundaries are distinct and easily defined at this stage of development. Volume measurements of these nuclei reveal an average size reduction of 50% in t r k B - / - ; trkC - / - double mutant mice (Table 1). Therefore, loss of the catalytic forms of the TrkB and TrkC receptor affects neuronal survival in the peripheral and central parts of the auditory and vestibular system. 2.2. Neuronal survival and target innervation are not affected in the inner ear o f homozygous trkA mutant mice

The results described above confirm that neuronal survival in the cochlear and vestibular ganglia is severely affected in t r k B - / - ; trkC - / - mutant mice. However, a substantial number of peripheral sensory neurons survive in these animals at birth. To analyse a potential role of the remaining Trk receptor, TrkA, during development of the inner ear we studied mice carrying a t r k A locus specifically targeted within its protein tyrosine kinase sequences (Smeyne et al., 1994). W e first examined cresyl violetstained sections of neonatal P1 mice containing cochlear and vestibular ganglia. Mutant animals show a normal cell density and neuronal morphology (Fig. 2B). Neuronal cell counts on serial sections reveal no differences between wild-type and t r k A - / - mice (Table 2). Likewise, combinations of the trkA mutant allele with trkB or trkC mutant alleles do not result in increased neuron loss compared with neuronal counts of single t r k B - / - or trkC - / - animals (Table 2). W e next studied target innervation of inner ear sensory epithelia in t r k A - / - mice. Antibodies directed against /3tubulin (Moskowitz et al., 1993) detect nerve fibres which

contact and enter the cochlear and vestibular epithelia as in wild-type animals (Fig. 2D,E). These results indicate that the TrkA signaling pathway is normally not required for formation of cochlear and vestibular ganglia and innervafion of their target epithelia. 2.3. Inner ear sensory neurons o f TrkB and TrkC double mutant mice do not maintain or upregulate TrkA expression during later development

Double mutant mice which are homozygous for a TrkB and TrkC mutation or homozygous mutant for one of these receptors and heterozygous for the other are severely affected in the survival of inner ear sensory neurons (Minichiello et al., 1995). Nevertheless, about 40% of cochlear and vestibular neurons survive in these animals at birth. In wild-type animals TrkA and N G F have only been detected during early development of these neurons (Pirvola et al., 1994; Schecterson and Bothwell, 1994; Staecker et al., 1996), but their expression may be maintained or upregulated during later development in the absence of TrkB or TrkC receptors and thereby permit neuronal survival. Alternatively, central neurons of TrkB and TrkC mutants may serve as a source supplying N G F to peripheral sensory neurons expressing TrkA. To investigate the potential role of TrkA for neuronal survival in this context we examined its expression in TrkB and TrkC double mutants. R N A in situ hybridisations were performed using a specific probe for the trkA receptor (see Section 4) on sections of wild-type and TrkB and TrkC double mutant mice. Whereas trigeminal ganglia which serve as a positive control on the same section show trkA transcripts (Martin-Zanca et al., 1990), no expression is detected in vestibular and cochlear ganglia of Table 2 Numbers of neurons in cochlear and vestibular ganglia of trk mutant mice Genotype

Mean no. of cochlear neurons per ganglion

%reduction

Wild-type trkA -ItrkA-/-; trkB +ltrkA-/-; trkB -/trkB -/trkA-I-; trkC -/trkC -/-

7950 + 236 (8) 8219 + 129 (3) 7611 + 429 (2) 6646 (1) 6765 + 119 (4) 4181 + 107 (2) 3901 + 157 (6)

0a 0 4b 16c 15a 47d 51a

Genotype

Mean no. of vestibular neurons per ganglion

%reduction

Wild-type trkA -/-

4819 + 115 (8) 4480 + 323 (3)

0a 7b

Neurons were counted from newborn mice (P1) as described in Schimmang et al., 1995. Mean numbers of neurons (±SEM) are listed parentheses. Differences were tested using Student's t-test. aData from Minichiello et al., 1995. ~Not significant compared to wild-t~_pe. CNot significant compared to trkB- -. aNot significant compared to trkC -/-.

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ganglion or the superior IX/X ganglion show strong TrkA immunoreactivity (Martin-Zanca et al., 1990), no expression is observed in cochlear or vestibular ganglia of TrkB and TrkC double mutant mice (Fig. 5). Therefore, the surviving neurons in these mutant mice apparently do not maintain or upregulate TrkA expression.

4

3. Discussion

3.1. TrkB and TrkC double mutant mice show defects in the peripheral and central parts of the auditory and vestibular system

Fig. 4. Expression of trkA RNA in wild-type and TrkB and TrkC double mutant animals. A specific probe for trkA detects transcripts in sections of the trigeminal ganglion in normal (A) and trkB -/ ; trkC +/- animals (C). On the same section no expression of trkA is observed in the vestibular ganglion of both wild-type and mutant animals (B,D, respectively). Scale bar in (A) corresponds to 25/~m in (A-D).

wild-type, trkB-t-; trkC +t- and trkB-/-; trkC -t- double mutant mice (Fig. 4 and data no t shown). To detect TrkA protein we used a highly specific antiserum raised against the extracellular domain of TrkA (Clary et al., 1994). Whereas positive control structures like the trigeminal

A recent study has described inner ear defects of the peripheral nervous system in trkB-t-; trkC -t- mutant mice (Minichiello et al., 1995). These animals show reduced numbers of inner ear sensory neurons and defects of their peripheral targets. The present study shows that the central targets of these neurons are also affected in trkB-/-; trkC -tmutants. Since TrkB and TrkC are detected in the central auditory and vestibular nuclei of rodents during postnatal development and in adulthood (Merlio et al., 1992; Zhou et al., 1993; Hafidi et al., 1996) the observed defects may be a direct consequence of the lack of TrkB and TrkC expression in central inner ear sensory neurons. However, several studies show an interdependence of the peripheral ganglia and their central targets. For example, postnatal ablation of the cochlea leads to a size reduction of 54% of the cochlear nucleus (Trune, 1982) which fits very well with the 50% volume loss of this nucleus observed in trkB-/-; trkC -/-

5 !~;~ ~:~,i~i~i~i!~!~i~i~i~~ ~i~ °~ ~ i i ~ , ~ ~: ~ i ~ i ?

~ i ~,i~ i ~ii~ ~:i~i~iliii~i~~!!:,i=~.~!::i~!~ii~i~:~il~i~ .~i~ii

iill i~i~i~!~i!~ ~y:~~%,~,~ ~ '

~!~'i~ !

~=

, ~ i ~ : ~~~

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Fig. 5. Expression of TrkA protein in TrkB and TrkC double mutant animals. A specific antibody raised against the extracellular domain of TrkA detects immunoreactivity in sections containing the trigeminal (A) and superior IX/X ganglion (C) of trkB-/-; trkC -/- animals. In the same animal no significant expression of TrkA is detected in the cochlear (B) and vestibular ganglion (D). Scale bar in (A) corresponds to 25/zm in (A-D).

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T. Schimmang et al. / Mechanisms of Development 64 (1997) 77-85

mutants. Consequently, the central auditory defects in these mice may be caused by an indirect mechanism due to the reduced number of neurons in the peripheral ganglia of trkB-/-; trkC -/- animals which may serve as a source of trophic factors for central targets. Vice versa, improved survival of cochlear neurons is demonstrated in cocultures with their central targets (Ard et al., 1985; Zhou and Van De Water, 1987). Therefore defects of the central nuclei in TrkB and TrkC double mutants may cause a reduction of peripheral ganglia. Similar defects like the ones seen in the auditory and vestibular system of TrkB and TrkC double mutant mice are also observed in knockout animals for BDNF and NT-3 (Erufors et al., 1995; Bianchi et al., 1996). Cochlear and vestibular ganglia and the afferent innervation towards their peripheral targets are severely affected or completely lost in these mice. The state of the central auditory and vestibular nuclei of bdnff/- and nt-3-/mutant animals has not been analysed in detail, but their afferent innervation from the peripheral sensory neurons is severely reduced (Ernfors et al., 1995). Therefore, signaling of BDNF and NT-3 via their high-affinity receptors TrkB and TrkC, respectively, constitute an essential component for the formation and innervation of the peripheral and central components of the inner ear. 3.2. Survival of inner ear sensory neurons without Trk receptors Several studies examining expression of NGF and TrkA and the effectiveness of NGF in bioassays suggest a function for this neurotrophin and its high-affinity receptor during inner ear development (Lefebvre et al., 1990, 1991; Pirvola et al., 1994; Schecterson and Bothwell, 1994; Staecker et al., 1996). The present study shows that mice carrying a mutation which inactivates the tyrosine kinase domain of TrkA have normal numbers of inner ear sensory neurons and innervation of the sensory epithelia is unaffected. Thus, signaling of NGF via TrkA does not play a role for ganglion formation and innervation, which only appear to require TrkB and TrkC receptors. However, in mice lacking TrkB and TrkC receptors a substantial number of neurons survive at birth (Minichiello et al., 1995). This situation may be explained by a potential signaling function of TrkA in this artificial situation. Sensory neurons are able to coexpress different Trk receptors or to switch neurotrophin requirements during development, which may lead to complex modes to regulate neuronal survival (Birren et al., 1993; Buchman and Davies, 1993; McMahon et al., 1994; Wright and Snider, 1995; Pifion et al., 1996). TrkA is induced or activated by other neurotrophins, like NT-3 in situations where its preferred receptor TrkC gets downregulated or is inactivated by homologous recombination. In the first case, NT-3 induces TrkA expression and acts as a survival factor in sympathetic neuroblasts (Verdi and Anderson, 1994). In the second example, NT-3 promotes the survival of trigeminal neurons signaling via TrkA in mice

carrying a mutation for the trkC gene (Davies et al., 1995). In the same mutant animals NT-3 is also able to promote survival of nodose neurons using TrkB as a receptor. In the inner ear NT-3, which is highly expressed in the peripheral sensory epithelia (Pirvola et al., 1992), may signal via TrkA, in mice where TrkB and/or TrkC have been inactivated. This may explain the more severe phenotype of nt-3 -lanimals compared to trkC -/- mice in the cochlear ganglion of the inner ear (Farifias et al., 1994; Schimmang et al., 1995). However, the sensory epithelia which are considered as the most important source of trophic support for peripheral inner ear sensory neurons (Ard et al., 1985) are affected in TrkB and TrkC double mutant mice and lose their peripheral innervation (Minichiello et al., 1995). Therefore, in these mice trophic support by neurotrophins via the peripheral targets is unlikely. A target independent mode of autocrine signaling may permit neuronal maturation of immature dorsal root ganglia sensory neurons and survival of these neurons during adulthood (Wright et al., 1992; Acheson et al., 1995; Davies and Wright, 1995). However, neonatal inner ear sensory neurons of P1 are passing through the phase of naturally occurring cell death, when their targets selectively support survival (Ard and Morest, 1984). Therefore, the central targets of cochlear and vestibular neurons may be considered as the most promising candidates to serve as a source for survival factors. NGF has been detected in the cochlear and vestibular nuclei (Nishio et al., 1994) and therefore appears to be an adequate candidate to regulate neuronal survival in mice lacking TrkB and TrkC receptors. However, the present study shows that no trkA expression is detected in the surviving peripheral sensory neurons of the inner ear of these mice. Since apoptotic figures are increased in trkB-/-; trkC -/- mutants compared to wild-type animals, more cochlear and vestibular neurons may eventually die during the postnatal period of cell death (Rueda et al., 1987). The hypothesis that trkB -/-; trkC-/- mice will lose more of their inner ear sensory neurons is supported by the fact that bdnf-/-; nt-3 -/- double mutant animals lack almost all of their inner ear sensory neurons at day 18 p.c. (Ernfors et al., 1995). On the other hand, the present results clearly show that neuronal cell death is delayed in trkB-I-; trkC-I- mice compared to bdnf-/-; nt-3 -/- double mutants. Therefore, neurotrophins may use other signaling mechanisms distinct from those exerted by the known catalytic Trk receptors to promote neuronal survival in the inner ear of trkB-/-; trkC -/- mice. In this context, there is a lack of knowledge about the role of the truncated forms of Trk receptors and the low-affinity receptor p75. In the inner ear truncated Trk receptors bind neurotrophins, yet their expression is only detected in non-neuronal cells (Pirvola et al., 1994; Biffo et al., 1995). Moreover, recent experiments have shown that truncated Trk receptor variants function as negative modulators of neurotrophin responsiveness (Eide et al., 1996; Ninkina et al., 1996; Palko et al., 1996). Therefore, they are unlikely to promote survival of inner ear sensory neu-

T. Schimmang et al. / Mechanisms of Development 64 (1997) 77-85

rons and are postulated to act as scavengers for excess ligand or as selective barriers for signaling by neurotrophins (Pirvola et al., 1994; Biffo et al., 1995). The p75 and TrkA receptor cooperate to increase NGF responsiveness, as demonstrated by p75-deficient trigeminal and dorsal root sensory neurons which display a decreased sensitivity to NGF (Davies et al., 1994; Lee et al., 1994). p75 is detected in the cochlear and vestibular ganglia throughout development and in adulthood (Schecterson and Bothwell, 1994; Ylikoski et al., 1993). However, in a situation where none of the known catalytic Trk receptors is expressed, like in the surviving neurons of TrkB and TrkC double mutants, promotion of neuronal survival by p75 on its own is rather

dorsal

wild-type

A

CB 13

83

unlikely. An analysis of the inner ear of p75 knockout mice (Lee et al., 1992) will help to clarify this issue. Next to BDNF and NT-3, NT-4, which binds to TrkB with high affinity (Berkemeier et al., 1991; Ip et al., 1992; Klein et al., 1992) promotes neuronal survival in isolated cochlear and vestibular neurons (Zheng et al., 1995a; Zheng et al., 1995b). However, analysis of neuronal survival in vestibular ganglia of NT-4 knockout animals produced different results (Conover et al., 1995; Liu et al., 1995; Bianchi et al., 1996) which need further clarification to delineate an essential role for this neurotrophin during inner ear development. In this context it is important to consider the possible influence of different genetic backgrounds on the phenotypes observed in neurotrophin and Trk receptor mutant mice. In summary, analysis of neurotrophin and Trk receptor mutant mice suggest that formation and innervation of inner ear sensory ganglia and their peripheral and central targets largely depend on TrkB and TrkC receptors, which are mainly activated by BDNF and NT-3 (Fig. 6).

1) Organ of

4. Experimental procedures

nuclei

j// //

4.1. Genotyping of animals

/

2) Peripheral processes

3) Neurons in the auditory ganglion

4)Central processes

dorsal

B

mutant

Genotyping of trkB and trkC mutant mice has been described in Schimmang et al. (1995). The trkA genotype was determined by PCR amplification using a common 5' primer (5'-GACCCTGCACTGTCGAGTTTGG-3') and either a 3' primer for the wildtype allele (5'-CGGACCTC A G T G T T G G A G A G C T G G - 3 ' ) or a 3' primer from the pgk- 1 promoter of the neo cassette (5'-GCTCCCGATTCGCAGCGCATC-3"). The mice utilized in this study have a mixed 129/J and C57B1/6 background.

4.2. Histological analysis

r

processes:

reduced or absent

3) Neurons in the

auditory ganglion: reduced in number or absent

4) Central processes: reduced or absent

Fig. 6. Summary of phenotypes observed in the auditory systems of neonatal neurotrophin and neurotrophin receptor mutant mice (structures are not drawn proportionally). Studies of the auditory system of BDNF, NT-3, TrkB and TrkC mutant mice (Ernfors et al., 1995; Fritsch et al, 1995; Minichiello et al., 1995; Schimmang et al., 1995) reveal defects in any of the five components of the peripheral auditory system: the organ of Corti, peripheral neurites, ganglionneurons, central processes and cochlear nuclei. Abbreviations:CB, cerebellum; chp, choroid plexus; CO, cochlear nuclei; cVIIIn, cochlear nerve; wm, white matter (includes part of the vestibulocochlear nerve, the inferior cerebellar peduncle and the trapezoid body).

Morphological observations, neuronal counts and volume measurements were performed with the help of a Q500MC video image analyser (Leica) on cresyl violet-stained serial sections as described in Schimmang et al. (1995). For the identification and nomenclature of anatomical structures we followed the atlas of Alvarez-Bolado and Swanson (1996). Immunohistochemistry was carded out using the ABC method (Vectastain Kit, Vector Laboratories) on sections cut at 25/~m on a cryostat as described earlier (Schimmang et al., 1995). Primary antibodies were diluted 1:500 (monoclonal antibody TuJ1, recognizing class III/3-tubulin; Moskowitz et al., 1993) and 1:800 (rat anti-TrkA rabbit polyclonal; Clary et al., 1994), respectively.

4.3. In situ hybridisation In situ hybridisation on sections of 25/~m cut on a cryostat was performed using the protocol by Nieto and Wilk-

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T. Schimmang et al. / Mechanisms of Development 64 (1997) 77-85

inson (1995). The trkA antisense probe is derived from a 398 bp long EcoRI-XbaI fragment derived from exons K4 to K6 of the mouse trkA gene.

Acknowledgements We would like to thank Carmen Valero for technical assistance and Kelly McNagny for critical reading of the manuscript. The TrkA antibody was a generous gift from Dr. Louis Reichardt (UCSF). L.M. is holder of an EMBO fellowship. This research was supported by a grant from DGCYT PM95/0087 to F.G. and FIS 94/1405 to J.R.

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