on survival and differentiation of dorsal root ganglion. (DRG), inner ear cochlear and vestibular sensory neurons. In most combinations of mutant trk alleles, the ...
Development 121, 4067-4075 (1995) Printed in Great Britain © The Company of Biologists Limited 1995 DEV1028
4067
Differential effects of combined trk receptor mutations on dorsal root ganglion and inner ear sensory neurons Liliana Minichiello1, Fredrik Piehl2, Esther Vazquez3, Thomas Schimmang3, Tomas Hökfelt2, Juan Represa4 and Rüdiger Klein1,* 1Differentiation Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany 2Department of Neuroscience, Karolinska Institutet, 17177 Stockholm, Sweden 3Departamento Bioquimica, Biologia Molecular y Fisiologia 4Departamento Anatomia, Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain
*Author for correspondence
SUMMARY We have generated double mutant mice deficient in pairs of two different Trk receptors and have analysed the effects on survival and differentiation of dorsal root ganglion (DRG), inner ear cochlear and vestibular sensory neurons. In most combinations of mutant trk alleles, the defects observed in double compared to single mutant mice were additive. However, double homozygous trkA−/−;trkB−/− DRG and trkB−/−;trkC−/− vestibular neurons showed the same degree of survival as single trkA−/− and trkB−/− mice, respectively, suggesting that those neurons required both Trk signaling pathways for survival. In situ hybridisation analysis of DRG neurons of double mutant mice revealed differential expression of excitatory neuropeptides.
INTRODUCTION During vertebrate development, many peripheral neurons depend for their survival on neurotrophins synthesised by the tissues that they innervate (Davies, 1994a). The effects of neurotrophins are mediated by the Trk family of receptor tyrosine kinases (Barbacid, 1993). To understand more precisely the function of neurotrophins during nervous system development, we and others have recently generated and analysed germline targeted mice deficient in either Trk receptors or neurotrophins (for reviews see Davies, 1994b; Klein, 1994; Snider, 1994). Mutant mice showed lesions of peripheral sensory neurons specific for the deleted receptor or ligand. Mutant mice deficient in either TrkA receptors or its high affinity ligand NGF, suffered from loss of dorsal root ganglion (DRG) sensory neurons responsive to temperature and pain (Crowley et al., 1994; Smeyne et al., 1994). Mice deficient in TrkB receptors or BDNF also had lesions in DRG neurons (Klein et al., 1993; Ernfors et al., 1994a; Jones et al., 1994). In addition, BDNF−/− mutants were found to have reduced numbers of inner ear vestibular and nodose neurons (Ernfors et al., 1994a; Jones et al., 1994). trkC−/− or NT-3 −/− mutant mice were found to lack DRG proprioceptive neurons, which resulted in abnormal movements and postures (Ernfors et al., 1994b;
Whereas calcitonin-gene-related peptide expression correlated with the trkA phenotype, substance P expression was detected in all combinations of double mutant mice. In the inner ear, TrkB- and TrkC-dependent neurons were shown to at least partially depend on each other for survival, most likely indirectly due to abnormal development of their common targets. This effect was not observed in DRGs, where neurons depending on different Trk receptors generally innervate different targets. Key words: Trk, neurotrophin receptor, tyrosine kinase, neuropeptide, sensory neuron
Farinas et al., 1994; Klein et al., 1994). In addition, NT-3−/− mice were found to lack the majority of trigeminal and inner ear cochlear neurons. At least two important conclusions could be drawn from the defects observed in these mice. First, sensory neurons subserving different functions require different Trk signaling pathways for their survival. Thus, neurotrophin dependence was confirmed to be modality specific (Snider, 1994), as previously suggested (Davies, 1987). Second, mutations of a single trk or neurotrophin gene causes dramatic cell loss of specific peripheral neurons, demonstrating that, in these neurons, one Trk receptor cannot compensate for the lack of another Trk receptor. Despite these important conclusions, many questions still remain unanswered. For example, do neurotrophins act sequentially during development (Davies, 1994c) so that the same population of neurons might be affected by the loss of two different Trk receptors? Can Trk receptors functionally compensate for each other, either directly within the cell, or indirectly via trophic interactions? Are the surviving sensory neurons in Trk receptor-deficient mice somehow affected by the mutation and, if so, do they show changes in expression of certain differentiation markers? In order to address these questions genetically, we have generated double mutant mice deficient in pairs of Trk
4068 L. Minichiello and others receptors by intercrossing single mutant mice lacking one of the three Trk receptors. We have focused on sensory neuron survival and differentiation to be able to derive some general conclusions on functional interactions between Trk signaling pathways. MATERIAL AND METHODS Histology and cell counting For the inner ear histology, entire temporal bone primordia were dissected from newborn mice and fixed in 4% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2) for 6-12 hours, dehydrated in ethanol, embedded in paraffin, serially sectioned at 8 µm and stained with 0.1% cresyl violet. For DRG histology and in situ hybridisation analysis, whole mouse bodies were mounted in tissue freezing medium (Tissue-Tek) and transversally sectioned at 14 µm in cranial direction on a cryostat. The correct ganglia levels were identified by carefully mapping the foramina intervertebralia beginning at the sacrum and by comparing the spinal cord morphology. For neuronal counts, series of every fifth section through the L4 ganglia were immersion fixed in 4% paraformaldehyde and counter stained with toluidine blue. All neurons having visible nuclei were counted. The raw counts were corrected according to Abercrombie (Abercrombie, 1946). Neuronal counts of cochlear and vestibular neurons were done essentially as described (Schimmang et al., 1995). Briefly, nuclei were counted at 200× magnification in 4 randomly chosen fields in every section of 8 µm thickness. Up to about 80 fields per ganglion in sections that were 40 µm apart were analysed. No corrections were done here, since neuronal size (max. 25 µm) was always smaller than the intersection interval, making it impossible to count the same neuron twice. In situ hybridisation In situ hybridisation analysis with oligonucleotide probes was performed as previously described (Dagerlind et al., 1992). The synthesised 48-mer oligonucleotide against substance P was complementary to nucleotides 145-192 of the rat preprotachykinin mRNA (Krause et al., 1987) and the 44-mer probe against CGRP was complementary to nucleotides 13-56 in the transcripts from the αCGRP/calcitonin and β-CGRP genes encoding the mature α- and βCGRP peptides (Amara et al., 1985). The synthesised 48-mer oligonucleotide probe against GAP-43 was complementary to nucleotides 70-117 of the rat GAP-43 gene (Karns et al., 1987). The probes were labelled at the 3′ end using 35S-labelled deoxyadenosinealpha(thio)triphosphate and terminal deoxynucleotidyl transferase (Amersham), then hybridised to the sections without pretreatment for 16-18 hours at 42°C. The hybridisation buffer contained 50% formamide, 4× SSC, 1× Denhardt’s, 1% sarcosyl (N-lauroylsarcosine; Sigma), 0.02 M phosphate buffer, 10% dextran sulfate, 250 µg/ml yeast tRNA, 500 µg/ml salmon sperm DNA and 200 mM DTT. Following hybridisation, the sections were washed several times in 1× SSC at 55°C, dehydrated in ethanol and dipped in NTB2 nuclear track emulsion (Kodak). After 3-4 weeks, the sections were developed in D-19 developer (Kodak) and cover-slipped. Control sections were hybridised with a 20-fold excess of cold oligonucleotide probe. SP expression was quantified by counting cells on sections from in situ hybridisation experiments. Cells were scored as positive, when the number of silver grains was 4-times over background (on average, 900 cells were counted). Immunohistochemistry Immunohistochemistry was carried out using the ABC Vectastain kit (Vector Labs) on 25 µm cryosections. Sections were incubated in TBS solution (50 mM Tris-HCl buffer pH 7.5, containing 0.1% sheep serum, 0.1% BSA, 0.1% Triton), quenched in 3% H2O2, blocked with
serum and left overnight at 4°C in TBS solution containing 2-4 µg/ml of a mouse anti-200K neurofilament (Boehringer Mannheim) and anti-β-tubulin antibodies. After incubation with a secondary biotinylated antibody and the ABC reagent, peroxidase was reacted with 0.05% diaminobenzidine tetrahydrochloride and 0.003% hydrogenperoxide.
RESULTS Survival of dorsal root ganglion and inner ear neurons in trk double mutant mice Double heterozygous mice carrying mutant alleles of two different trk genes were crossed and the offspring analysed at postnatal day 1 (P1), since none of the possible combinations of double homozygous mutant mice (trkA−/−;trkB−/−, trkA−/−; trkC−/−, and trkB−/−;trkC−/−) survived to later stages. To assess the effects of combined trk mutations on dorsal root ganglion (DRG) neurons, we calculated neuron numbers in serial sections of lumbar level 4 DRGs (Table 1). As recently described (for review see Snider, 1994), each of the single trk mutant mice showed reduced survival of DRG neurons to varying degrees, with loss of TrkA receptors being more severe (73%), than loss of either TrkB (20%) or TrkC (17%) receptors. Double mutant trkA−/−;trkC−/− mice had phenotypes that were most consistent with the sum of the individual phenotypes (93% combined DRG neuron loss compared to 90%, the sum of individual trkA and trkC phenotypes). Likewise, the combination of trkB and trkC mutant alleles was found to have an additive effect. However, combinations involving trkA and trkB mutant alleles did not result in significantly increased neuron loss compared to the single trkA mutant phenotype (78% combined neuron loss versus 73% single trkA mutant mice). The differTable 1. Numbers of neurons and substance P (SP) expression in L4 dorsal root ganglia of wild-type and trk mutant mice Genotype wild-type trkA−/− trkB−/− trkC−/− trkA−/−;trkB+/− trkA+/−;trkB−/− trkA−/−;trkB−/− trkA−/−;trkC+/− trkA+/−;trkC−/− trkA−/−;trkC−/− trkB−/−;trkC+/− trkB+/−;trkC−/− trkB−/−;trkC−/−
n=6 n=6 n=4 n=6 n=4 n=4 n=6 n=6 n=6 n=6 n=4 n=4 n=6
No. of neurons ± s.e.m.
% Reduction
% SPpositive
3839±114 1024±144 3080±85 3172±121 1023±62 3008±152 828±67 1063±124 2664±90 268±23 2792±111 2697±140 2257±118
73a 20b 17b 73a 22c 78d 72a 31e 93f 27c 30g 41h
39 16 53 51 18 50 14 16 61 8 51 60 62
The numbers of neurons (expressed as mean ± s.e.m.) were calculated from newborn (P1) mice. Sample numbers are indicated. Statistical analysis was carried out using Student’s t-test. aP
