Absence of sensory neurons before target innervation in brain-derived neurotrophic factor-, neurotrophin 3-, and TrkC-deficient embryonic mice

Abstract

Gene-targeting experiments of Trk receptors and neurotrophins has confirmed the expectation that embryonic sensory and sympathetic neurons require neurotrophin function for survival. They have further revealed correlation between a specific neurotrophin requirement and eventual sensory modality. We have analyzed embryonic and neonatal mice with mutations in the BDNF, neurotrophin 3 (NT-3), and TrkC genes. Our data confirm an unexpectedly high proportion of sensory neuron losses in NT-3 (>70%), BDNF (>20%), and TrkC (>30%) mutants, which encompass populations thought to be NGF-dependent. Direct comparison of TrkC and NT-3 mutants indicates that only a subset of the NT-3-dependent neurons also requires TrkC. The observed losses in our TrkC mutant, which is null for all proteins encoded by the gene, are more severe than those previously reported for the kinase-negative TrkC mutation, implicating additional and important functions for the truncated receptors. Our data further indicate that mature NGF-requiring neurons undergo precocious and transitory requirements for NT-3 and/or BDNF. We suggest that neurotrophins may function in creating early heterogeneity that would enable ganglia to compensate for diverse modality requirements before the period of naturally occurring death.

Fig. 1.

Generation of BDNF mutant mice. a, Schematic showing the replacement vector and strategy used to inactivate the BDNF gene. The filled bar indicates the BDNF coding region. Restriction enzyme sites are as indicated:B, BglII; E,EcoRl; S, Smal;X, Xbal. b, Southern blot analysis of tail DNA from a litter obtained intercrossing two BDNF +/− mice. BglII restriction enzyme digestion and the 5′ external probe indicated in a were used to detect rearrangement in the mouse BDNF locus. The 14 kb wild-type (WT) and 15.6 kb rearranged (mt) DNA bands are indicated.

Fig. 2.

TrkB (A) and TrkC (B) expression in trigeminal gangila (TG) and dorsal root ganglia (DRG) of wild-type and BDNF and/or NT-3 mutant neonates. q andr show representative examples of positive and negative Trk-expressing trigeminal and DRG neurons, respectively. In a–p arrows indicate small groups of TrkB- or TrkC-expressing neurons still present in mutant mice. In q and r black arrowheads indicate “positive” Trk-expressing neurons; white arrowheads indicate negative Trk-expressing neurons.

Fig. 3.

Quantitation of TrkA-, B-, and C-expressing neurons in the TG (A, B) and DRG (C, D) of neonatal mice. A, C, Percentage of neurons expressing Trk receptor mRNA compared with the total number of neurons present in wild-type ganglia. B,D, Relative percentage of neurons expressing Trk mRNA as a function of the total number of neurons in their respective ganglia.Open bar, Wild type; hatched bar, BDNF−/−; dotted bar, NT-3−/−; solid bar, BDNF/NT-3 DKO. Data and statistical analysis are from Table 1.

Fig. 4.

Analysis of peripherin-positive L4 DRG neurons of wild-type (A, C) and NT-3 mutant (B, D) E13–E13.25 embryos. Hematoxylin and eosin (H&E)-stained sections (A, B) were used for quantification of total cell numbers, whereas antibodies to peripherin (B, D) identified neurons. The ganglia are outlined by a dotted line.

Fig. 5.

Ia afferents are absent in TrkC and NT-3 mutants but not in BDNF or TrkA mutants. Hemisection view of DiI (see Materials and Methods) retrogradely labeled sensory afferents and motor neurons in the P0.5 spinal cord. A, Schematic diagram of the spinal cord indicating the dorsal horn (DH) and associated afferent laminae. The ventral horn (VH) indicates where motoneuron nuclei are retrogradely labeled. B–F, Samples of cords from mutant pups as indicated. The arrowheads point to the location of Ia afferents.

Fig. 6.

Schematic diagram of dorsal root ganglion neuronal subpopulations present in newborn animals as a consequence of neurotrophin knock-outs. In wild-type ganglia (WT), the red stippled circles are presumed to be TrkA- and NGF-dependent neurons based on other studies (Crowley et al., 1994; Smeyne et al., 1994) and as undergoing transitory NT-3 requirements based on the present study and others (Tessarollo et al., 1994; Airaksinen et al., 1996; Fariñas et al., 1996; White et al., 1996). Shape indicates receptor type (circle, TrkA; triangle, TrkB;square, TrkC). Color indicates neurotrophin requirements (blue, NGF;green, BDNF; red, NT-3), and the relative numbers indicate approximate percentages.