Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate

Abstract

Tactile information is perceived by a heterogeneous population of specialized neurons. Neurotrophin receptors (the receptor tyrosine kinases, Trks) mark the major classes of these sensory neurons: TrkA is expressed in neurons that sense temperature and noxious stimuli, and TrkC is expressed in proprioceptive neurons that sense body position. Neurotrophin signaling through these receptors is required for cell survival. To test whether neurotrophins have an instructive role in sensory specification, we expressed rat TrkC from the TrkA (also known as Ntrk1) locus in mice. The surviving presumptive TrkA-expressing neurons adopted a proprioceptive phenotype, indicating that neurotrophin signaling can specify sensory neuron subtypes.

Figure 1

Mice engineered to express TrkC protein from the TrkA locus. (a) Schematic representation of the TrkA genomic locus, the gene targeting construct and the final TrkA^TrkC targeted allele. An IRES assures translation of rat TrkC transgenic cDNA independent of the endogenous TrkA start site. The positive selection marker, neo, is flanked by Cre recognition sites (loxP, shown as gray triangles). The removal of the selection cassette ensures proper transcription of the transgene. The locations of HindIII sites, PCR primers and the Southern blot probe are marked. (b) Southern blot analysis of offspring from a TrkA^TrkC heterozygous intercross. HindIII-digested genomic DNA was hybridized with the 5′ external probe shown in a. The genotype of the mice is shown at the top, where (+) designates a wildtype allele and (-) a TrkA^TrkC allele. (c) RT-PCR analysis of transgene expression in a TrkA^TrkC heterozygous mouse. The PCR primers specifically recognized spliced transcripts from either the endogenous TrkA locus or the TrkC-expressing TrkA locus. The transgene expression was confined to DRG neurons, demonstrating proper tissue-specificity. GAPDH primers were used as a positive control.

Figure 2

Mice engineered to express tau-lacZ (τlacZ) fusion protein from the TrkA locus, while inactivating TrkA. (a) Schematic representation of the TrkA genomic locus, the gene targeting construct and the final TrkA^τlacZ targeted allele. The τlacZ-SV40 polyA selection cassette was inserted at the endogenous TrkA start site (exon 1). The positive selection maker, neo, was flanked by Cre recognition sites (loxP, shown as gray triangles). The removal of the selection cassette ensured proper transcription of the transgene. The locations of HindIII and EcoRI sites and the Southern blot probe are marked. Dashed line in the top diagram indicates that the 5′ EcoRI site is not to drawn to scale. (b) Southern blot analysis of TrkA^τlacZ heterozygous mice compared to wild type. EcoRI-digested genomic DNA was hybridized with the 3′ external probe shown in a. The genotype of the mice is shown at the top, where (+) designates a wildtype allele and (-) a TrkA^τlacZ allele. (c) Analysis of transgene expression in TrkA^τlacZ heterozygous mice using whole-mount X-gal staining. At P2, the transgene was expressed in DRG cell bodies (arrows) and TrkA-positive DRG projections in dorsal spinal cord (top) and skin (bottom). Robust expression of transgene was detectable at E13.5.

Figure 3

DRG neurons from TrkA^TrkC/TrkC mice do not express markers specific for the TrkA population, but a greater number of neurons express markers for the TrkC population. (a-l) Nociceptive-thermoceptive markers were expressed in wild-type DRG neurons (a,d,g,j) but were completely absent in the TrkA^TrkC/TrkC (b,e,h,k) and TrkA^{τlacZ/τlacZ} (c,f,i,l) mice at birth. TrkA^{τlacZ/τlacZ} mice are phenotypically identical to TrkA-null mice. (m-u) In L5 DRGs, the number of neurons expressing proprioceptive markers were markedly higher in the TrkA^TrkC/TrkC mice (n,q,t) than in wild-type (m,p,s) and TrkA^{τlacZ/τlacZ} (o,r,u) mice. The number of neurons positive for proprioceptive markers in TrkA^{τlacZ/τlacZ} sections seemed to be intermediate between wild type and TrkA^TrkC/TrkC. This apparent increase is likely due to the small size of the sensory ganglia, which juxtaposes proprioceptive neurons in close proximity in these sections. Indeed, total counts from wild-type and TrkA^{τlacZ/τlacZ} DRGs show similar numbers of parvalbumin-positive neurons (Table 1). TrkA, CGRP, TrkC and parvalbumin were visualized by antibody staining, and the remaining markers by in situ hybridization. Scale bar is 50 μM.

Figure 4

Increased innervation of proprioceptive target tissues in TrkA^TrkC/TrkC mice. (a,b) Parvalbumin antibody staining marks the central projections of DRG neurons in wild-type and TrkA^TrkC/TrkC mice. The axonal projections converging toward the intermediate and ventral spinal cord (arrows) are denser in the L5 region of TrkA^TrkC/TrkC mice. (c-f) Hematoxylin- and eosin- stained paraffin sections through the soleus muscle show an increased number of spindles (arrows) in the TrkA^TrkC/TrkC (d) and TrkA^TrkC/TrkC;Myo/NT3 (f) mice at P5 (c,d) and P0 (e,f). (g,h) Parvalbumin-positive muscle spindles within the back muscles at the L5 level of newborn Myo/NT3 (g) and TrkA^TrkC/TrkC;Myo/NT3 (h) mice. Scale bars are 50 μM.

Figure 5

Mechanism of the phenotypic switch in TrkA^TrkC/TrkC mice. (a-d) LacZ-derived activity is apparent in cell bodies (a) and central projections (c) of nociceptive-thermoceptive neurons in mice carrying a single copy of TrkA^τlacZ. This staining is absent in TrkA^τlacZ/TrkC compound heterozygous mice (b,d). (e-h) In situ hybridization analysis shows that DRG neurons from TrkA^TrkC/TrkC;TrkC^-/- double homozygous mice do not express the proprioceptive marker parvalbumin (h), similarly to TrkC^-/- mice (f), indicating that endogenous TrkC is required for the phenotype observed in TrkA^TrkC/TrkC mice (g). (i-k) A model of the mechanism underlying the phenotypic switch described in this study. Neurons in TrkA^TrkC/TrkC mice that initially activate the TrkA promoter now instead express TrkC. This leads to a Trk switch, including the downregulation of the TrkA promoter and upregulation of the endogenous TrkC promoter. (l) Levels of TrkC transgene expression were assessed by semiquantitative RT-PCR at two different time points. GAPDH and peripherin transcripts were used as controls for the cDNA samples. Consistent results were obtained in three different experiments. In agreement with the above model, the transgene was expressed at E13.5 in both TrkA^TrkC and TrkA^TrkC/TrkC mice. Postnatally, the transgene was expressed in heterozygous, but not homozygous, mice. TrkA was absent in all homozygous samples, as expected.