In addition, ∼85% of Rx3 neurons expressed Pv, and ∼90% of Pv+ DRG neurons expressed Rx3 (Figures 1D–1F and Figure S1 available online; Table S1). Thus, the composite expression of TrkC, Rx3, and Pv defines four neuronal subsets: two large populations of TrkC+Rx3offPvoff and TrkC+Rx3+Pv+ neurons, and two small subsets of TrkC+Rx3+Pvoff and TrkCoffRx3offPv+ DRG neurons. In marked contrast to the profile of endogenous TrkC expression, analysis of a TrkC:GFP BAC transgenic line ( Gong Alisertib purchase et al., 2003) revealed GFP expression only in TrkC+Rx3+Pv+

and TrkC+Rx3+Pvoff neurons ( Figures 1G, 1H, and S2), a restriction we use in studies described below. Which of these subsets represent pSNs? Many TrkC+Rx3offPvoff neurons expressed Ret, TrkB, and/or TrkA (Figure S2, data not shown), indicating that expression of TrkC in the absence of Rx3 or Pv marks cutaneous sensory neurons. To determine the sensory modalities associated with the remaining three neuronal populations we compared

cell body marker status and axonal projection pattern in transgenic mice carrying reporter genes directed by tamoxifen-activated Rx3:CreER or Pv:Cre driver alleles (see PF-01367338 clinical trial Table S2 for mice used in this study). Bicistronic mGFP/nuclear LacZ (nLZ), or tdTomato (tdT) reporters were used to label Rx3+ or Pv+ sensory neuron cell bodies, along with their central and peripheral axons ( Figures 1I, 1J, S1, and S3) ( Hippenmeyer et al., 2005; Madisen et al., 2010). In Rx3:CreER-directed mGFP-nLZ reporter crosses we found that all mGFP+ DRG neurons expressed nuclear Rx3 protein ( Figure S3). Only ∼10% of all Rx3+ neurons expressed mGFP, presumably a reflection

of the inefficiency of tamoxifen-triggered Cre recombination of target genes in DRG neurons ( Zhao et al., 2006). Nevertheless, Rx3:CreER-directed mGFP reporter expression was observed in both MS and GTO pSN sensory endings in limb, axial and hypaxial muscles ( Figure 1I; data not shown). Pv:Cre-directed reporter expression was restricted to Pv+ neurons and was detected in ∼98% of DRG neurons that expressed endogenous Pv ( Figure S1). old mGFP-labeled axons innervated virtually all MSs and GTOs in axial, hypaxial, and hindlimb muscles ( Figures 1J and S1). These data, together with the fact that all MS- and GTO-innervating pSNs are eliminated in TrkC and Rx3 mutant mice ( Klein et al., 1994; Kramer et al., 2006; J.C.d.N. and T.M.J., unpublished data) suggest that the larger TrkC+Rx3+Pv+ neuronal population represents authentic pSNs. We next examined the profile of Etv1 expression with reference to the TrkC+Rx3+Pv+ pSN population. At neonatal stages, Etv1 expression was detected in all TrkC+Rx3+Pv+ neurons (Figures 1D–1F). Nevertheless, ∼60% of Etv1+ neurons lacked Rx3 and/or Pv expression, indicative of sensory neuron classes other than proprioceptors (Figure S2).