Fig 2: Sparse Mrgprd+ nociceptor labeling in untreated 3–4 pw MrgprdCreERT2; RosaiAP mice.(A and B) Whole mount AP DRG staining of thoracic (A) and L5 (B) DRGs. (C) AP+ cells / DRG for cervical (C), thoracic (T), and lumbar (L) DRGs, n = 47 DRGs from three animals. (D–H) Whole mount DRG immunostaining plus AP fluorescent staining. Sparse AP+ cells express non-peptidergic nociceptor markers peripherin (D), RET (F) and PAP (H) but not large diameter neuron maker NF200 (E) or peptidergic marker CGRP (G). (I) Whole mount EGFPf immunostaining plus AP fluorescence staining of untreated MrgprdCreERT2/EGFPf; RosaiAP DRGs. AP+ neurons are Mrgprd+ nociceptors. Quantification of overlap (% of AP+ cells that co-express MrgprdEGFPf, n = 126 neurons from three animals). Scale bars = 50 µm.

Fig 3: Nociceptor populations are reduced in Prdm12AvilCKO mice(A) RNAscope using exon-V-specific probes confirmed knockout of Prdm12 from mutant DRGs. Scale bar, 50 µm.(B) Prdm12AvilCKO mice have a reduction of Tom+ neurons, p = 0.0326.(C and D) The remaining neurons in Prdm12AvilCKO are larger, on average (C) (p = 0.0466), because of selective loss of cells = 300 µm2 (D).(E) Representative image showing number of IB4+ (arrows) and CGRP+ (arrowheads) nociceptors are reduced in Prdm12AvilCKO DRGs. Scale bar, 100 µm.(F and G) Quantification of IB4+ (F) (p < 0.0001) and CGRP+ (G) (p = 0.027) nociceptors as a percentage of the total Tom+ population of sensory neurons.(H) Representative image of TRKA+ nociceptors and NF200+ myelinated neurons in Prdm12AvilCKO and control mice. Scale bar, 100 µm.(I) Quantification showing significant reduction of TRKA+ nociceptors, p = 0.0135.(J) Quantification of NF200+ neurons showing a wide range in Prdm12AvilCKO mice, but no significant change from control littermates, p = 0.278.(K and L) Representative image (K) and quantification (L) of the reduction in number of TRPV1+ nociceptors, p = 0.0025. Scale bar, 100 µm.(M) qRT-PCR results plotted showing —log(p) versus log2 (fold change) reveal significant reductions in many nociceptive gene transcripts, including Prdm12, but a significant increase in Trpm3, and no change in touch and proprioceptive genes. The dotted line represents p = 0.05; points above it differ significantly between control and mutant mice. Each point represents the analysis of significance and fold change from average values of n = 3 biological replicates.All analysis was completed using DRGs from lumbar levels 2 through 5. Each data point in (B), (C), (F), (G), (I), (J), and (L) represents the average count across three DRGs from control (n = 3) or Prdm12AvilCKO (n = 4) mice taken after behavior analysis around 10 weeks of age. All quantification analyzed by two-tailed Student’s t test; results are presented as means ± SEM.

Fig 4: Representative confocal images of the right knee of a Ccr2RFP mouse 8 weeks after DMM surgery, immunostained with CGRP. a CGRP/CCR2 overlay of the lateral compartment of the knee; b magnified image of a showing CCR2, CGRP signals and the overlay of both images. White arrows indicate an example of overlap between CGRP and CCR2 signals in the lateral synovium (CCR2 receptors present on CGRP+ nerve fibers). c CGRP/CCR2 overlay of the medial compartment of the knee; d, e magnified of c showing CCR2, CGRP signals and the overlays of both images. c, d White arrows indicate an example of an overlap between CGRP and CCR2 signal in the medial synovium (CCR2 receptors present on CGRP+ nerve fibers). c, e Blue arrows point to a CGRP/CCR2-positive channel in the medial subchondral bone. f, g Quantification of the CGRP+ and CCR2+/CGRP+ channels in the subchondral bone of naïve and DMM knees. Unpaired two-tailed t test. Scale bar for a, c, d = 100 µm. Scale bar for b, e = 25 µm. **p < 0.01. Mean ± SEM

Fig 5: Loss of adipose mTORC2 reduces sensory neurons in the proximity of adipocytes. (A) Low magnification projection of inguinal WAT (iWAT) four weeks after tamoxifen treatment co-immunostained with tyrosine hydroxylase (TH, yellow) and calcitonin gene-related peptide (CGRP, magenta) (N = 15; 18). Scale bar = 500 µm. (B) High magnification projection of iWAT four weeks after tamoxifen treatment co-immunostained with TH (yellow) and CGRP (magenta) (N = 21; 22). Scale bar = 100 µm. (C) Subsequent sections (P1–P6; 6.48/6.28 µm interval, respectively) of control and iAdRiKO iWAT immunostained with TH (yellow) and CGRP (magenta) illustrating single nerve fibers innervating the periphery. Region of interest: Nerve ending and potential synapses (arrows). Tissue autofluorescence = green. Scale bar = 100 µm.