Fig 1: Mesenchyme homeobox protein 2 (MEOX2) deficiency in the dorsal root ganglia (DRG) causes deregulation of a number of known pain modulator and sensory neuron developmental genes. (A) Levels of mRNA in Meox2+/− (red bars) DRG for nociceptor markers Calca, Ntrk1 and TrpV1 compared to Meox2+/+ littermate DRG controls (black bars) relative to Gapdh. Levels of mRNA for all RT‐qPCR experiments are normalised to an average of Meox2+/+ samples. (B) Levels of Ntrk2 and Ntrk3 mRNA in Meox2+/− (red bars) compared to Meox2+/+ littermate DRG controls (black bars) relative to Gapdh. (C) Levels of mRNA in Meox2+/− (red bars) DRG for nociceptor‐specific voltage‐gated sodium channels Scn9a, Scn10a and Scn11a compared to Meox2+/+ littermate DRG controls (black bars) normalised to Gapdh. (D) Volcano plot of differentially regulated genes depicts significantly up‐ and down‐regulated genes in pooled DRG isolated from three adult Meox2+/− animals as compared to their control littermates, n = 3. X‐axis represents log2 fold change and the y‐axis represents –log10 (q‐values). Genes with q‐value (FDR) of less than 0.1 were assigned differentially regulated, and coloured in blue. Top pain‐ or itch‐related genes are labelled. (E) Levels of Actn3 mRNA in Meox2+/− (red bars) compared to Meox2+/+ littermate DRG controls (black bars) normalised to Gapdh, as one of the top upregulated hits in the transcriptome of Meox2+/− DRG. For all RT‐qPCR experiments three littermate animals per genotype were used; mean ± SEM; Unpaired Student’s t‐test, P‐values * < 0.05, or as indicated. (F) The top up‐ and down‐regulated gene ontology (GO) terms for cellular process. Enriched GO terms were analysed using GOrilla, and those with a P‐value < 0.001 were considered significant. Top eight GO terms are plotted both the upregulated (right side, marked in red) and downregulated (left side, in blue) with bars representing –log10(P‐value) for particular GO term.
Fig 2: Loss of Prdm12 in nociceptor of adult mice does not alter diversity of DRG neuronal subpopulations. (A) Representative images of DRG sections of Avil icKO and Rosa26 iKO mice injected with tamoxifen or with corn oil as control immunolabelled for Peripherin, CGRP, Nav1.8, and NF-200 or stained with IB4-Alexa594, with quantification showing that the number of cells positive for the different markers is similar in DRG sections of icKO and control mice. Scale bar: 100 µm, inset scale bar: 25 µm. (B) Proportion of CGRP+ neurons and of IB4+ neurons among Peripherin+ neurons in DRG are similar in both icKO and control mice. (C) Proportions of DRG neurons positive for Nav1.8 and NF200 are unchanged in DRG sections of both icKO. (D) Left panel, βIII-Tubulin immunostainings on skin tissue of TAM-injected Rosa26 icKO and control mice. Arrows point skin epidermis innervation (E = epidermis; D = dermis). Scale bar: 50 µm. Right panel, Quantification showing the percentage of labelled sensory terminals invading the epidermis in TAM-injected Rosa26 icKO mice compared to controls. In all experiments, n ≥ 3 for both genotypes. All quantifications were submitted to the 2 tailed student's t test or two-way ANOVA test. Values are represented as mean ± SD. DRG, dorsal root ganglia; icKO, inducible conditional knockout.
Fig 3: Mesenchyme homeobox protein 2 (MEOX2) is expressed in both central and peripheral nervous tissues. (A) Western blot analysis of wild‐type tissue shows relatively ubiquitous but variable expression levels of MEOX2 in kidney, liver, lung and heart, and (B) throughout the peripheral and central nervous systems; DRG – dorsal root ganglion, SC – spinal cord; CBL – cerebellum; Hipp. – hippocampus; Hypo. – hypothalamus; WB – whole brain, n = 1. (C) Representative sections of adult wild‐type lumbar DRG co‐immunolabelled with antibodies against MEOX2 (green) and CGRP (red); (D) TrkA (red), (E) Nav1.8 (red) and (F) IB4 (red). DAPI nuclear staining in blue for all panels; white arrowheads indicate double positive cells rightmost merged image for all. For CGRP, TrkA and IB4 staining, n = 3 WT; 2‐3 sections per 5 DRG/animal; for Nav1.8 staining n = 2 WT; 2 section per 5 DRG/animal. Quantification of colocalisation on the right, expressed as percentage of single or double stained DAPI+ cells. Scale bar is 20 µm.
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