<i>TrkC^GFP</i> BAC line shows enriched expression in proprioceptors.

<p>(a) Immunohistochemisty showing expression overlap between GFP and genes expressed by proprioceptors in p0 L5 DRG of <i>TrkC^GFP</i> BAC transgenic mouse line (top row: Runx3 and Pvalb; bottom row: TrkC and Pvalb; overlay of all three channels shown to the right; scale bar = 60 µm). (b) From left to right: Quantification of percentage of Runx3^on neurons co-expressing the TrkC^GFP allele (86.4% ± SEM), percentage of TrkC^GFPon cells co-expressing Runx3 (97.6% ± SEM; of neurons with Isl1^on nucleus on section), percentage of TrkC^on cells co-expressing TrkC^GFP (49.6% ± SEM) and of percentage of TrkC^GFPon cells co-expressing TrkC (grey bar: 98.5% ± SEM). Data from n = 4 p0 <i>TrkC^GFP</i> mice; total >20 L5 DRG sections. (c) Example of FACS scatterplot (left) and histogram (right) on dissociated lumbar DRG cells isolated from p0 <i>TrkC^GFP</i> mice gated by fluorescence (GFP^off and GFP^on cells are indicated; red-f: red fluorescence; green-f: green fluorescence). Four examples of genes (<i>Runx3</i>, <i>TrkC</i>, <i>Etv1</i>, <i>Pvalb</i>) with known enriched expression in proprioceptors (TrkC^GFPon) when compared to non-proprioceptors (TrkC^GFPoff). Each panel shows Affymetrix expression values to the left (y-scale raw expression values; ± SEM).</p

Isolation of genes with enriched expression in proprioceptors.

<p>(a) Affymetrix gene expression profiling data showing probes enriched in TrkC^GFPon proprioceptors and TrkC^GFPoff non-proprioceptors isolated from p0 mice. Diagonal lines indicate cut-off for probes with expression values >5 fold change (outermost dotted lines), >2 fold change (middle dotted lines) and ≤2 fold change (grey squares around central diagonal line). TrkC^GFPon proprioceptor data points with enrichment ≥2 fold are displayed in turquoise and TrkC^GFPoff non-proprioceptors with the same criteria in purple. (b) Venn diagram illustrating the number of probes enriched ≥2 fold in proprioceptors and non-proprioceptors isolated from p0 mice respectively. (c) Analysis of the 25 probes with highest fold changes displayed in detail. Values of two samples of each p0 TrkC^GFPon proprioceptors (left) and TrkC^GFPoff non-proprioceptors (right) are shown. Grey scale values represent row z-score values and log unit average expression values are shown to the right of each probe (scales plotted bottom left). Probe names are displayed to the left of each row. (d–g) Four examples of individual genes with highly enriched expression in proprioceptors (TrkC) when compared to non-proprioceptors (non-TrkC) are displayed. Each panel shows raw Affymetrix expression values to the left (y-scale expression values; ±SEM) and verification by either <i>in situ</i> hybridization on wild-type and <i>TrkC</i> mutant lumbar DRG sections (d–f; scale bar = 50 µm) or immunohistochemistry in ventral spinal cord lamina IX (g; green: Cx36; red: vGlut1; scale bar = 2 µm) to the right.</p

Genes with anticorrelation in NT3 regulation profiles.

<p>(a) Flow diagram to illustrate analysis of gene expression data extracted from proprioceptors and non-proprioceptors of various mouse strains. To isolate genes with anticorrelative expression profile, genes with expression changes in opposing directions in proprioceptors of <i>NT3^−/−Bax^−/−</i> mice and <i>mlc^NT3</i> mice were isolated (purple arrows 1 and 2). (b, c) Detailed expression analysis of three individual genes following anticorrelation scheme 1 (<i>Gas6</i>, <i>Ndst4</i>, and <i>Crim1</i>) and three genes following scheme 2 (<i>Mrg1</i>, <i>Nova1</i>, and <i>P2rx1</i>). Affymetrix analysis: y-scale displays raw expression values; ± SEM.</p

<i>Gabrg1</i> expression in proprioceptors in rostro-caudal gradient and regulated by NT3.

<p>(a) <i>In situ</i> hybridization experiments demonstrating downregulation of <i>Gabrg1</i> expression in p0 <i>TrkC</i> mutant L5 DRG when compared to wild-type. (b) Affymetrix expression value analysis of <i>Gabrg1</i> in proprioceptors (TrkC) and non-proprioceptors (nonTrkC) of wild-type (left), <i>mlc^NT3</i> (middle) and <i>NT3^−/−Bax^−/−</i> (right) mice (y-scale displays raw expression values; ±SEM). Note selective upregulation of <i>Gabrg1</i> in proprioceptors of <i>mlc^NT3</i> and downregulation in <i>NT3^−/−Bax^−/−</i> mice compared to wild-type values, in absence of gene expression changes in non-proprioceptors. (c–e) <i>In situ</i> hybridization analysis of <i>Gabrg1</i> expression on p0 wild-type DRG, displaying representative images from lumbar levels L1, L3, L5 and L6 (c) and quantification of cell numbers at lumbar (d) and cervical (e) rostro-caudal levels (n = 3 animals; ±SEM). (f) Affymetrix expression value analysis of <i>Gabrg1</i>, <i>TrkC</i>, and <i>Pvalb</i> at L1 and L5 (y-scale displays raw expression values; ±SEM). (g, h) Analysis of <i>Gabrg1</i> expression in adult DRG by <i>in situ</i> hybridization (g: L1 and L5; scale bar = 80 µm), Affymetrix gene expression values (h; n = 3; ±SEM), and quantitative PCR (h; n = 3; ±SEM). In comparison, Affymetrix expression values of <i>TrkC</i> are not significantly different between L1 and L5 adult DRG (h; n = 3; ±SEM).</p

Surplus skeletal muscle NT3 alters proprioceptor gene expression.

<p>(a, b) Analysis of 25 upregulated (a) or downregulated (b) probes in <i>mlc^NT3</i> mice is displayed. Average values of p0 TrkC^on proprioceptors (left; TrkC) and TrkC^off non-proprioceptors (right; non-TrkC) isolated from wild-type and <i>mlc^NT3</i> mice are shown. Grey scale values represent row z-score values and log unit average expression values are shown to the right of each probe (scales plotted top right of each panel). Probe names are displayed to the left of each row. The number of probes regulated in proprioceptors (p≤0.02; regulation ≥1.5 fold) is shown below the plots. (c, d) Detailed expression analysis of two individual genes upregulated (<i>Igf1</i> and <i>Shc4</i>) and two genes downregulated (<i>Tacr3</i> and <i>Myb</i>) in proprioceptors of <i>mlc^NT3</i> mice is shown (Affymetrix analysis: y-scale displays raw expression values; ±SEM).</p