<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

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

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

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

<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

Progressive Neuronal Specification Is Paralleled by a Developmental Shift in Response to ETS Transcription Factor Signaling

<p>Schematic summary diagram illustrating the importance of temporally appropriate upregulation of transcription factor expression during specification of DRG neurons for late aspects of neuronal differentiation and circuit assembly.</p> <p>(A–D) Expression of <i>EWS-Pea3</i> from the endogenous <i>Er81</i> locus can rescue anatomical defects observed in <i>Er81^−/−</i> mice, and no change in expression of TrkC (green) or Calretinin (CR; grey) is observed in proprioceptive afferents (A, B, and D). In contrast, precocious ETS signaling leads to severe defects in the establishment of DRG neuronal projections accompanied by inappropriate gene expression changes (C; upregulation of CR (red) and downregulation of TrkC [grey]).</p> <p>(E) Precocious ETS signaling (red) during progressive specification of proprioceptive sensory neurons leads to aberrant neuronal differentiation (red dashed line). In contrast, the isochronic, target-induced (green; peripheral signal) onset of ETS transcription factor signaling (black) induces appropriate terminal neuronal differentiation (blue).</p

Rescue of Ia Proprioceptive Afferent Projections into the Ventral Spinal Cord in <i>Er81^EWS-Pea3</i> Mutants

<p>(A–F) Morphological analysis of central projections at lumbar level L3 of PV⁺ DRG neurons (A–C) or all DRG sensory afferents after application of fluorescently labeled dextran to individual dorsal roots (D–F) in P0.5 (A–C) or P5 (D–F) wild-type (A and D), <i>Er81^−/−</i> (B and E), and <i>Er81^EWS-Pea3/−</i> (C and F) mice. Red dotted line indicates intermediate level of spinal cord.</p> <p>(G–I) Analysis of vGlut1 immunocytochemistry in the ventral horn of P0.5 wild-type (G), <i>Er81^−/−</i> (H), and <i>Er81^EWS-Pea3/−</i> (I) mice. Yellow dotted box in (A) indicates size of images shown in (G–I).</p> <p>(J–L) Schematic summary diagrams of the morphological rescue of Ia proprioceptive afferent projections (blue) into the ventral spinal cord observed in wild-type (J), <i>Er81^−/−</i> (K), and <i>Er81^EWS-Pea3/−</i> (L) mice. DRG indicated by dotted grey line; motor neurons are shown in black.</p> <p>Scale bar: (A–C), 150 μm; (D–F), 160 μm; (G–I), 70 μm.</p

Gene Expression Analysis upon Induction of Precocious or Isochronic ETS Signaling

<p>(A–H) Analysis of <i>TrkC</i> expression by in situ hybridization (A–D), or Calretinin (red) and LacZ (green) expression by immunohistochemistry (E–H), on E16.5 lumbar DRG of wild-type (A and E), <i>Tau^EWS-Pea3/+ Isl1^Cre/+</i> (B and F), <i>Er81^EWS-Pea3/−</i> (C and G), and <i>Tau^EWS-Pea3/+ PV^Cre/+</i> (D and H) embryos.</p> <p>(I) Summary diagram illustrating deregulation of TrkC (red arrows, downregulation) and Calretinin (green arrows, upregulation) expression upon precocious (B and F) induction of EWS-Pea3 expression in DRG neurons (B and F; E10–E11, i.e., shortly after cell cycle exit, E9.5–E10). In contrast, activation of EWS-Pea3 from the endogenous <i>Er81</i> locus (C and G; E12.5–E13) or via Cre recombinase expression from the <i>PV</i> locus activating late expression from the <i>Tau</i> locus (D and H; E14.5) does not interfere with the normal expression of <i>TrkC</i> and Calretinin (shown in grey).</p> <p>Scale bar: (A–D), 65 μm; (E–H), 80 μm.</p

Replacement of <i>Er81</i> by <i>EWS-Pea3</i>

<p>(A) Generation of <i>Er81^EWS-Pea3</i> mutant mice. Above is the organization of the <i>Er81</i> genomic locus in the region targeted by homologous recombination in analogy to [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030159#pbio-0030159-b14" target="_blank">14</a>]. Exons 1–4 are shown as light blue boxes, and the <i>Er81</i> start codon in exon 2 is indicated as ATG. The probe used to detect homologous recombination is shown as a grey box. Below is replacement of <i>Er81</i> by <i>EWS-Pea3</i> through the integration of <i>EWS-Pea3</i> in frame with the endogenous start codon of the <i>Er81</i> locus in exon 2 (in analogy to [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030159#pbio-0030159-b14" target="_blank">14</a>]).</p> <p>(B) PCR and Southern blot analysis of <i>Er81^EWS-Pea3</i> wild-type (<i>+/+</i>), heterozygous (<i>+/−</i>), and homozygous (<i>−/−</i>) genomic DNA to detect the mutant allele. PCR primer pairs (EWS-Pea3ki) were used to detect specifically the recombined allele, and a primer pair in exon2 was used to detect the presence of the wild-type allele [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030159#pbio-0030159-b14" target="_blank">14</a>].</p> <p>(C–E) Analysis of Er81 expression in lumbar DRG neurons of E16.5 wild-type (C), <i>Er81^−/−</i> (D), and <i>Er81^EWS-Pea3/−</i> (E) embryos. Inset in lower right corner of each panel shows Isl1 expression in the respective DRG.</p> <p>(F–H) PV expression in lumbar DRG of E16.5 wild-type (F), <i>Er81^−/−</i> (G), and <i>Er81^EWS-Pea3/−</i> (H) embryos. Confocal scans were performed with equal gain intensity.</p> <p>(J) Transcriptional transactivation of luciferase expression from a minimal reporter construct containing five consensus ETS DNA-binding sites ( GCCGGAAGC; [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030159#pbio-0030159-b18" target="_blank">18</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030159#pbio-0030159-b19" target="_blank">19</a>]) and a minimal TK promoter upon transient transfection of Er81 (<i>n</i> ≥ 7; 3.03 ± 0.66) or EWS-Pea3 (<i>n</i> ≥ 7; 20.3 ± 2.7). Relative luciferase activity normalized to control (Con). </p> <p>Scale bar: 80 μm.</p

Loss of Trk Receptor Expression and Increased Survival in DRG Neurons upon Precocious ETS Signaling

<p>(A–C and G–I) In situ hybridization analysis of <i>TrkA</i> (A and G), <i>TrkB</i> (B and H), and <i>TrkC</i> (C and I) expression in E16.5 lumbar DRG of wild-type (A–C) and <i>Tau^EWS-Pea3/+ Isl1^Cre/+</i> (G–I) embryos.</p> <p>(D–F and J–L) Analysis of lumbar DRG of wild-type (D), <i>Tau^mGFP/+ Isl^Cre/+</i> (E and F), and <i>Tau^EWS-Pea3/+ Isl1^Cre/+</i> (J, K, and L) embryos for (1) neuronal cell death at E13.5 by TUNEL (green; D and J), (2) cell survival and proliferation at E16.5 by LacZ (blue) wholemount staining (E and K; lumbar levels L1 and L2 are shown), and (3) BrdU (green)/LacZ (red) double labeling (F and L).</p> <p>(M and N) Quantitative analysis (<i>n</i> ≥ 3 independent experiments) of the mean number of apoptotic events relative to wild-type levels is shown in (M) and neuronal survival in (N) as percent of wild-type of DRG at lumbar levels L1 to L5 as quantified on serial sections.</p> <p>(O) Western blot analysis of protein extracts isolated from lumbar DRG of E16.5 wild-type (wt) and <i>Tau^EWS-Pea3/+ Isl1^Cre/+</i> (mut) embryos using the following antibodies: Akt, p-Akt (Ser473), CREB, p-CREB (Ser133), Bax, Bcl2, and Bcl-xl.</p> <p>(P) Quantitative analysis of protein levels relative to wild-type in percent is shown on the right (<i>n</i> = 3 independent experiments).</p> <p>Scale bar: (A–C and G–I), 35 μm; (D and J), 40 μm; (E and K), 200 μm; (F and L), 50 μm.</p