Replacing <i>TrkA</i> with <i>TrkAC</i> is compatible with grossly normal survival of sensory neurons in <i>TrkAC-KI</i> mice.

<p>(A,B) An <i>in situ</i> probe recognizing endogenous <i>TrkA</i>, but not chimeric <i>TrkAC</i> is labeling the majority of wild type E15.5 DRG neurons, while mutant DRGs lack any staining. (C,D) An <i>in situ</i> probe specific to both <i>TrkC</i> and chimeric <i>TrkAC</i> is labeling few TrkC-positive neurons in wild type embryos and the majority of neurons in the E15.5 mutant DRGs. (E,F) Immunostaining with a TrkA antibody recognizing both TrkA and TrkAC (red) and a TrkC-specific antibody (green) showing normal pattern of TrkC expression and comparable TrkA antibody immunoreactivity in E15.5 mutant DRG. (G,H) There is normal TrkA antibody immunoreactivity in DRGs from P0 <i>TrkAC-KI</i> mutant mice. (I) Total number of lumbar (L3–4) and thoracic (T11–12) DRG neurons is reduced by 16% and 18% respectively in <i>TrkAC-KI</i> mice. Data represent mean ± s.e.m. Lumbar counts: 9 mutant and 8 wild type DRGs from 4 animals for each genotype, p = 0.041; thoracic counts: 8 mutant and 6 wild type DRGs from 3 and 2 animals respectively, p = 0.0019. * p<0.05, ** p<0.01. Scale bar is 50 µm.</p

Peripheral, but not central innervation is drastically reduced in TrkAC-KI mice.

<p>(A–D) Peptidergic (CGRP-positive) and total (PGP9.5-positive) fiber innervation is decreased in thick glabrous skin of adult <i>TrkAC-KI</i> hindlimbs. (E) Free nerve endings (FNE) counts in thick glabrous skin. Shown are means ± s.e.m. from 8–10 sections from two animals per genotype (* p>0.05, ** p<0.01). (F–I) Peptidergic (CGRP-positive) and total (PGP9.5-positive) fiber innervation is decreased in thin glabrous skin of adult <i>TrkAC-KI</i> hindlimbs. (J) Free nerve ending (FNE) counts in thin glabrous skin. Shown are means ± s.e.m. from 8–10 sections from two animals per genotype (* p>0.05, ** p<0.01). (K,L) Central projections are normal in <i>TrkAC-KI</i> mice. Peptidergic (CGRP-positive, green) and nonpeptidergic(IB4-positive, red) fibers normally innervate adult spinal cord in <i>TrkAC-KI</i> mice. Scale bar is 50 µm.</p

Abnormal mechanical and chemical pain response in <i>TrkAC-KI</i> mice.

<p>(A) Latency to mechanical stimulation using Von Frey apparatus was significantly lower in control mice one day after CFA injection, while <i>TrkAC-KI</i> mice did not show this response. Of note, the baseline latency to mechanical stimulation was lower in mutant mice (n = 9 for wild type and 13 for <i>TrkAC-KI</i>). (B) Lack of mechanical hypersensitivity after inflammation was also evident from a Dynamic Weight Bearing test. For <i>TrkAC-KI</i> mice, the weight distribution between inflamed and non-inflamed hindpaws was equal one day after CFA injection, while control mice favored the non-injected paw (n = 10 for wild type and 9 for <i>TrkAC-KI</i>). (C) Both <i>TrkAC-KI</i> and control mice developed thermal hyperalgesia one day after CFA injection (n = 8 for wild type and 6 for <i>TrkAC-KI</i>). The CFA effect (difference in latency between Day0 and day CFA+1) was significantly different between <i>TrkAC-KI</i> and wild type mice for Von Frey and DWB tests (A and B), but not for Hargreaves test (C). (D) <i>TrkAC-KI</i> mice exhibited severe deficit in pain from chemical injury when tested for nociceptive response after intraplantar injection of 10 µl of 2% formalin. Comparing to wild type littermates, <i>TrkAC-KI</i> mice had drastically reduced time of hindpaw shaking and biting during the first (0–10 min) and second (15–60 min) pain phases (n = 7 for each genotype). Data represent mean ± s.e.m * p<0.05, ** p<0.01.</p

Microarray screen for differentially expressed genes in DRGs from E14.5 <i>TrkAC-KI</i> embryos reveal a number of genes potentially responsible for NGF-dependent axonal growth.

<p>(A) Results of microarray experiment comparing gene expression between DRGs from <i>TrkAC-KI</i> and control E14.5 embryos. 28% of identified genes encoded for cell-cell interaction and cell adhesion molecules, and 21% for trafficking and post-translational modification proteins. Non-coding genes, as well as genes with both up- and down-regulated probes, were excluded from the data set presented in this figure (123 genes). Ret is downregulated 2 fold in our microarray. (B–G) Ret expression in small neurons is delayed <i>in TrkAC-KI</i> mice, as shown by <i>in situ</i> hybridization (B,C) and antibody labeling of E14.5 DRGs (D,E) and <i>in situ</i> hybridization of P0 DRGs (F,G) from mutant and control animals. Scale bar is 50 µm.</p

Peripheral innervation defect is evident during embryonic development.

<p>(A–D) Sections of E14.5 <i>TrkAC-KI</i> and control embryos stained with anti-TrkA antibody recognizing both TrkA and TrkAC proteins. Labeling of DRGs and projections to the spinal cord (central projections) is similar in both mutant and control animals while epidermal innervation is greatly decreased. (E and F) There are less PGP9.5 positive fibers in the skin of <i>TrkAC-KI</i> comparing to control embryos. (G and H) Skin innervation by TrkB-positive fibers is not changed in <i>TrkAC-KI</i> embryos. Scale bar is 50 µm.</p

Anti-C12orf4 intrabody inhibits mast cell degranulation.

<p>Analysis of stable clone 5H4: a) measurement of Annexin-V staining; b) β-hexosaminidase release; c) calcium flux and d) TNFα secretion. T-: Irrelevant intrabody. S: IgE/DNP stimulated. NS: unstimulated. Boxplot whiskers extend to the most extreme data point that is no more than 1.5 times the interquartile range. e) Measure of β-hexosaminidase release by retroviral infected populations. Clone R_8 is identical to the intrabody expressed by the plasmid clone 5H4. Sequences of the clones are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998.s006" target="_blank">Fig. S6</a>. Boxplot whiskers extend to the most extreme data point. f) Specific binding of 5H4-VH to C12orf4. Top panel: pull-down assay using 5H4-VH as capture agent and a commercial anti-C12orf4 polyclonal serum to reveal the protein. Irr: Irrelevant VH fragment, differing from 5H4 VH only by its CDR3 sequence. Low panel: subcellular localization of C12orf4 analyzed by confocal laser microscopy after double staining. Top left: Hoechst; top right: 5H4-VH-Fc fusion; bottom left: anti-C12orf4 commercial antibody; bottom right: merge. *: p<0.05; **: p<0.01; ***: p<0.001 (Student t-test).</p

Selection of intrabodies that inhibit mast cell degranulation.

<p>a) Schematic view of the selection method. The scFv/intrabody library previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998-Philibert1" target="_blank">[13]</a> was cloned in plasmid and retroviral vectors and used to transfect the RBL-2H3 cell line in order to induce a phenotypic diversity in a collection of cells. Clones displaying the desired phenotype, measured by inhibition of degranulation, were selected and the couple constituted by the inhibitory intrabody and its target antigen was identified and characterized. b) Annexin-V staining of cell populations from the library selection rounds is illustrated as the ratio of the geometric mean (MFI) of the FcεRI-stimulated (S) to the unstimulated (NS) cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104998#pone.0104998.s001" target="_blank">Fig. S1</a>).</p

Radiological features of the patients F2-IV.3 and F1-IV.3.

<p>Radiographs of patient <b>F2-IV.3</b> at 9 months (<b>A, C, E</b>) and <b>F1-IV.3</b> at birth (<b>B, F</b>) and at 3 months (<b>D</b>) show platyspondyly, square iliac bones, and delayed epiphyseal ossification.</p

The missense <i>MAGMAS</i> mutation c.226A>G (p.Asn76Asp).

<p>(<b>A</b>) Sequencing chromatograms showing the segregation of the c.226A>G transition in <i>MAGMAS</i> with the disease in both families F1 and F2. n: heterozygous peak. (<b>B</b>) Multiple alignments between human MAGMAS protein and several orthologs showing that the Asparagine at codon 76 in MAGMAS is well conserved among mammals and vertebrates.</p

Yeast strains and plasmids used in this study.

<p>Yeast strains and plasmids used in this study.</p