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Glycerophospholipid regulation of modality-specific sensory axon guidance in the spinal cord

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Science  28 Aug 2015:
Vol. 349, Issue 6251, pp. 974-977
DOI: 10.1126/science.aab3516
  • Fig. 1 Intraspinal localization of PtdGlc and chemorepulsive activity toward DRG afferents.

    (A and B) Transverse sections of st. 28 (A) and st. 34 (B) chick lumbar spinal cord with hematoxylin and eosin (H & E) stain (scale bar, 100 μm), double immunofluorescence of PtdGlc and either TrkA or TrkC, and grayscale images showing the unilateral dorsal cord from green/magenta merges at higher magnification (scale bar, 50 μm). The arrow and arrowhead indicate the dorsal root entry zone (DREZ) and the primordial dorsal funiculus, respectively. (C) Collagen gel explant assay. Dorsomedial (DM) or dorsolateral (DL) st. 34 spinal cord explants were cocultured with a DRG explant enriched for either TrkA or TrkC neurons by adding NGF or NT-3 to the medium, respectively. DRG axons were visualized with antibody to β-tubulin. Images are composites of three or four photomicrographs. Scale bar, 500 μm. (D) Explants were scored using a scale of 0 to 10, with 0 representing the greatest chemorepulsion (see the supplementary methods for the scoring protocol). Bars represent mean chemorepulsion score ± SEM, and numbers in boxes represent cultures per group. ***P < 0.001; Kruskal-Wallis test.

  • Fig. 2 LysoPtdGlc repels NGF-responsive but not NT-3–responsive DRG axons.

    (A) Dissociated st. 36 chick DRG neurons were cultured with either NGF or NT-3 and exposed to a concentration gradient of purified LysoPtdGlc. The number in each panel indicates minutes after initiation, and the arrowhead shows the direction of LysoPtdGlc gradient; scale bar, 10 μm. (B) Quantification of axon turning assay. Bars represent mean turning angle ± SEM with positive and negative values indicating attraction and repulsion, respectively. Numbers in parentheses are growth cones tested per group. The y-axis labels indicate the lipid or neurotrophin gradient, and in parentheses the neurotrophin added to the culture medium. Abbreviations and in-pipette concentrations used are as follows: LysoPtdGlc (purified; 1 μM); LysoPtdCho, lysophosphatidylcholine (1 μM); low LPA (1 μM); LPA (20 μM); NGF (50 μg/ml); NT-3 (50 μg/ml). ***P < 0.001, **P < 0.01, *P < 0.05; Kruskal-Wallis test. (C) Antibody to LysoPtdGlc blocked the chemorepulsive turning of NGF-responsive st. 28 DRG axons induced by LysoPtdGlc. Bars represent mean turning angle ± SEM. Numbers in parentheses are growth cones tested per group. In-pipette concentrations used are as follows: LysoPtdGlc (synthetic; 10 μM); LPA (20 μM); Sema3A (10 μg/ml). ***P < 0.001; Kruskal-Wallis test.

  • Fig. 3 LysoPtdGlc regulates nociceptive afferent projections in developing spinal cord.

    Injection of function-blocking antibody into the spinal cord in ovo attenuated LysoPtdGlc signaling. (A) Transverse lumbar spinal cord sections showing unilateral DiI-labeled putative nociceptive afferents in embryos injected with control antibody, anti-LysoPtdGlc, or a cocktail of anti-LysoPtdGlc and anti-NRP1. Scale bar, 75 μm. (B) Method of evaluating normal and dorsomedial projection phenotypes. The dorsolateral white matter known as the oval bundle of His is composed of the DREZ and the primordial dorsal funiculus (PDF). For quantification, the oval bundle of His was divided into dorsal and ventral halves as indicated by the broken line. Sections showing DiI- or TrkA antibody–labeled afferents only in the ventral half were considered normal, whereas those showing positive labeling in the dorsal half were classed as dorsomedial projections. (C) Quantification of dorsomedial projection of DiI-labeled afferents after antibody injection. (D) TrkA immunofluorescence in control antibody or anti-LysoPtdGlc–injected spinal cords. Scale bar, 50 μm. (E) Quantification of TrkA+ dorsomedial projection after antibody injection. [(C) and (E)] Bars represent mean percentage ± SEM of sections showing dorsomedial projection of all embryos in each test group. Numbers within each bar indicate the number of embryos examined per group. ***P < 0.001, **P < 0.01, *P < 0.05; Kruskal-Wallis test.

  • Fig. 4 GPR55 is a receptor for LysoPtdGlc-mediated axon guidance.

    (A) LysoPtdGlc chemorepels NGF-responsive DRG axons of wild-type (WT) but not Gpr55−/− mice. The number in the panels indicates minutes after initiation, and the arrowheads show the direction of the gradient; scale bar, 10 μm. (B) Quantified turning responses of WT or Gpr55−/− axons to gradients of LysoPtdGlc, LPA, LysoPtdIns, or Sema3A. NGF or NT-3 was added to the culture medium as indicated in parentheses. Bars represent mean turning angle SEM; numbers in parentheses indicate growth cones tested. **P < 0.01; one-way analysis of variance with Tukey’s post-test. (C) TrkA immunofluorescence in WT or Gpr55−/− mouse lumbar spinal cord. Solid circles indicate the midline; scale bar, 200 μm. (D) Method of evaluating TrkA immunofluorescence. Gray-shaded area represents the TrkA+ domain, and the broken line indicates the midline. If the TrkA+ domain in the medial quarter (MQ) of the spinal cord was equal to or greater in thickness than the TrkA+ domain in the DREZ (arrowheads), the section was classified as a dorsomedial projection. (E) Quantification of TrkA+ dorsomedial afferent projection in WT and Gpr55−/− mice. Bars represent mean percentage SEM of sections showing dorsomedial projection of all embryos in each test group. Numbers within each bar indicate the number of embryos examined per group. *P < 0.05; t test.

Supplementary Materials

  • Glycerophospholipid regulation of modality-specific sensory axon guidance in the spinal cord

    Adam T. Guy, Yasuko Nagatsuka, Noriko Ooashi, Mariko Inoue, Asuka Nakata, Peter Greimel, Asuka Inoue, Takuji Nabetani, Akiho Murayama, Kunihiro Ohta, Yukishige Ito, Junken Aoki, Yoshio Hirabayashi, Hiroyuki Kamiguchi

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Additional Author Notes
    • Supplementary Text
    • Figs. S1 to S28

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