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First published online November 26, 2007
doi: 10.1242/10.1242/dev.011452

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Semaphorin and neuropilin co-expression in motoneurons sets axon sensitivity to environmental semaphorin sources during motor axon pathfinding

Université de Lyon, F-69003, France, Université Lyon1, F-69003, France. CNRS, UMR5534, Centre de Génétique Moléculaire et Cellulaire, Villeurbanne, F-69622, France.

View larger version (65K): [in this window] [in a new window]   Fig. 1. Sema3A and Nrp1 coexpression in spinal motoneurons. (A) In situ hybridization of Sema3A mRNA on brachial and thoracic transverse sections of chick spinal cord from HH18-26 embryos. Lim3 and Islet1/2 immunostainings performed on adjacent sections are provided to show that Sema3A expression coincides with the medial motor column (MMCm: black arrowhead) and not with the lateral motor column (LMC: white arrowhead) at HH24. In situ hybridization and immunostaining of Nrp1 (NP1) show that both MMCm (black arrowhead) and LMC (white arrowhead) neurons express Nrp1 (NP1). (B) Schematic of the Sema3A expression pattern (gray) at HH24 and HH26 in spinal motoneurons and in peripheral tissues, in parallel with motor projections at the brachial level. sc, spinal cord; dm, dermamyotome; drg, dorsal root ganglion; nt, notochord; ao, aorta. Sema3A expression is observed at HH24 in MMCm neurons forming the dorsal ramus, but not in LMC neurons extending to the ventral ramus, and is then expanded at HH25-26 in most motoneurons. Scale bars: 100 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 2. Sema3A overexpression in motoneurons disrupts nerve projections. (A-F) Effects of control EGFP, Sema3AiresEGFP overexpression on spinal nerve fasciculation (A,B), on motor projections (arrowhead, B,C) towards the dermamyotome (outlined), the DRG (asterisk, D), and on dorsal ramus length (E, double arrowhead). A shows NgCAM immunostaining on whole-mount HH24 embryos. Immunolabeling (indicated in horizontal frames) was performed on transverse cryostat sections (B,D,E) or on vibratome sections (C,F) and observed by confocal microscopy. Scale bars: 100 µm. (G) Schematic of the misrouting produced by Sema3A overexpression (defects, right side, in green; normal pattern, left side).

View larger version (42K): [in this window] [in a new window]   Fig. 3. In vivo Sema3A silencing in motoneurons alters motor axon tracts. (A) In toto NgCAM immunostaining (lateral view) on pShSema3A-EGFP embryo showing the reduction of MMC axon length (bracket) and ventral nerve thickness (arrowheads) on the electroporated side compared with the control side. (B) Horizontal cross sections at the cervical-brachial level of spinal nerves labeled with NF160kD or NgCAM from pShSema3A-EGFP or pShScramble-EGFP embryos. (C) Histogram of normalized spinal nerve thickness in control and pShSema3A-EGFP HH25 embryos (spinal nerve sections from three pShScramble-EGFP embryos and eight pShSema3A-EGFP embryos; *, P<0.0001 with Mann-Whitney test). (D) Transverse sections at the cervical-brachial level of pShSema3A-EGFP and pShScramble-EGFP HH24 embryos stained with NF160kD. (E) Histogram of the ratio of the length of EGFP+ fibers to NF+ fibers within the dorsal ramus, as illustrated (dorsal rami from five pShScramble-EGFP and seven pShSema3A-EGFP embryos; *, P<0.0001 with Mann-Whitney test). (F) Histogram of the length of NF+/EGFP- fibers of dorsal rami in pShScramble and pShSema3A conditions (arbitrary units). Error bars indicate s.e.m. Scale bars: 100 µm.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Intrinsic Sema3A cell-autonomously sets the growth cone sensitivity to exogenous Sema3A. (A) Histogram showing the percentage of EGFP+ or EGFP- collapsed growth cones of Sema3AiresEGFP or control EGFP ventral spinal cord explants following exposure to Sema3A or control supernatant. Axons were counted from at least three independent experiments. ***, P<0.001 with X2 test; n.s., non significant. (B) Morphology of ctrlEGFP+ (collapsed) or Sema3AiresEGFP+ (non-collapsed) axons emerging from ventral spinal cord explants after Sema3A supernatant exposure. (C) Comparison of the collapse response (%) of pShScramble-EGFP+, pShScramble-EGFP-, pShSema3A-EGFP+ and pShSema3A-EGFP- axons following exposure to decreasing doses of Sema3A. *P<0.01 with X2 test between pShSema3A-EGFP+ and all other conditions. At least 150 growth cones from two embryos were examined in each condition.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Intrinsic Sema3A cell-autonomously modulates the Nrp1 level at the growth cone surface. (A) In situ hybridization of Nrp1 mRNA (NP1) on spinal transverse sections from Sema3AiresEGFP and EGFP embryos. Brackets indicate the electroporated side. Scale bar: 100 µm. (B,C) Nrp1 and NgCAM immunostaining of EGFP+ and EGFP- growth cones of Sema3AiresEGFP and ctrlEGFP explants, with membrane permeabilization (B), and without permeabilization (C). Scale bars: 10 µm. (D-G) Histograms showing fluorescence intensities per unit of surface at the growth cone. Fluorescence intensities were normalized to EGFP+ conditions in D-F. Fluorescence intensities of pShScramble-EGFP+ and pShSema3A-EGFP+ growth cones were normalized to the pShScramble-EGFP- or pShSema3A-EGFP- conditions, respectively (G). **, P<0.01; ***, P<0.001 with Mann-Whitney test. The number of growth cones analyzed is indicated for each condition. (H) Nrp1 and NgCAM immunostaining of EGFP+ and EGFP- growth cones of pShScramble-EGFP and pShSema3A-EGFP explants without permeabilization.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Non cell-autonomous effects of motoneuronal Sema3A in conditions of forced overexpression suggests that intrinsic Sema3A acts via secretion. (A) Myc immunostaining on transverse sections of Sema3AiresEGFP HH25 embryo showing that Myc-tagged Sema3A is targeted to the defasciculated spinal nerve (arrowhead). (B) Histogram showing the percentage (±s.d.) of EGFP+ or EGFP- collapsed growth cones following Sema3A and control supernatant exposure, of Sema3AiresEGFP, or control ventral spinal cord explants. **, P<0.01; ***, P<0.001 with Student's t-test; n.s.: non significant. The total number of axons from three independent experiments is indicated on the bars. (C,D) Histograms showing Nrp1 (NP1) and NgCAM immunofluorescence intensities per unit of surface at the growth cone in conditions of increased level of Sema3A overexpression. Immunolabeling of cell surface Nrp1 and NgCAM was performed on non-permeabilized explants. Fluorescence intensities were normalized to EGFP+ conditions. **, P<0.01; ***, P<0.001 with Mann-Whitney test. The number of axons examined is indicated on the bars.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Model of action of intrinsic Sema3A. (A) Mode of action of intrinsic Sema3A, showing that Sema3A expressed in neurons inhibits the response of their growth cones to environmental Sema3A through regulation of cell availability of Nrp1 (NP1). Predominant autocrine effect of neuronal semaphorin is indicated with an arrow. (B) Mechanism of interplay between intrinsic and extrinsic Sema3A in the development of motoneuron projections. Nrp1+ LMC axons are guided ventrally by the repulsive effects of Sema3A expressed in the DRG, the notochord and the dermamyotome. MMC axons are attracted dorsally by cues including ephrinA5 and FGFs. Intrinsic Sema3A in Nrp1 + MMCm neurons decreases their sensitivity to environmental repulsion by Sema3A, which enables their axons to project dorsally but to avoid the DRG and the dermamyotome.

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