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Fig. 1. Immunolabeling of Xenopus NMJs.
Whole mounted X. tropicalis NF48 embryos at the medial ventral region (A) and longitudinal sections of Tibialis anterior longus muscles obtained from NF66 post-metamorphic X. laevis froglets (B) were immunostained to reveal neurons (3A10 plus SV2 antibodies, red) and AChR aggregates (BTX staining, green), and subsequently imaged by confocal microscopy. Scale bars: 50 μm (A); 30 μm (B).
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Fig. 2. Early steps of NMJ development in X. tropicalis embryos.
X. tropicalis embryos obtained at stages NF20 (A), 26 (B), and 31 (C) were stained by immunodetection of neurons (using 3A10 plus SV2 antibodies, red) along with staining of AChR aggregates with BTX (green), and subsequently imaged by confocal microscopy. Magnified images of the region labelled with a white dotted line square in A, B and C are shown in their respective insets (merge in A; upper: AChR, lower: neuronal staining in B and C). Magnified images of the rostral region labelled with a cyan square in B are shown in B' (left: merge, middle: neuronal, and right: AChR staining). Magnified images of the regions labelled with cyan and yellow squares in C are shown in C' and C'', respectively (left: merge, middle: neuronal, and right: AChR staining in C' and C"). Orientation of embryos: anterior (left), posterior (right), dorsal (up), ventral (down). Arrowheads in the insets of B and C label the most ventral detection of presynaptic (yellow) and postsynaptic (cyan) staining. Scale bars: 200 μm (A,B,C); 50 μm (B'); 40 μm (C',C").
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Fig. 3. NMJ formation in X. tropicalis myotomal muscles.
X. tropicalis embryos obtained at stages NF35/36 (A) and 53 (B) were stained by immunodetection of neurons (using 3A10 plus SV2 antibodies, red) along with staining of AChR aggregates with BTX (green), and imaged by confocal microscopy. Magnified images of the regions labelled with cyan and yellow line squares in A are shown in A' and A", respectively (left: merge, middle: neuronal, and right: AChR staining in A' and A"). Cyan arrowheads in A" label the most ventral detection of postsynaptic staining. Magnified images of the region labelled with a white dotted line square in B are shown in the inset of B (left: merge, and right: AChR staining in the insets). Magnified images of the regions labelled with cyan, purple and yellow line squares in B are shown in B', B'', and B''', respectively (left: merge, middle: neuronal, and right: AChR staining in B' B'', and B'''). Orientation: anterior (left), posterior (right), dorsal (up), ventral (down). Scale bars: 200 μm (A); 1000 μm (B); 40 μm (A',A", B',B'',B''').
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Fig. 4. NMJ analyses of X. laevis tadpoles subjected to sham spinal cord injury operation.
(A) Scheme of the sham operation procedure in NF50 X. laevis embryos, where only the skin and the superficial muscle tissues were injured. (B-D) Sham tadpoles were collected 2 (B), 10 (C) and 18 (D) days post sham (DPS). Embryos were immunostained to label neurons (using 3A10 plus SV2 antibodies, red) along with staining of AChR aggregates with BTX (green), and subsequently imaged by confocal microscopy. Magnified images of B, C and D are shown in B', C' and D', respectively. Orientation of embryos: anterior (left), posterior (right), dorsal (up), ventral (down). Scale bars: 200 μm (B,C,D); 100 μm (B',C',D').
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Fig. 5. NMJ regeneration after SCI in X. laevis tadpoles.
(A) Scheme of the SCI procedure in NF50 X. laevis embryos, where the skin, superficial muscles and the spinal cord were injured (total transection, red asterisk). Injured larvae were collected 2 (A), 10 (B) and 18 (C) days post transection (DPT), immunostained to reveal neurons (using 3A10 plus SV2 antibodies, red) along with staining of AChR aggregates with BTX (green), and imaged by confocal microscopy. Asterisks in B, C and D label the transection site. In B (2DPT embryos), magnified images of the regions labelled with cyan (rostral to the SCI site), yellow (immediately caudal from the SCI site), and purple (more caudal to the SCI site) squares are shown in B', B'', and B''', respectively. Arrowheads in B'' show denervated AChR aggregates. In C (10DPT), magnified images of the regions labelled with cyan (rostral to the SCI site) and yellow (immediately caudal from the SCI site) squares are shown in C' and C'', respectively. In C (18DPT), magnified images of the regions labelled with yellow (immediately caudal from the SCI site) and purple (more caudal from the SCI site) squares are shown in D' and D", respectively. Arrows in C'', D' and D" indicate axonal branching. Orientation of embryos: anterior (left), posterior (right), dorsal (up), ventral (down). Scale bars: 500 μm (A); 200 μm (B–C); 30 μm (A'''); 50 μm (B',B'',C',C''); 10 μm (A',A").
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Fig. 6. Proposed mechanisms for axon collateral sprouting after SCI.
Previous to SCI, motor axons (red) project from the spinal cord to innervate AChR aggregates (green) of myotomal muscles to form NMJs (yellow) (A). Following SCI (asterisk), motor axons within the lesion site will undergo Wallerian degeneration (red dotted line). After 10DPT, collateral projections from rostral axons migrate towards caudal denervated muscle fibres. We envision two possible mechanisms for these observations. (B) These projections are branches of axons located rostral to the injury site, resulting in the formation of transiently bigger motor units. (C) These projections are “escape fibres†that protrude from a rostral innervated NMJ to contact caudal denervated postsynaptic domains. In both cases, collateral axons will be guided by projections of terminal Schwann cells (light blue), which protrude from the denervated NMJs. Schemes at the right of B and C represent the origin of the motor axon collaterals (gray lines) from a pre-existing axon (black line).
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