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Fig. 8. Motoneuron growth cones in the spinal cord extend invadosome-like protrusions toward the peripheral myotome, which are necessary for proper axon extension into the periphery. (A,B) Maximum z-series projected images of a whole-mount embryo (lateral view, anterior left) with descending motoneuron growth cones on the ventral fascicle labeled with GFP by using targeted blastomere injection. This embryo was immunolabeled for βI+II tubulin (A) and GFP (B). (C) Merged image of motoneurons labeled for tubulin (red) and GFP (green) showing a robust protrusion that extends toward the notochord (n) from the central domain of the lead growth cone. Note that a MT has polymerized into this invadosome-like protrusion (A, arrowhead) that extends diagonally away from the spinal cord, as seen in an x-z view (C, inset). (D) Maximum z-series projected image (inverted contrast) of a whole-mount embryo (lateral view, anterior left) immunolabeled with the Znp-1 antibody. Note several peripheral axons and fine protrusions extend from motoneurons (arrows). (E) Magnified image from the boxed region in D shows a terminal motoneuron growth cone with many fine protrusions. (F) x-z view resampled along the dashed line in E shows an invadosome-like protrusion that extends deep into the lateral tissue (arrow). (G,H) Maximum z-series projected images of whole-mount embryos (lateral view, anterior left) expressing GFP (G) or δPX-Tks5-GFP (H) in motoneurons. Embryos were immunolabeled for βI+II tubulin (red) and GFP (green). Note that several peripheral GFP-expressing motoneuron axons with growth cones (G, arrowheads) have exited the spinal cord en route to the peripheral myotome, whereas motoneurons expressing δPX-Tks5-GFP remain within the spinal cord (H, arrows). (I) The percentage of tubulin-positive peripheral motoneuron axons that express δPX-Tks5-GFP is significantly less than that expressing GFP in control embryos (see Materials and Methods). *P<0.05, Fisher's exact test. The n values for numbers of peripheral axons (tubulin-positive, GFP-positive), image z stacks and embryos, respectively, are 64, 14, 28, 7 for control and 140, 16, 32, 8 for experimental conditions. Scale bars: 10 µm (A-C); 30 µm (D,G,H). |
