Figure 8.
Sytx1A function is required for the navigation of dorsal commissural axons toward and across the floor plate in vivo. A, C, E, and G are transverse sections. B, D, F, H, and I show open-book preparations of the spinal cord that were obtained by cutting the roof plate and flipping the cord open as indicated by the arrows (J). Rostral is at the top of the panels. A, B, Dorsolateral commissural neurons extend their axons toward the floor plate in a Netrin-1-dependent manner. By stage 25, axons have crossed the floor plate and turned into the longitudinal axis of the spinal cord, and are still in contact with the floor plate. C, D, The injection of the pIRES-EGFP construct did not interfere with commissural axon pathfinding. E–H, Perturbation of Sytx1A function either by expression of the dominant-negative variant Sytx1AH3TM (E, F) or by its downregulation using in ovo RNAi (G, H) interfered with commissural axon navigation to and across the floor plate. A large number of axons failed to reach the floor plate (E, F, H, arrows) or stopped at the ipsilateral floor plate border (F, H, arrowhead). As would be expected in the absence of Netrin-1-mediated axon guidance, axons were found to extend ventrally along the pial surface of the neural tube rather than along the correct pathway (E, G, open arrowhead). Axons were never found to extend along the spinal cord margin in control embryos (A, C, open arrowhead). I, As a control for the specificity of RNAi, we used dsRNA from NgCAM. NgCAM is a cell adhesion molecule of the Ig superfamily expressed by commissural axons. NgCAM was shown to be required for fasciculation of commissural axons but not for their midline crossing. K, Quantification of the axon guidance phenotype in open-book preparations of embryonic chicken spinal cords. Phenotypes were assessed in open-book preparations (black bars, strong phenotype; gray bars, weak phenotype; white bars, normal phenotype). We analyzed at least 10 embryos per condition (except for pIRES) with 6–10 dye injection sites per spinal cord. A failure to reach and cross the floor plate was found at 85% of the injection sites of embryos electroporated with the Sytx1H3TMEGFP fusion protein (91 injection sites in 10 embryos with 48% strong and 36% weak phenotypes). Similarly, the expression of pIRES-Sytx1AH3TMEGFP resulted in a phenotype at 87% of the injection sites (94 injection sites in 12 embryos with 56% strong and 31% weak phenotypes). In contrast, using the same criteria, embryos expressing the EGFP under the control of the β-actin promoter (74 injection sites in 11 embryos with 11% strong and 35% weak phenotypes) or from the pIRES construct (51 injection sites in 7 embryos; 71% of them normal; 29% with a weak phenotype) did not show a significant increase in the failure of commissural axons to reach and cross the floor plate. The same result was also found for the expression of the Sytx1ATMEGFP fusion protein (85 injection sites in 11 embryos with 9% strong and 40% weak phenotypes). We found a weak but significant increase in axon guidance errors in embryos expressing the Sytx1AH3EGFP fusion protein. A phenotype was observed at 60% of the injection sites (80 injection sites in 11 embryos with 29% strong and 31% weak phenotypes) in Sytx1AH3EGFP-expressing embryos. Downregulation of Sytx1A by in ovo RNAi resulted in the failure of commissural axons to reach and cross the floor plate at 64% of the injection sites (96 injection sites in 12 embryos with 44% strong and 20% weak phenotypes). Perturbation of NgCAM function was shown to interfere with fasciculation of commissural axons but not with their midline crossing. Accordingly, we found successful crossing of the floor plate at 77% of the injection sites (87 injection sites in 12 embryos with only 6% of the injection sites exhibiting a strong phenotype). Scale bars: A, 150 μm; I, 50 μm.