• Thu. Sep 21st, 2023

(Fig, 7D, E)


May 4, 2023

(Fig, 7D, E). labels (B, D) show that Slit1 is expressed in the floor plate and Rabbit polyclonal to CD14 Slit2 is expressed in the medial floor plate and spinal motor neurons. (E) -galactosidase reporter gene labels on E12.5 spinal cord sections, showing Netrin-1 is mainly expressed on the ventral spinal cord. Dotted white lines in B, WEHI-345 D, and E show the spinal cord. Scale bars: ACE, 100 m. NIHMS912561-supplement.pdf (323K) GUID:?B06F5E4D-E980-447B-AE75-34C9992E78BA Abstract In the spinal cord, motor axons project out the neural tube at specific exit points, then bundle together to project toward target muscles. The molecular signals that guide motor axons to and out of their exit points remain undefined. Since motor axons and their exit points are located near the floor plate, guidance signals produced by the floor plate and adjacent ventral tissues could influence motor axons as they project toward and out of exit points. The secreted Slit proteins are major floor plate repellents, and motor neurons express two Slit WEHI-345 receptors, Robo1 and Robo2. Using mutant mouse embryos at early stages of motor axon exit, we found that motor exit points shifted ventrally in Robo1/2 or Slit1/2 double mutants. Along with the ventral shift, mutant axons had abnormal trajectories both within the neural tube toward the exit point, and after exit into the WEHI-345 periphery. WEHI-345 In contrast, the absence of WEHI-345 the major ventral attractant, Netrin-1, or its receptor, DCC caused motor exit points to shift dorsally. Netrin-1 attraction on spinal motor axons was demonstrated by explant assays, showing that Netrin-1 increased outgrowth and attracted cultured spinal motor axons. The opposing effects of Slit/Robo and Netrin-1/DCC signals were tested genetically by combining Netrin-1 and Robo1/2 mutations. The location of exit points in the combined mutants was significantly recovered to their normal position compared to Netrin-1 or Robo1/2 mutants. Together, these results suggest that the proper position of motor exit points is determined by a push-pull mechanism, pulled ventrally by Netrin-1/DCC attraction and pushed dorsally by Slit/Robo repulsion. experiments showed that cultured spinal motor axons can respond to Slit signals (Bai et al., 2011). Interestingly, however, they reported that explanted spinal cord motor axons were unresponsive to Netrin-1 because of an activity of the Presenilin (PS1) secretase complex that cleaves DCC receptors (Bai et al., 2011). A recent report showed that Nkx2.9 controls spinal accessory motor axon projections to their lateral exit from the spinal cord by regulating Slit/Robo2 signaling (Bravo-Ambrosio et al., 2012). Together, these findings imply that guidance cues within the ventral spinal cord have the potential to set the precise position of motor exit points. We have therefore investigated whether Slit and Netrin-1 signals are involved in guiding ventral motor exits in the spinal cord, by analyzing mouse embryos with mutations in these cues or their receptors, and assaying direct effects on cultured spinal motor axons. Our results suggest that the proper position of motor axon exits is determined by a balance between a ventral pull by Netrin-1/DCC attraction and a dorsal push by Slit/Robo repulsion. Materials and Methods Mouse embryos Mouse experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, by protocols approved by the University of Nevada, Reno Institutional Animal Care and Use Committee. Embryonic day 9.5 (E9.5), 9.75 (E9.75), 10 (E10), 10.5 (E10.5),.