Figures S9–S11 . " width="100%" height="100%">
Journal: iScience
Article Title: The WAVE regulatory complex interacts with Boc and is required for Shh-mediated axon guidance
doi: 10.1016/j.isci.2024.111333
Figure Lengend Snippet: Nckap1 and Cyfip1/2 are required for Shh-mediated growth cone turning (A) Dunn chamber schematic from top (left) and side (right) views from Yam et al. 2009. The inner well is filled with media containing no chemoattractant, whereas the outer well is filled with media containing chemoattractant. Diffusion of the chemoattractant from the outer well to the inner well forms a gradient of the chemoattractant over the bridge region. The neurons cultured on a coverslip and exposed to the gradient in the bridge region are imaged. (B and G) Time-lapse imaging of commissural neurons electroporated either with scrambled shRNA or shRNA targeting Nckap1 or Cyfip1/2 and exposed to a Shh gradient in a Dunn chamber. Axons of commissural neurons electroporated with scrambled shRNA turned toward high concentrations of Shh, whereas axons of Nckap1 or Cyfip1/2 knockdown neurons did not change their direction of growth. The Shh gradient increases along the y axis in the images. Scale bar: 20 μm. (C) Scatterplots of the angle turned versus the original angle between the axons and the direction of the Shh gradient for neurons under the indicated conditions. Positive angles represent turning of axons toward the Shh gradient. (D) The mean angle turned (±SEM) of axons of commissural neurons in a Shh gradient. Nckap1 knockdown inhibits the turning of axons up a Shh gradient. Welch’s t test. n = 100 and n = 134 axons for scrambled and Nckap1 shRNA electroporated commissural neurons, respectively. (E) Scatterplots of the angle turned vs. the original angle and (F) the mean angle turned (±SEM) of axons of commissural neurons in a Shh gradient under the indicated conditions. Expression of NCKAP1 sm -Flag rescues the inhibitory effect of Nckap1 knockdown on the turning of axons up a Shh gradient. One-way ANOVA, n = 84, n = 46, and n = 45 axons for scrambled shRNA + empty vector, Nckap1 shRNA + empty vector, and Nckap1 shRNA + NCKAP1 sm -Flag electroporated commissural neurons, respectively. (H) Scatterplots of the angle turned vs. the original angle and (I) the mean angle turned (±SEM) of axons of commissural neurons in a Shh gradient under the indicated conditions. Cyfip1/2 knockdown inhibits the turning of axons up a Shh gradient. Unpaired t test, n = 114 and n = 101 axons for scrambled and Cyfip1/2 shRNA electroporated commissural neurons, respectively. (J) Net extension (±SEM) of the axons during the 2 h exposure to a Shh gradient. Mann-Whitney test, n = 100 and n = 134 axons for scrambled and Nckap1 shRNA electroporated commissural neurons, respectively. Kruskal-Wallis test, n = 84, n = 46, and n = 45 axons for scrambled shRNA + empty vector, Nckap1 shRNA + empty vector, and Nckap1 shRNA + NCKAP1 sm -Flag electroporated commissural neurons, respectively. Mann-Whitney test, n = 114 and n = 101 axons for scrambled and Cyfip1/2 shRNA electroporated commissural neurons, respectively. Error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01; n.s., not significant. See also Figures S9–S11 .
Article Snippet: Gradients were generated in the Dunn chamber with 0.1–0.2 μg/mL Shh (R&D Systems, 1845-SH or 8908-SH) in the outer well.
Techniques: Diffusion-based Assay, Cell Culture, Imaging, shRNA, Knockdown, Expressing, Plasmid Preparation, MANN-WHITNEY
Figures S9–S11 . " width="250" height="auto" />