Figure 7. PlxnA1_Y1815F commissural growth cones have an increased exploratory behavior exerted through morphological split.

(A) Analysis of growth cone behaviors with fast time-lapse sequences. Schematic representation of growth cone categories and microphotographs of representative growth cones. (B) Histogram of the quantification of exploratory growth cones in PlxnA1^WT and PlxnA1_Y1815F open-books (pHluo-PlxnA1_WT, N = 5 electroporated embryos, 89 growth cones; pHluo-PlxnA1_Y1815F, N = 3 electroporated embryos, 73 growth cones). Chi-squared test has been applied, ***: p<0.001. (C) Histogram depicting the mode of exploration adopted by PlxnA1^WT and PlxnA1_Y1815F growth cones. The percentage was calculated over the total growth cone population (pHluo-PlxnA1_WT, N = 5 electroporated embryos, 89 growth cones; pHluo-PlxnA1_Y1815F, N = 3 electroporated embryos, 73 growth cones). Chi-squared test has been applied between non-exploratory and split populations (**: p<0.01), and between non-exploratory and turned (ns: non-significant). (D) Time-lapse sequences of individual growth cones navigating the floor plate (FP). The right panels show growth cone positions at time 0 and time 26. Time interval: 8 min. (E) Quantification of growth cone width reporting their exploratory behavior during the FP navigation in PlxnA1_WT and PlxnA1_Y1815F open-books (pHluo-PlxnA1_WT, N = 4 electroporated embryos, 23 growth cones; pHluo-PlxnA1_Y1815F, N = 3 electroporated embryos, 25 growth cones). Data are shown as the mean ± s.e.m., Student's t-test has been applied, ***: p<0.001. (F) Histograms of individual growth cone width (μm) from t = 0 min to t = 122 min (pHluo-PlxnA1_WT, N = 4 electroporated embryos, 23 growth cones; pHluo-PlxnA1_Y1815F, N = 3 electroporated embryos, 25 growth cones). (G) Current and proposed novel model of the mechanisms ensuring proper midline crossing of spinal cord commissural axons. Scale bars: 10 μm in (A and D, left), 50 μm in (D, right).

Figure 7—figure supplement 1. PlxnA1_Y1815F mutation confers to commissural growth cones navigating the floor plate (FP) a complex morphology.

(A) Microphotograph of spinal cord open-books from chick embryo electroporated with pHluo-PlxnA1_WT or pHluo-PlxnA1_Y1815F, and immunolabeled with anti-BEN antibody and Atto-647 GFP nanobodies. BEN labeling delineates the FP. (B) Comparative analysis of the proportion of complex growth cones observed after PlxnA1_WT and PlxnA1_Y1815F electroporation (PlxnA1_WT, N = 5 embryos, 367 growth cones; PlxnA1_Y1815F, N = 4 embryos, 199 growth cones). Spinal-cord open-books were subdivided in three fragments and observed by confocal microscopy as a dorso-ventral stack (27 stacks). A representative image for each condition is shown. Data are shown as the mean ± s.e.m., Student's t-test has been applied, ***: p<0.001. (C) Microphotographs of representative growth cones from STEP microscopy of atto-647N-GFP nanobodies-labeled chick open-books. Scale bar: 5 μm. (D) Microphotographs of cultured PlxnA1_{Y1815F/Y1815F} commissural neuron illustrating representative growth cone morphologies exposed to different treatments. (E) Histogram depicting the proportion of collapsed growth cones in the different experimental conditions (NB, N = 403; GDNF, N = 215; Sema3B, N = 215; SlitC, N = 197; GDNF+Sema3B, N = 406; GDNF+SlitC, N = 363). The intermediate condition represents growth cones with a shrinked central domain but still having filopodia as shown in (D) with white arrows. Chi-squared test has been applied, ***: p<0.001. Scale bars: 5 μm in (C), 15 μm in (D), 50 μm in (A).