Figure 10. Too much or too little of Endoglycan impaired the timing and morphology of single dI1 growth cones migrating in the floor plate.

Data at the single axon level extracted from 24 hr time-lapse recordings of tdTomato-positive dI1 axons crossing the floor plate. (A₁) The time of floor-plate crossing was measured for the entire floor plate, for the first and for the second half for each condition. (A₂) The average growth cone area was measured in the first half, the second half and at the exit site of the floor plate for each condition. (B) Overexpression of Endoglycan significantly decreased the time axons needed to cross the entire floor plate compared to control and dsEndo conditions (Kruskal-Wallis test with Dunn’s multiple-comparisons test). (C) The average time of crossing the first half and the second half of the floor plate was compared. Interestingly, there was a highly significant difference in the spinal cords unilaterally electroporated with dsEndo, as axons spent much longer in the first compared to the second half of the floor plate. There was no difference between the two halves of the floor plate in the control condition, but there was a significant decrease in growth cone speed between the first (electroporated) half of the floor plate and the second half, where only axons were overexpressing Endoglycan (Wilcoxon test). (D) and (E) The ratios of the time axons spent in the first half (D) or the second half (E) of the floor plate divided by the time they needed to cross it entirely were compared between conditions. Unilateral knockdown of Endoglycan resulted in a significant increase of the ratio in the first half and a decrease in the second half compared to both control and overexpression conditions (one-way ANOVA with Sidak’s multiple-comparisons test). (F-H) The average dI1 growth cone area at each position of the floor plate (as depicted in A₂) was compared across all conditions. (F) Overexpression of Endoglycan induced a significant reduction in the average growth cone area compared to the control condition (Kruskal-Wallis test with Dunn’s multiple-comparisons test) but not compared to Endoglycan knockdown (p value = 0.08). (G) In the second half of the floor plate, the average growth cone area was reduced in both Endoglycan knockdown and overexpression condition compared to control (one-way ANOVA with Sidak’s multiple-comparisons test). (H) At the floor-plate exit site, overexpression of Endoglycan induced a significant decrease in the average growth cone area compared to both control and knockdown conditions (one-way ANOVA with Sidak’s multiple-comparisons test). Error bars represent standard deviation. p<0.0001 (****), p<0.001 (***), p<0.01 (**), p<0.05 (*), and p≥0.05 (ns) for all tests. N(embryos)=3 for each condition; n(axons, panel C-E)=161 (Endo Ctrl), 168 (dsEndo), 234 (Endo OE); n(axons, panel F-H)=70 (Endo Ctrl), 78 (dsEndo), 83 (Endo OE). See Table 2 for detailed results. Ent, entry; Mid, midline; Ex, exit; ipsi, ipsilateral; contra, contralateral; FP, floor plate; GC, growth cone. Source data and statistics are available in Figure 10—source data 1 spreadsheet.