Fig. 6: Both Trio-GEF domains are required for IN migration, and nucleokinesis dynamics defects are partly attributable to disruptions of actin remodeling.

A–E’ Confocal images of (A-A’) Trio^WT INs in MGE explants cultures electroporated with a control Dlx5/6::mCherry construct (mCherry), (B-B’) Trio^cKO INs in MGE explant cultures electroporated with mCherry or Trio^cKO INs in MGE explant cultures electroporated with mCherry together with (C-C’) full-length Trio cDNA (Trio^F-L), (D-D’) a mutated version of the Trio cDNA lacking the GEFD1 (Trio^ΔGEFD1) or (E-E’) a mutated version of the Trio cDNA lacking the GEFD2 (Trio^ΔGEFD2). Histograms of (F) the soma area, (G) the number, (H) complexity and (I) length of leading neurites, as well as (J) the number, (K) complexity and (L) length of trailing neurites, following rescue with Trio^F-L (yellow bars), Trio^ΔGEFD1 (blue bars) or Trio^ΔGEFD2 cDNA (pink bars) (n = 82 GFP + MGE-INs from 5 Trio^WT brains electroporated with mCherry, 49 GFP + MGE INs from 4 Trio^cKO brains electroporated with mCherry, 42 GFP + MGE-INs from 3 Trio^cKO brains co-electroporated with mCherry and Trio^F-L, 52 GFP + MGE-INs from 6 Trio^cKO brains co-electroporated with mCherry and Trio^ΔGEFD1 and 34 GFP + MGE-INs from 4 Trio^cKO brains co-electroporated with mCherry and Trio^ΔGEFD2). M–Y Both GEF domains are required to fully rescue IN migration. M Schematic of the experimental procedure to rescue IN migration and branching. N Time-lapse color-coded sequences showing a Trio^WT GFP + MGE-IN electroporated with mCherry, (O) a Trio^cKO GFP + MGE-IN electroporated with mCherry, (P) a Trio^cKO GFP + MGE-IN co-electroporated with mCherry and Trio^F-L, (Q) a Trio^cKO GFP + MGE-IN co-electroporated with mCherry and Trio^ΔGEFD1 and (R) a Trio^cKO GFP + MGE-IN co-electroporated with mCherry and Trio^ΔGEFD2. S–Y Histograms showing the rescue of (S) total migration speed (velocity), (T) net displacement (U) persistence ratio (directionality), (V) nucleokinesis cycle speed (W) nucleokinesis cycle duration and (X) nucleokinesis cycle amplitude (n = 81 GFP + MGE-INs from 5 Trio^WT brains electroporated with mCherry, 88 GFP + MGE-INs from 6 Trio^cKO INs electroporated with mCherry, 65 GFP + MGE-INs from 3 Trio^cKO brains co-electroporated with mCherry and Trio^F-L, 38 GFP + MGE-INs from 3 Trio^cKO brains co-electroporated with mCherry and Trio^ΔGEFD1 and 76 GFP + MGE-INs from 4 Trio^cKO brains co-electroporated with mCherry and Trio^ΔGEFD2). (Y) Histogram showing the rescue of the growth cone splitting rate (n = 42) GFP + MGE-INs from 3 Trio^WT brains electroporated with mCherry, 51 GFP + MGE-INs from 4 Trio^cKO brains electroporated with mCherry, 52 GFP + MGE-INs from 3 Trio^cKO brains co-electroporated with mCherry and Trio^F-L, 29 GFP + MGE-INs from 3 Trio^cKO brains co-electroporated with mCherry and Trio^ΔGEFD1 and 69 GFP + MGE-INs from 4 Trio^cKO brains co-electroporated with mCherry and Trio^ΔGEFD2. Z–CC Conditional deletion of Trio in GABAergic INs reduces actin remodeling. Z Schematic of the experimental design used to live-track actin dynamics in MGE-INs. AA-BB” Time-lapse sequences showing the distribution of F-actin (fire mode) in a Trio^WT (AA-AA”) and a Trio^cKO (BB-BB”) GFP + MGE-IN after electroporation of the Lifeact construct. CC Histogram showing the distribution of F-actin in the soma of GFP + MGE-INs (n = 13 GFP + MGE-INs from 3 Trio^WT brains and 15 GFP + MGE-INs from 4 Trio^cKO brains). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparisons tests (F–L, S–Y), or two-way ANOVA followed by Bonferroni’s multiple comparisons test (CC). Note that, with the exception of (S, V), whenever Trio^cKO MGE-INs electroporated with Trio^F-L, Trio^ΔGEFD1 or Trio^ΔGEFD2 were statistically different from Trio^cKO MGE-INs electroporated with mCherry (statistical differences currently showed on the figure), they were also not different from Trio^WT MGE-INs electroporated with mCherry. Thus, for better clarity, only comparisons with Trio^cKO INs electroporated with mCherry (black column) are shown in (F–L) and (S–Y), but p-values for all comparisons can be found in Supplementary Table 1. Scale bars: 25 µm (E, BB”) and 50 µm (R).