Article Figures & Data
Figures
- Figure 1.
JNK signaling regulates the dynamic migratory properties of MGE interneurons. A, Schematic diagram of MGE explant cortical cell coculture assay with pharmacological inhibition of JNK signaling. B, C, Individual cell tracks (pseudo-colored by time) from four interneurons in control (B) or 20 μm SP600125 (C) treated cultures imaged live for 12 h. D-G, Quantification of interneuron migratory properties revealed significant disruptions in migration speed (D), speed variation (E), and displacement (F), but not straightness (G), during JNK inhibition. For each condition, a minimum of 10 cells were tracked from n = 11 movies (127 cells/condition) obtained over four experimental days. Data are presented as Gardner–Altman estimation plots. The values of both groups are plotted on the left axes with the mean difference between groups plotted on the right axes as a bootstrap resampling distribution. The mean difference is depicted as a large black dot with the 95% confidence interval indicated by the ends of the vertical error bar; ****p < 0.0001, ***p < 0.001, Student’s t test. Time in hours. Scale bar: 50 µm. In addition, a subset of MGE interneurons in control and JNK inhibited conditions were analyzed to determine whether JNK signaling influenced migratory properties of cells traveling at the same average speed (Extended Data Fig. 1-1).
- Figure 2.
Migrating MGE interneurons require intact JNK signaling for proper leading process branching. A, B, Time series depicting growth cone (GC) splitting from control (A) or JNK-inhibited (B) MGE interneurons. Closed arrowhead = GC, open arrowhead = new GC branch. C, Quantification of leading process length; n = 10 cells were measured from eight movies/condition obtained over four experimental days. D, Quantification of GC splitting frequency; n = 19 control cells from eight movies and n = 19 SP600125 cells from 10 movies were measured. E, F, Interstitial side branching from control (E) or JNK-inhibited (F) interneurons. Closed arrowhead = new side branch. G, Quantification of interstitial side branch frequency of control and SP600125-treated interneurons; n = 19 control cells from eight movies; n = 19 SP600125 cells from 10 movies. H, Quantification of interstitial side branch duration in control and JNK-inhibited conditions; n = 52 branches from 14 control cells and 18 SP600125 cells were measured from 10 movies/condition. All branching data were from movies obtained over five experimental days. Data are presented as Gardner–Altman estimation plots; *p < 0.05, Student’s t test. Time in minutes. Scale bar: 15 µm.
- Figure 3.
Pharmacological inhibition of JNK signaling impairs nucleokinesis in migrating MGE interneurons. A, B, Time series of a control (A) and SP600125-treated (B) interneuron undergoing a single cycle of nucleokinesis. Closed arrowhead = leading process swelling, n = nucleus. C–E, Cortical interneurons treated with JNK inhibitor have significantly shorter somal translocation distances (C), decreased frequency of nucleokinesis (D), and increased pause duration (E) compared with controls. C, Cartoon showing how the distance (d) that an interneuron cell body translocates over time was measured. In each condition, 50 cells were measured from n = 10 movies obtained over four experimental days. F, Cartoon showing how the distance (d) that a swelling extends from a cell body was measured. JNK-inhibited cells display significantly decreased distance of swelling extension. G, Swelling duration is significantly increased in JNK-inhibited interneurons. A total of 43 control cells were measured from n = 10 control movies and 53 treated cells were measured from n = 6 SP600125 (SP) movies, each obtained over four experimental days. H, Histogram showing nuclear translocation over time for a single cell in each condition. Distance traveled between two points is plotted and every movement above 5 µm (gray dashed line) is considered to be a nucleokinesis event. Data in C–G are presented as Gardner–Altman estimation plots; ****p < 0.0001, ***p < 0.001, Student’s t test. Time in minutes. Scale bar: 15 µm.
- Figure 4.
Genetic removal of JNK signaling impairs migratory properties and leading process dynamics of MGE interneurons. A, Diagram of MGE explant assay with Dlx5/6-CIE+ WT or JNK cTKO explants cultured on WT cortical feeder cells. B, C, Four individual cell tracks (pseudo-colored by time) from WT or cTKO interneurons imaged live for 12 h. D–G, Quantification of migratory properties reveals no alterations in migratory speed (D), but significant disruptions to speed variation (E), displacement (F), and straightness (G) between control and cTKO interneurons. A total 120 WT cells were measured from n = 13 control movies and 130 cTKO cells were measured from n = 12 cTKO movies, each obtained over four experimental days. H, I, cTKO interneurons have significantly decreased growth cone split frequency (H) without changes in interstitial side branch frequency (I); n = 11 cells measured from six movies/condition collected over four experimental days. J, Side branches from cTKO interneurons are significantly shorter-lived than controls; n = 34 branches were measured from 10 WT cells and n = 28 branches were measured from 10 cTKO cells recorded from six movies/condition obtained over four experimental days. Data are presented as Gardner–Altman estimation plots; *p < 0.05, Student’s t test. Time in hours. Scale bar: 50 µm.
- Figure 5.
Genetic removal of Jnk disrupts nucleokinesis in migrating MGE interneurons. A, WT cortical interneuron undergoing a single nucleokinesis event. B, cTKO cortical interneuron completing two nucleokinesis events over the same interval of time. Closed arrowhead = leading process swelling, n = nucleus. C–E, cTKO interneurons have significantly decreased translocation distance (C), increased translocation frequency (D), and decreased pause duration (E) compared with WT interneurons. In each condition, 50 cells were measured from n = 10 movies obtained over four experimental days. F, cTKO interneurons have decreased swelling duration compared with WT interneurons. A total of 37 WT cells were measured from n = 6 WT movies and 38 cTKO cells were measured from n = 6 cTKO movies, each obtained over four experimental days. Data are presented as Gardner–Altman estimation plots; ***p < 0.001, **p < 0.01, *p < 0.05, Student’s t test. Time in minutes. Scale bar: 15 µm.
- Figure 6.
Centrosomes are mislocalized in MGE interneurons during JNK inhibition. A, Diagram depicting ex vivo electroporation of MGE tissue and subsequent culture of MGE explants on cortical feeder cells. B, An interneuron expressing a fluorescently tagged centrosome protein (Centrin2; Cetn2-mCherry) shows translocation of the centrosome into the cytoplasmic swelling before nucleokinesis in control conditions. C, A Cetn2-mCherry expressing interneuron treated with SP600125 shows aberrant rearward movement of the centrosome into the trailing process. Arrowhead = Cetn2-mCherry. D, Scatter plot of centrosome distribution over time (centrosome: two-way ANOVA: F(2,114) = 13.82; p < 0.0001; p < 0.0001). Error bars represent mean ± SEM, post hoc by Fisher’s LSD ***p < 0.001, **p < 0.01, *p < 0.05. E, Scatter plot depicting centrosome occupancy of a formed swelling over time (χ2 test; ****p < 0.0001). Error bars represent mean ± SEM. F, Average maximum distance the centrosome traveled from the soma front (Student’s t test; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). In each condition, n = 20 cells were measured from 11 movies obtained over five experimental days. Data in F are presented as Gardner–Altman estimation plots. Time in minutes. Scale bar: 7.5 µm.
- Figure 7.
Primary cilium localization in MGE interneurons is disrupted during JNK inhibition. A, An interneuron expressing a fluorescently tagged primary ciliary marker (Arl13b-tdTomato) shows translocation of the primary cilium into the cytoplasmic swelling before nucleokinesis in control conditions. B, An interneuron expressing Arl13b-tdTomato shows aberrant rearward movement of the primary cilium into the trailing process when treated with SP600125. Arrowhead = Arl13b-tdTomato. C, Scatter plot of primary cilium distribution over time (two-way ANOVA: F(2,114) = 12.13; p < 0.0001). Error bars represent mean ± SEM, post hoc by Fisher’s LSD ***p < 0.001, **p < 0.01, *p < 0.05. D, Scatter plot depicting primary cilium occupancy of a formed swelling over time (χ2 test; ****p < 0.0001). E, Average maximum distance the primary cilium traveled from the soma front (Student’s t test; **p < 0.01). In each condition, n = 20 cells were measured from 15 movies obtained over six experimental days. F, Average ciliary length measured over time. In each condition, n = 17 control and n = 15 SP600125 (SP) cells were measured over 13 movies and six experimental days. Data in E, F are presented as Gardner–Altman estimation plots. Student’s t test; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Time in minutes. Scale bar: 7.5 µm.
Tables
Estimation statistics Figure Measurement Datastructure Type of test Comparison Statisticalvalue Two-sidedpermutationt test value Mean DifferenceWith 95% ConfidenceInterval Fig. 1D Maximum migration speed Normal Unpaired t test Control vs SP p = 1.68E-10; t(20) = 11.86 0.0 –54.2 [–63.1, –46.0] Fig. 1D Mean migration speed Normal Unpaired t test Control vs SP p = 1.68E-09; t(20) = 10.38 0.0 –28.1 [–33.8, –23.6] Fig. 1D Minimum migration speed Normal Unpaired t test Control vs SP p = 0.0000717; t(20) = 4.98 0.0 –4.68 [–6.76, –3.08] Fig. 1E Speed variation Normal Unpaired t test Control vs SP p = 0.000188; t(20) = –4.56 0.0002 0.136 [0.0827, 0.196] Fig. 1F Displacement Normal Unpaired t test Control vs SP p = 4.73E-07; t(20) = 7.29 0.0 –81.2 [–1.06e+02, –63.6] Fig. 1G Straightness Normal Unpaired t test Control vs SP p = 0.451; t(20) = 0.769 0.389 –0.0282 [–0.0955, 0.03] Fig. 2C Maximum leadingprocess length Normal Unpaired t test Control vs SP p = 0.977; t(18) = –0.0294 0.975 0.177 [–10.7, 11.7] Fig. 2C Mean leading processlength Normal Unpaired t test Control vs SP p = 0.947; t(18) = 0.0668 0.947 –0.257 [–6.85, 7.48] Fig. 2C Minimum leadingprocess length Normal Unpaired t test Control vs SP p = 0.911; t(18) = –0.112 0.917 0.413 [–6.12, 7.32] Fig. 2D Frequency of growthcone splits Normal Unpaired t test Control vs SP p = 0.0169; t(34) = 2.51 0.0146 –0.681 [–1.13, –0.121] Fig. 2G Frequency of side branches Normal Unpaired t test Control vs SP p = 0.900; t(34) = 0.126 0.897 0.0347 [–0.479, 0.499] Fig. 2H Side branch duration Normal Unpaired t test Control vs SP p = 0.016; t(102) = 2.46 0.0124 –7.58 [–14.2, –2.12] Fig. 3C Nucleokinesis distance Normal Unpaired t test Control vs SP p = 2.36E-10; t(18) = 12.58 0.0 –6.36 [–7.3, –5.42] Fig. 3D Nucleokinesis frequency Normal Unpaired t test Control vs SP p = 1.92E-08; t(18) = 8.96 0.0 –0.765 [–0.912, –0.598] Fig. 3E Pause duration Normal Unpaired t test Control vs SP p = 1.45E-07; t(18) = –7.89 0.0 9.5 [7.44, 11.8] Fig. 3F Soma-swelling distance Normal Unpaired t test Control vs SP p = 0.001698; t(18) = 3.68 0.0022 –1.79 [–2.64, –0.824] Fig. 3G Swelling duration Normal Unpaired t test Control vs SP p = 0.00047; t(14) = –4.53 0.0014 7.04 [4.33, 9.76] Fig. 4D Maximum migration speed Normal Unpaired t test WT vs cTKO p = 0.981; t(23) = 0.0236 0.982 –0.117 [–9.81, 9.2] Fig. 4D Mean migration speed Normal Unpaired t test WT vs cTKO p = 0.105; t(23) = 1.69 0.11 –5.59 [–12.0, 0.867] Fig. 4D Minimum migration speed Normal Unpaired t test WT vs cTKO p = 0.260; t(23) = 1.16 0.256 –1.24 [–3.27, 0.771] Fig. 4E Speed variation Normal Unpaired t test WT vs cTKO p = 0.022; t(23) = –2.46 0.0208 0.057 [0.0166, 0.107] Fig. 4F Displacement Normal Unpaired t test WT vs cTKO p = 0.048; t(23) = 2.09 0.0492 –29.1 [–54.8, –0.875] Fig. 4G Straightness Normal Unpaired t test WT vs cTKO p = 0.027; t(23) = 2.37 0.0284 –0.0651 [–0.118, –0.0133] Fig. 4H Frequency of growthcone splits Normal Unpaired t test WT vs cTKO p = 0.0454; t(20) = 2.13 0.0318 –0.618 [–1.29, –0.183] Fig. 4I Frequency of sidebranches Normal Unpaired t test WT vs cTKO p = 0.658; t(20) = 0.448 0.654 –0.113 [–0.542, 0.385] Fig. 4J Side branch duration Normal Unpaired t test WT vs cTKO p = 0.046; t(60) = 2.04 0.044 –8.83 [–17.7, –1.83] Fig. 5C Nucleokinesis distance Normal Unpaired t test WT vs cTKO p = 0.028; t(18) = 2.39 0.0298 –0.92 [–1.61, –0.17] Fig. 5D Nucleokinesis frequency Normal Unpaired t test WT vs cTKO p = 0.00203; t(18) = –3.60 0.0012 0.458 [0.262, 0.752] Fig. 5E Pause duration Normal Unpaired t test WT vs cTKO p = 0.000464; t(18) = 4.27 0.0002 –5.09 [–7.84, –3.27] Fig. 5F Swelling duration Normal Unpaired t test WT vs cTKO p = 0.00257; t(10) = 3.99 0.0012 –2.61 [–3.94, –1.54] Fig. 6D Centrosome localization Normal Two-way ANOVA(post hoc Fisher’sexact test) Control vs SP p < 0.0001; F(2,114) = 13.82 NA NA Fig. 6E Centrosome presencein swelling Normal χ2 Control vs SP p < 0.0001; Χ2(331.1, 1) =18.20 NA NA Fig. 6F Centrosome maximumdistance forward Normal Unpaired t test Control vs SP p = 0.028; t(38) = 2.30 0.0256 –3.2 [–5.82, –0.79] Fig. 6F Centrosome maximumdistance behind Normal Unpaired t test Control vs SP p = 0.000015; t(38) = 4.97 0 –10.3 [–14.6, –6.49] Fig. 7C Cilia localization Normal Two-way ANOVA(post hoc Fisher'sexact test) Control vs SP p < 0.0001; F(2,114) = 12.13 NA NA Fig. 7D Cilia presence in swelling Normal χ2 Control vs SP p < 0.0001; Χ2(314.2,1) = 17.72 NA NA Fig. 7E Cilia maximum distanceforward Normal Unpaired t test Control vs SP p = 0.0094; t(38) = 2.76 0.0094 –4.16 [–7.0, –1.61] Fig. 7E Cilia maximum distance behind Normal Unpaired t test Control vs SP p = 0.017; t(38) = 2.51 0.0152 –6.38 [–11.6, –1.97] Fig. 7F Cilia length Normal Unpaired t test Control vs SP p = 0.558; t(30) = 0.592 0.575 –0.0983 [–0.439, 0.195] Extended Data Fig. 1-1A Mean migration speed all cells Normal Unpaired t test Control vs SP p = 4.65E-53; t(252) = 19.74 0.0 –28.4 [–31.1, –25.5] Extended Data Fig. 1-1B Maximum migration speed Normal Unpaired t test Control vs SP p = 0.0053; t(25) = 3.05 0.0042 –24.0 [–41.8, –10.6] Extended Data Fig. 1-1B Mean migration speed Normal Unpaired t test Control vs SP p = 0.559; t(25) = 0.592 0.545 –0.299 [–1.28, 0.636] Extended Data Fig. 1-1B Minimum migration speed Normal Unpaired t test Control vs SP p = 0.209; t(25) = –1.29 0.210 1.39 [–0.708, 3.25] Extended Data Fig. 1-1C Speed variation Normal Unpaired t test Control vs SP p = 0.0038; t(25) = 3.19 0.0028 –0.187 [–0.317, –0.0848] Extended Data Fig. 1-1D Displacement Normal Unpaired t test Control vs SP p = 0.158; t(25) = –1.45 0.172 15.6 [–5.9, 35.7] Extended Data Fig. 1-1E Straightness Normal Unpaired t test Control vs SP p = 0.146; t(25) = –1.49 0.162 0.104 [–0.0304, 0.238] Extended Data Fig. 1-1F Soma-swelling distance Normal Unpaired t test Control vs SP p = 0.0017; t(25) = 3.51 0.0008 –2.3 [–3.7, –1.19] Extended Data Fig. 1-1G Average nucleokinesis distance Normal Unpaired t test Control vs SP p = 8.26E-06; t(25) = 5.58 0.0 –3.94 [–5.36, –2.6] Extended Data Fig. 1-1H <10-µm nucleokinesis distance Normal Unpaired t test Control vs SP p = 0.239; t(25) = –1.20 0.236 0.259 [–0.113, 0.719] Extended Data Fig. 1-1I >10-µm nucleokinesis distance Normal Unpaired t test Control vs SP p = 0.00094; t(25) = 3.75 0.0004 –2.73 [–4.26, –1.43] Extended Data Fig. 1-1J Nucleokinesis frequency Normal Unpaired t test Control vs SP p = 0.382; t(25) = 0.890 0.381 –0.111 [–0.345, 0.123] Extended Data Fig. 1-1K Pause duration Normal Unpaired t test Control vs SP p = 0.805; t(25) = –0.250 0.805 0.363 [–2.62, 2.85] Extended Data Fig. 1-1L >10-µm nucleokinesis frequency Normal Unpaired t test Control vs SP p = 0.087; t(25) = 1.78 0.098 –0.233 [–0.458, 0.0489] Extended Data Fig. 1-1M Frequency of growth cone splits Normal Unpaired t test Control vs SP p = 0.00398; t(8) = 3.99 0.0096 –1.33 [–1.81, –0.612] Extended Data Fig. 1-1N Frequency of side branches Normal Unpaired t test Control vs SP p = 0.324; t(8) = 1.05 0.329 –0.44 [–1.1, 0.369] Extended Data Fig. 1-1O Side branch duration Normal Unpaired t test Control vs SP p = 0.529; t(25) = 0.639 0.556 –3.16 [–14.8, 4.59] All statistical measurements performed in the study, organized by figure panel. The type of measurement, data structure, type of test, group comparison, and statistical values are included for each analysis. Estimation statistics are provided, where applicable. SP = SP600125.
Movies
- Movie 1.
Live imaging of MGE interneurons under control conditions. Movie clip 1, E14.5 Dlx5/6-CIE MGE explant cortical cell co-culture imaged live for 12 h in control conditions. Interneurons robustly migrate from the margin of the MGE explant to fill the field of view. Movie clip 2, Three MGE interneurons tracked for the duration of the recording.
- Movie 2.
Live imaging of MGE interneurons under 20 μm SP600125 conditions. Movie clip 1, E14.5 Dlx5/6-CIE MGE explant cortical cell co-culture imaged for 12 h in SP600125 conditions. Interneurons slowly migrate from the margin of the explant, sparsely populating the field of view. Movie clip 2, Three MGE interneurons tracked for the duration of the recording.
- Movie 3.
Leading process branching dynamics of MGE interneurons under control conditions. Movie clip 1, Interneuron undergoing growth cone splitting over the course of 1 h in control conditions. First growth cone split is marked with an open arrowhead. Movie clip 2, MGE interneuron extending an interstitial side branch (open arrowhead) over the course of 1 h. Only side branches persisting for 10 min were measured (see Materials and Methods).
- Movie 4.
Leading process branching dynamics of MGE interneurons under 20 μm SP600125 conditions. Movie clip 1, SP600125-treated interneuron undergoing one growth cone splitting event (marked by open arrowhead) over the course of 1 h. Movie clip 2, SP600125-treated interneuron extending a short-lived interstitial side branch (open arrowhead) over the course of 1 h.
- Movie 5.
Nucleokinesis of MGE interneurons in control and JNK-inhibited conditions. Movie clip 1, Interneuron undergoing multiple nucleokinesis events in control conditions. Movie clip 2, SP600125-treated interneuron engaging in nucleokinesis at a slower rate than control. Open arrowheads mark translocating cell body in both clips.
- Movie 6.
Leading process branching dynamics of WT MGE interneurons. Movie clip 1, WT interneuron undergoing multiple growth cone splitting events (open arrowhead) over the course of 1 h. Movie clip 2, WT interneuron extending an interstitial side branch (open arrowhead) over the course of 1 h.
- Movie 7.
Leading process branching dynamics of cTKO MGE interneurons. Movie clip 1, cTKO interneuron undergoing fewer growth cone splits (open arrowhead) over the course of 1 h compared to control. Movie clip 2, cTKO interneuron extending a short-lived interstitial side branch (open arrowhead) over the course of 1 h.
- Movie 8.
Nucleokinesis in WT and cTKO MGE interneurons. Movie clip 1, WT interneuron undergoing nucleokinesis. Movie clip 2, cTKO interneuron engaging in nucleokinesis at a higher rate than control. Open arrowheads mark translocating cell body in both clips.
- Movie 9.
Centrosome dynamics in MGE interneurons under control and SP600125-treated conditions. Movie clip 1, Control interneuron electroporated with centrosomal marker Cetn2- mCherry. The centrosome moves from the cell body into the cytoplasmic swelling under control conditions. Movie clip 2, SP600125-treated interneuron electroporated with Cetn2-mCherry. The centrosome moves from the cell body into the trailing process under SP600125 conditions. Movie clip 3, A second SP600125-treated interneuron electroporated with Cetn2-mCherry. The centrosome separates into two centrioles and moves from the cell body into the trailing process and back under SP600125 conditions. Open arrowheads mark Cetn2-mCherry expressing interneurons in all clips.
- Movie 10.
Dynamics of primary cilia in MGE interneurons under control and SP600125-treated conditions. Movie clip 1, Interneuron electroporated with primary ciliary marker Arl13b-tdTomato. The cilium moves from the cell body into the cytoplasmic swelling under control conditions. Movie clip 2, Interneuron electroporated with Arl13b-tdTomato in SP600125-treated conditions. The cilium moves from the cell body into the trailing process under SP600125 conditions. Movie clip 3, A second interneuron expressing Arl13b-tdTomato under SP600125-treated conditions. The primary cilium moves from the cell body into the trailing process and returns to the cell body without ever entering the swelling. Open arrowheads mark Arl13b-tdTomato expressing interneurons in all clips.
Extended Data Figure 1-1
JNK inhibition results in migratory deficits of cortical interneurons regardless of average migratory speed. A, Average migratory speed of individual interneurons in control and JNK inhibited conditions. Red dashed lines highlight cells migrating at the same average speed (35–40 µm/h). Data are individual data points with 5000 bootstrap sampling distribution with the mean difference between control and SP600125-treated conditions on the right y-axis. In each condition, n = 127 cells from 11 movies obtained over four experimental days. Quantification of migratory properties in interneurons migrating at 35–40 µm/h revealed significant disruptions in maximum migration speed (B), and speed variation (C), but not displacement (D), or straightness (E) during JNK inhibition. A total of n = 13 control cells and n = 14 SP600125-treated cells collected from six to seven movies over four experimental days were within the 35–40 µm/h average speed. F–L, Quantification of nucleokinesis dynamics in control and JNK-inhibition interneurons traveling at the same average migratory speed. Cortical interneurons treated with 20 µm SP600125 have significantly shorter average swelling distances (F), smaller average translocation distances (G), no change in short translocation distances (H), and a significant reduction in large translocation distances (I) compared to controls. SP600125 had no effect on average nucleokinesis frequency (J), pause duration (K), or large translocation distance frequency (L) when compared to controls. M–O, Quantification of leading process branching dynamics in control and JNK-inhibited interneurons traveling at the same average migratory speed. Interneurons treated with SP600125 have significantly reduced growth cone split frequencies (M), with no disruptions in side branch frequency (N) or duration (O). In each condition, n = 5 cells were analyzed from five movies collected over four experimental days with n = 13 control and n = 14 SP600125 side branches. Data are presented as Gardner–Altman estimation plots (Student’s t test; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). Download Figure 1-1, TIF file.
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