|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
| ||||||||||||||||||||
Files in this Data Supplement:
Fig. S1. Leading process branching is common to many neurons in the developing brain. (A-D) Serial sections through the brain (A,B) and spinal cord (D) of Gad65-Gfp embryos at E12.5 (D) and E13.5 (A,B). In Gad65-Gfp embryos, Gfp expression is restricted to GABAergic neurons, some of which have typical morphologies of migrating cells. The schematic drawinsg in C depict the approximate localization of sections (A,B,D). (E-H) High-magnification images of cells migrating in the developing cortex (E), thalamus (F), midbrain reticular formation (G) and ventral spinal cord (H). All these cells display similar morphologies characterized by the presence of a branched leading process (arrowheads). H, hippocampus; Dh, dorsal horn of the spinal cord; dTh, dorsal thalamus; dRa, dorsal raphe nucleus; FP, floor plate; GP, globus pallidus; Hyp, hippothalamus; NCx, neocortex; NLL, nucleus of the lateral lemniscus; RF, reticular formation; RP, roof plate; Rt, reticular nucleus; SC, superior colliculus; Str, striatum; LC, locus coeruleus; Vh, ventral horn of the spinal cord. Scale bars: 300 µm in A,B,D; 10 µm in E-H.
Fig. S2. Nuclear movement in tangentially migrating interneurons. (A-A′′) An E13.5 tangentially migrating interneuron electroporated with dsRed and nuclear Gfp (nGfp). (B) Time-lapse sequence of an E13.5 dsRed-electroporated interneuron migrating from the subpallium to the cortex in a slice culture. Time is depicted in hours. Note that nuclear movement is preceded by the formation of a swelling in front of the nucleus (arrowhead). Scale bar: 10 µm
Fig. S3. Leading process branch selection during tangential migration is preceded by coordinated growth cone structural modifications. (A) Time-lapse sequence of an E13.5 dsRed-electroporated interneuron migrating from the subpallium to the cortex in a slice culture. Time is depicted in hours. Only selected frames from the entire sequence are shown. Color-coded arrowheads indicate individual growth cones tipping each of the branches of the leading process. (B,C) High-magnification images of the tips of the leading process branches shown in A, illustrating the morphology of individual growth cones as the branch growths (B) or retracts (C; t=0:30, t=0:42). (D) Drawings illustrating the morphology of the migrating neuron for each of the frames shown in A. Scale bars: 10 µm in A; 5 µm in B,C.
Fig. S4. Leading process branches do not steer much during elongation. (A) Time-lapse sequence of a dsRed-electroporated interneuron migrating from the subpallium to the cortex in a slice culture. Time is depicted in hours (h). (B) Superimposed images of the frame t=0:00 h (green) and t=0:52 h (red). Despite prominent growth, there is very little variation in the direction followed by both leading process branches. (C) Schematic of an average cell, showing the angular shift of a process between incipient (green) and final stages (red). A total of 27 processes were measured, corresponding to 14 nucleokinetic phases in 10 different cells. The average angular shift of a process during its lifetime (birth to retraction/branching) ±s.e.m. was 8.04±1.46°. The average angular shift of a process after 10-15 minutes was only 1.86±0.31°. Scale bar: 10 µm.
Fig. S5. The migration of cortical interneurons depends on ROCK function. (A) Experimental paradigm for the pharmacological inhibition of ROCK in organotypic slices. Gfp-electroporated interneurons have migrated for 12 hours before the addition of vehicle solution or inhibitor to the culture medium. (B,C) Representative examples of Gfp-electroporated interneurons migrating from the medial ganglionic eminence (MGE) to the neocortex (NCx) in organotypic slices cultured in medium containing vehicle solution (B) or the ROCK inhibitor Y27632 (30 µM) (C). After 36 hours in culture, migrating interneurons reach the cortex in large numbers in control slices than in slices treated with Y27632 (control: 75% of electroporated slices with at least 100 cells in the cortex, n=8; Y27632: 21% of electroporated slices with at least 100 cells in the cortex, n=19). (D,E) High-magnification images of neurons migrating in slices treated with vehicle solution (D) or the ROCK inhibitor Y27632 (E). Images were obtained from the boxed regions in B and C. Arrowheads indicate representative cells for each case. (D′,E′) Drawings of representative neurons migrating in slices cultures treated with vehicle solution (D′) or the ROCK inhibitor Y27632 (E′). (F) Quantification of the effect of the pharmacological inhibition of ROCK in the relative abundance of neurons with a branched leading process (n=35). χ2 test, **P<0.01. H, hippocampus; LGE, lateral ganglionic eminence. Scale bar: 300 µm in B,C; 100 µm in D,D′,E,E′.
Fig. S6. ROCK function is required for nucleokinesis in migrating interneurons. (A,B) Time-lapse sequences of Gfp-electroporated interneurons migrating in organotypic slices cultured with vehicle solution (A) or in the presence of the ROCK inhibitor Y27632 (30 µM) (B). Superimposed images of selected cumulative frames from a recording lasting 1:35 hours for both conditions. Selected individual frames are also shown for each case. Arrowheads indicate the position of the nucleus at the end of discrete active periods of nucleokinesis. (C) Quantification of the distance migrated by the nucleus in each nucleokinesis. Histograms show mean±s.e.m.: 13.66±0.75 µm (control, n=5), 13.27±0.46 µm (Y27632, n=6); t-test, P=0.412. (D) Quantification of the nucleokinesis frequency. Histograms show mean±s.e.m.: 2.91±0.31 µm (control, n=5), 0.77±0.30 µm (Y27632, n=6); t test, **P<0.01. (E) Quantification of the migration speed. Histograms show mean±s.e.m. 23.34±7.13 µm/hour (control, n=5), 12.52±4.33 µm/hour (Y27632, n=6); t-test, **P<0. 01. Scale bar: 10 µm.
Fig. S7. ROCK inhibition perturbs leading process branching in migrating interneurons. (A,B) Time-lapse sequences of E13.5 dsRed-electroporated interneurons (asterisks) migrating from the subpallium to the cortex in slice cultures. Time is depicted in hours. The broken lines indicate the position of the bifurcations from frame to frame, and each leading process branch is coded with color arrowheads. In A, vehicle solution containing Alexa 488 was applied through a glass micropipette from t=0:25 hours to t=1:25 hours, whereas in B, 3 mM Y27632 was applied through a glass micropipette from t=0:10 hours to t=1:10 hours. (C) Quantification of the relative number of branches in control and Y27632-treated neurons during micropipette application, represented in relative proportion to the initial state for each case. Histograms show mean±s.e.m. t-test, *P<0.05 (n=6). (D) Quantification of the number of new branching points in the leading process of control and Y27632-treated neurons during micropipette application. Histograms show mean±s.e.m.: 1.33±0.52 (control, n=6), 0.33±0.52 (Y27632, n=6); t-test, **P<0.01.
Movie 1. Migratory dynamics of a tangentially migrating interneuron. Migration of an E13.5 Gfp-electroporated interneuron migrating from the subpallium to the cortex in a slice culture. Images were acquired every 15 minutes. The total duration of the movie is 7:25 hours. The movie shows successive translocations of the nucleus along one of the branches of the leading process from one bifurcation point to the next one.
Movie 2. Migratory dynamics of a population of tangentially migrating interneurons. Tangential migration of Gad65-Gfp interneurons through the developing cortex in an E13.5 slice culture. Images were acquired every 15 minutes. The total duration of the movie is 6:30 hours. Interneurons moving through different cortical zones display similar cellular dynamics.
Movie 3. Branch dynamics in migrating neurons following relatively constant trajectories. Interneurons were imaged while migrating through the subpallium after 12 hours of electroporating the MGE of a E13.5 slice with dsRed. This migration is characterized by the generation of new processes forming an angle of 45° and the biased selection of one of them in every migratory cycle.
Movie 4. Branch dynamics in migrating neurons rapidly changing direction. An E16.5 Gad65-Gfp interneuron migrating through the cortical plate in a slice culture. Images were acquired every 6 minutes. The total duration of the movie is 2:18 hours. This cell (arrowhead) changes its orientation by means of a wide leading process bifurcation angle (75°; arrow).
Movie 5. Branch dynamics during rapid chemotaxis induced by Nrg1. Interneurons were imaged while migrating through the subpallium (LGE) after 12 hours of electroporating the MGE of an E13.5 slice with dsRed. The artificial gradient created with Ig-Nrg1 out of the migratory pathway induce the generation of large angles that let the cell reorients its trajectory very fast to migrate towards the pipette.
Movie 6. ROCK inhibition perturbs leading process branching in migrating interneurons. Effect of accute application of the ROCK inhibitor Y27632 in the migration of an E13.5 dsRed-electroporated interneuron in a slice culture. Images were acquired every 10 minutes. The total duration of the movie is 2:30 hours. After the application of Y-27632 (green fluorescence), one of the branches retracts, the other one extends and nucleokinesis is temporary inhibited.
| ||||||||||||||||||||
