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First published online 19 May 2004
doi: 10.1242/dev.01162

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Distinct roles of Rac1/Cdc42 and Rho/Rock for axon outgrowth and nucleokinesis of precerebellar neurons toward netrin 1

Frédéric Causeret^1,*, Matias Hidalgo-Sanchez^1,*, Philippe Fort², Stéphanie Backer¹, Michel-Robert Popoff³, Cécile Gauthier-Rouvière² and Evelyne Bloch-Gallego^1,

¹ Institut Cochin, GDPM, INSERM U567, CNRS 8104, Université Paris V, CHU Cochin, 24 rue du Faubourg Saint Jacques, 75014 Paris, France
² CRBM/CNRS FRE2593, 1919 Route de Mende, 34293 Montpellier Cedex, France
³ Institut Pasteur, Unité des Bactéries Anaérobies et Toxines, 28 rue du Dr Roux, 75724 Paris Cedex 15, France

View larger version (70K): [in a new window]   Fig. 2. Rac and Cdc42 GTPases play an essential role in axon outgrowth of PCN toward a netrin 1 source. E11 rhombic lip explants were faced with netrin 1-secreting cells (Net) and cultured for 3 days. Axon outgrowth and nuclear migration were then analyzed after Tuj1 and DAPI staining, respectively. In control conditions, axon growth developed mainly toward the netrin 1 source (A) and nuclear migration occurred almost exclusively within the netrin 1-attracted neurites (C). Addition of 1 ng/ml of lethal toxin, to specifically inhibit Rac and Cdc42, resulted in a severe impairment of axon growth (B) but did not affect nuclear migration (D). Measurement of the surface covered by migrating nuclei (E), or quantification of their number (F), represented as cumulative distributions and histograms (mean±s.e.m.), revealed no significant difference between control (n=24) and drug-treated (n=24) explants. (G) Axon outgrowth toward (proximal) and away (distal) from the netrin 1 source (proximal and distal quadrants are represented in A; *P<0.001 compared with control; error bars represent s.e.m.). (H) Migration/outgrowth ratio in control and drug-treated explants (*P<0.001). (I,J) High magnification of axons stained with rhodamine-conjugated phalloidin to visualize F-actin structures. Whereas control growth cones showed F-actin enrichment (arrows in I), lethal toxin-treated axons lacked phalloidin staining at their distal tip (arrows in J). Scale bars: A, 500 µm (for A,B); C, 200 µm (for C,D); I,J, 10 µm.

View larger version (94K): [in a new window]   Fig. 1. Expression of Cdc42, Rac1 and Rhoa/b in mouse at embryonic day 11-13. At E11, Cdc42, Rac1 and Rhoa expression was located in the ventricular (vz in A; short arrows in A',C,E) and subventricular zone (svz in A; large arrows in A',C,E), whereas Rhob transcripts were present in the early migratory stream in the marginal zone (mz in A; short arrows in G,G'), as well as in the submarginal zone (smz in A; large arrows in G,G'). At E13, Cdc42, Rac1 and Rhoa/b genes were strongly expressed by migrating LRN located in the marginal stream (arrowheads in B,D,F,H). The ION (asterisks in B,D,F,H) showed strong levels of Cdc42 and Rhob expression (B,H), and lower levels of Rac1 and Rhoa expression (D,F). In addition, all four transcripts were present in the developing floor plate (fp; A-H). The roof plate (rp) at E11-E13 and the ventral ventricular zone of the hindbrain at E13 (arrows in B,D,F) showed different levels of Cdc42, Rac1 and RhoA expression. Note the presence of Rhob transcripts in the hypoglossal nucleus (XII; G,H). (I,J) Western blot illustrating the presence of Cdc42, Rac1 and RhoA proteins in rhombic lip at E11 (delimited ventrally by dotted line in A) and E12 (I), as well as in the LRN and ION domains (into the dotted square in B) at E13 (J). (K) Schematic drawings of the spatio-temporal changes in the Cdc42, Rac1 and Rhoa/b expression at E11 and E13. Scale bars: in A, 200 µm for A,C,E,G; in A', 140 µm; in B, 400 µm for B,D,F,H.

View larger version (62K): [in a new window]   Fig. 3. RhoA/B/C GTPases are strictly required for nuclear migration to occur. (A) RhoA modification by TAT-C3 through ADP-ribosylation at E11. In vitro ADP-ribosylation of explants pre-treated with TAT-C3 showed that the radiolabeled Rho band was markedly decreased, indicating that TAT-C3 efficiently modified Rho substrate in neurons. (B,C) When E11 rhombic lip explants were treated with 20 µg/ml TAT-C3, neurites lost their preferential orientation toward the netrin 1 (Net) source (B) and cell nuclei failed to translocate within those neurites (C). (D,E) Quantification analysis of (D) migration (cumulative distributions and histograms) and (E) axon outgrowth (n=13 in TAT-C3-treated explants; n=19 in control explants). (F) Migration/ougrowth ratio of control and TAT-C3-treated explants. Error bars in D, E and F represent s.e.m. *P< 0.001. Scale bars: B, 500 µm; C, 200 µm.

View larger version (45K): [in a new window]   Fig. 4. TAT-C3 treatment affects axonal morphology. The individual morphology of neuronal processes from E12 explants could be visualized after GFP electroporation. Control neurons showed straight axons (A), whereas TAT-C3-treated neurons exhibited tortuous axons (B). For quantification analysis, the trajectory of entire axons in control (C, n=11) and TAT-C3 treated explants (E, n=11) was drawn, and the mean deviation from a straight line measured (D). Data are presented using arbitrary units; *P<0.001. Scale bar in A: 100 µm for A,B.

View larger version (57K): [in a new window]   Fig. 5. Localization of active Rho GTPases in vivo at E11-E13. (A) Rhombic lips from E11 were lyzed and incubated with GST-RBD-Rhotekin protein. GTP-bound RhoA (active) was detected by western blot using an anti-RhoA antibody and compared with total RhoA contained in cell lysates before the incubation with GST-RBD-Rhotekin. (B) Section through the caudal hindbrain at E11 after incubation with RBD-Rhotekin fused to the GST and then treatment with an anti-GST antibody. A strong RhoA/B/C GTPase activity was observed in the ventricular zone (vz) and the dorsal border (arrow) of the rhombic lip. The marginal (mz) and submarginal (smz) zones also showed GST-RBD-Rhotekin labeling. No staining could be observed in the subventricular zone (svz). (C) Higher magnification of image in B, showing scattered GST-RBD-Rhotekin-positive cells (arrowheads) leaving the ventricular zone (large arrow) toward the early migrating stream (short arrows). (D) At E13, migrating LRN located in the marginal stream showed strong GST-RBD-Rhotekin binding (arrowhead). ION (asterisk) reaching the vicinity of the floor plate showed faint Rho GTPase activity. (E,F) Control experiments (CTRL) were performed with a non-relevant GST-tagged protein (M-Cadherin) at E11 (E) and E13 (F). No staining could be observed. (G) The expression patterns of RhoA/B GTPases and active RhoA/B proteins at E11 and E13. Scale bars: B,E, 200 µm; C, 500 µm; D,F, 260 µm.

View larger version (54K): [in a new window]   Fig. 6. Expression of Rock in mouse at E11-E13. At E11 (A,A'), Rock1 mRNA could be detected by in situ hybridization in the ventricular (short arrow) and subventricular zone (large arrow) of the rhombic lip. Note the presence of Rock1-positive cells in the early migratory pathway (arrowheads in A'). At E13 (B), Rock1 expression could be detected in ION (asterisk) and in migrating LRN (arrowhead). The floor plate (fp) showed strong Rock1 expression (A,B). (C,D) Western blot illustrating the presence of Rock1 and Rock2 proteins in rhombic lip at E11 and E12 (C), as well as in the LRN and ION domains at E13 (D). Scale bars: A, 180 µm; A', 90 µm; B, 100 µm.

View larger version (67K): [in a new window]   Fig. 7. Rock1/2 inhibition impairs the orientation of axon outgrowth toward netrin 1 and the nuclear migration of PCN. (A) When E11 rhombic lip explants were exposed to 20 µM Y-27632 to inhibit Rock1/2 signaling, axons exhibited a tortuous aspect and grew toward and away from the netrin 1 source (Net). (B) Nuclear migration within those axons failed to occur. (C,D) Quantification revealed a gradual inhibition of nuclear migration from 5 to 100 µM Y-27632 (C; *P<0.001; P<0.01 compared with control), and a significant increase in proximal and distal axon growth at 20 µM (D; *P<0.001; P<0.01 compared with corresponding orientation in control; n=42 control, n=24 Y-27632 treated explants). (E) Migration/ougrowth ratio of treated and control explants (*P<0.001 compared with control). Error bars in C, D and E represent s.e.m. Scale bars: A, 500 µm; B, 200 µm.