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First published online 15 August 2006
doi: 10.1242/jcs.03080

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Mechanism of polarized protrusion formation on neuronal precursors migrating in the developing chicken cerebellum

Department of Cell Biology, University of Virginia, P.O. Box 800732, 1300 Jefferson Park Avenue, Charlottesville, VA 22908-0732, USA

View larger version (50K): [in a new window]   Fig. 1. In situ imaging of chicken embryonic neuronal migration. (A) Image of stage 28 chicken embryo. Organotypic explants were prepared from embryonic cerebellum (CB). (B) Experimental design for electroporation. A cerebellar explant is placed on the culture insert membrane with its pial surface facing the membrane insert. In this photograph, rostral is on top. The cathode (-) was placed near the left side of the cerebellar rhombic lip. The anode (+) was touched to the right side of the rhombic lip. 2 µl of plasmid solution were applied between the cathode and rhombic lip (arrow), followed by electroporation five times using 30 V pulses for 50 milliseconds at 100 millisecond intervals. (C) Schematic view of in situ imaging of neuronal precursors migrating within the organotypic cerebellar explant. An open-book style flat-mount explant was prepared from stage 28 chicken embryonic cerebellum; cross section images of the cerebellar tissue and explant are shown (top and bottom). The explant was electroporated as shown in Fig. 1B. Fluorescent images of GFP-expressing cells were acquired from the pial-side of the explant through the culture insert and dish by using a 20x long-working-distance objective lens (bottom). (D-E) In situ imaging of cultured cerebellar explants. Bright field (D) and EGFP fluorescence (E) images of 24-hour cerebellar explant cultures are shown. In these micrographs, the left margin of the explant is the rhombic lip, the ventral midline is along the right end of the micrographs, and rostral is to the top. Neuron-specific -tubulin promoter-driven EGFP fluorescence is detected around the electroporated rhombic lip. Rhombic-lip-derived EGFP-positive cells have an elongated, single long leading process directed toward the ventral midline. (F) Fluorescent time-lapse imaging of leading protrusions elongating from rhombic-lip-derived neuronal precursors expressing EGFP. Fluorescent images were acquired every 2 minutes for 2 hours (see supplementary material Movie 1). Images at 0, 40, 80, 120 minutes are shown. In these micrographs, the rhombic lip is to the left, the ventral midline is to the right, and rostral is at the top. The same orientation is used in all the following micrographs and movies. The leading protrusions from EGFP-expressing cells show highly directed extension toward the ventral midline. Bar, 2 mm (A,B); 200 µm (D,E); 10 µm (F).

View larger version (22K): [in a new window]   Fig. 2. Simplified network of putative signaling intermediates that regulate cell polarization and protrusion formation in directionally migrating cells. Regardless of the receptor type, localized activation of receptor by stimulant is thought to elicit subsets of signaling events similar to those depicted here and regulate cell polarization and directed protrusion formation.

View larger version (105K): [in a new window]   Fig. 3. Functional analysis of Cdc42 signaling and microtubules in polarized protrusion formation of directionally migrating neuronal precursors. (A) Dominant-negative mutant of Cdc42 (N17-Cdc42) was co-expressed with EGFP in rhombic-lip-derived neuronal precursors. EGFP fluorescent images were captured every 2 minutes for 2 hours (see supplementary material Movie 3), and images from 0, 40, 80, 120 minutes time points are displayed. N17-Cdc42-expressing cells showed a bipolar morphology and some were rounded up. (B) Function-interfering mutant of Par6 (Par6-NT) altered orientation of TLPs and frequently induced branching (arrowheads) (see supplementary material Movie 4). (C) Neuronal precursors expressing a constitutively active mutant of GSK3ß (S9A-GSK) showed a branching phenotype that was somewhat weaker than that seen in Par6-NT-expressing cells (arrowheads). (D) Microtubule binding-deficient mutant of APC (APCCT) produced branched TLPs (arrowheads). (E) Cerebellar explants electroporated with EGFP were pre-cultured for 24 hours before treatment with 5 µM nocodazole (microtubule-depolymerization drug), for 15 minutes, followed by in situ fluorescent imaging. The same drug-containing medium was used during the time-lapse imaging. Disruption of microtubule structure by nocodazole induced fragmentation of leading processes (see supplementary material Movie 5). In some cases, altered orientation of TLPs were observed (arrows). (F) Fluorescent images of leading processes visualized by microtubule plus-end binding protein EB3-GFP. EB3-GFP showed a broad localization that increased toward the TLPs. These processes displayed non-directional extension of the polarized process (see supplementary material Movie 6). Bar, 10 µm.

View larger version (112K): [in a new window]   Fig. 4. Rac is required for generation of the protrusion on the tip of the leading process. (A) Dominant-negative mutant of Rac (N17-Rac) was co-expressed with EGFP in cerebellar rhombic-lip-derived neuronal precursors. EGFP fluorescent images were captured every 2 minutes for 2 hours (see supplementary material Movie 7), and images at 0, 40, 80, 120 minutes are shown. N17-Rac suppressed the formation of the protrusion on the TLPs and inhibited their extension. (B) Overexpression of Rac induced the formation of multiple protrusive structures at the TLP, whose extension was inhibited (see supplementary material Movie 8). Bar, 10 µm.

View larger version (91K): [in a new window]   Fig. 5. PIP3 signaling controls direction and magnitude of protrusion formation. (A) Class IA PI3K mutant, p85, was co-expressed with EGFP in cerebellar rhombic-lip-derived neuronal precursors. EGFP fluorescent images were captured every 2 minutes for 2 hours (see supplementary material Movie 9), and images at 0, 40, 80, 120 minutes are shown. p85-expressing neuronal precursors do not form long leading process and show random migration. Since these cells no longer extend their leading process directionally toward the ventral midline, images of cell soma-rich region near the rhombic lip are shown. (B) Dynamics of Class IB PI3K mutant, KD-p110-expressing neuronal precursors visualized with co-expressed EGFP. These cells do not show any apparent defect in directed extension of leading processes (see supplementary material Movie 10). (C) Overexpression of PTEN reduced the protrusive activity on the tip of the leading processes. However, it had no apparent effect on rate and direction of process extension. (D) Phosphatase-dead PTEN mutant, C124A-PTEN, upregulated the protrusive activity at the process tips. This significantly perturbed the extension of leading process (see supplementary material Movie 11). Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 6. PAK, Rho and actomyosin system regulate spreading of protrusions. (A) A kinase-dead mutant of PAK (KD-PAK) was co-expressed with EGFP in cerebellar rhombic-lip-derived neuronal precursors. EGFP fluorescent images were captured every 2 minutes for 2 hours (see supplementary material Movie 12), and images at 0, 40, 80, 120 minutes are shown. KD-PAK created unstable branches (arrowheads) and caused frequent changes in the growing direction of the leading process. (B) Overexpression of PAK induced a large, spread growth-cone-like protrusion at the TLPs and inhibited their extension. These large protrusions were relatively stable (see supplementary material Movie 13). (C) Cerebellar explants electroporated with EGFP were pre-cultured for 24 hours, before treatment with 100 µM blebbistatin, a myosin II ATPase inhibitor, for 15 minutes. Inhibition of myosin II reduced formation of spread lamella on the TLPs and decreased the rate of their extension (see supplementary material Movie 14). (D) C3-toxin-expressing neuronal precursors showed reduced spreading of leading protrusions. Termini of their leading process often displayed an angular shape and irregularly extended protrusion in non-directional manner (arrows; see supplementary material Movie 15). (E) Expression of DN-Rho-kinase reduced spreading of leading protrusions and perturbed their coordinated extension. Bar, 10 µm.

View larger version (98K): [in a new window]   Fig. 7. ErbB4 signaling is essential for the extension of leading process on migrating cerebellar rhombic lip cells. (A) Cerebellar explants electroporated with EGFP were pre-cultured for 24 hours, before treatment with 100 µM AG1478, an RTK inhibitor, for 2 hours, followed by fluorescent in situ imaging. Fluorescent images were captured every 2 minutes for 2 hours, and images from 0, 40, 80, 120 minute time points are shown. Inhibition of RTK suppressed the protrusion formation at the TLP. Bar, 10 µm. (B) Expression of dominant-negative mutant ErbB4IC inhibited extension of TLPs (see supplementary material Movie 16).

View larger version (27K): [in a new window]   Fig. 8. Schematic summary of morphological changes on polarized processes of neuronal precursors observed in situ. (A) Control EGFP-expressing cells elongated a long leading process. The tip of the process formed protrusions in a spatially restricted manner. (B) Expression of N17-Cdc42 induced retraction of the major existing process and abrogated the monopolar morphology. (C) Perturbation of polarity effectors of Cdc42 (Par6-NT, S9A-GSK, APCCT) generated branched protrusions on the terminus of the leading process. Expression of KD-PAK also induced branching of the terminal protrusion. The duration of these branches were short; but the resulting processes often displayed a curved shape at the points the branches formed. (D) Depolymerization of microtubules by nocodazole caused fragmentation of the leading process. Altered orientation of TLP was often observed during the fragmentation. (E) Overexpression of microtubule plus-end binding protein EB3-GFP induced non-directional extension of the leading process. (F) Dominant-negative mutant of class IA PI3K (p85 PI3K) promoted random migration of rhombic-lip-derived cells. This mutant disturbed formation of long leading process as well as perturbed directional migration. (G) Kinase-dead mutant of class IB PI3K (KD-p110 PI3K) affected directional extension but somewhat reduced the size of TLP. Overexpression of PTEN also reduced the size of TLP. The resulting TLPs extended normally compared with control EGFP cells. (H) Inhibition of actomyosin contraction by blebbistatin reduced the degree of spreading of TLP. Perturbation of the Rho-Rho-kinase pathway by expression of C3 toxin or DN-Rho-kinase also suppressed spreading of TLP. (I) C124A-PTEN increased the terminal spreading of leading process. Overexpression of PAK also enhanced the terminal protrusion and resulted in a well-developed growth cone-like structure. (J) Overexpression of Rac produced an aberrant multiply protrusive structure on the TLP. This structure displayed rapid extension and retraction of microspikes rather than lamella. (K) Perturbing the function of Rac (N17-Rac) as well as RTK (AG1478, ErbB4IC) suppressed generation of protrusions at the TLP.

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