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First published online May 23, 2008
doi: 10.1242/10.1242/dev.016725

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C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment

¹ Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.
² Department of Medical Biology, University of Melbourne, Parkville 3010, Victoria, Australia.
³ Howard Florey Institute, Parkville, 3010 Victoria, Australia.

View larger version (90K): [in this window] [in a new window]   Fig. 1. C3G expression and histology of the developing cerebral cortex of the C3Ggt/gt mutant. (A-F) In situ hybridisation using a C3G-specific cRNA probe (A,B,D,E) or sense control probe (C,F) on adjacent sections of wild-type mouse cerebral cortex at E12.5 (A-C) and E15.5 (D-F). Bright-field (A,D) and corresponding dark-field (B,E) images. Silver grains representing C3G mRNA distribution appear white in the dark-field image (B,E,C,F). (G-N) Haematoxylin and Eosin stained paraffin sections of wild-type (G,I,K,M) and C3Ggt/gt mutant (H,J,L,N) developing cerebral cortex at E12.5 (G,H), E13.5 (I,J) and E14.5 (K-N). Images were taken using differential interference contrast optics, which reveal unstained or weakly stained structures. Arrows in G indicate basement membrane and arrowheads in H the lack thereof. Stippled line in I indicates the demarcation between the neuroepithelium and the pericerebral tissue; arrowheads in J indicate neuroepithelial cells invading the pericerebral space. Dashed line in M indicates ventricular-to-pial orientation of cortical plate cells. CNE, cortical neuroepithelium; CP, cortical plate; IZ, intermediate zone; LV, lateral ventricle; MZ, marginal zone; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone. Scale bar: 197 µm in A-C; 94 µm in D-F; 13 µm in G,H; 32 µm in I,J; 38 µm in K,L; 18 µm in M,N.

View larger version (68K): [in this window] [in a new window]   Fig. 2. Abnormal cortical plate development in C3Ggt/gt mutant mouse embryos. (A-I) Wild-type (A,C,F,H) and C3Ggt/gt mutant (B,D,E,G,I) developing cerebral cortex, at E13.5 and E14.5 as indicated, stained for the neuronal marker Map2 (brown in A,B; green in F-I) and/or laminin (red in C-I) and counterstained with bisbenzimide (blue in C-I). C3Ggt/gt mutants show disorganisation of the cortical plate and invasion of the pericerebral space by neurons (arrow in B). E13.5 developing cortex shows continuity of laminin staining along the basement membrane (asterisk) in the wild type (C) and the disorganisation (D) and discontinuity (arrows in E) of laminin staining in the E13.5 C3Ggt/gt mutant, as well as nuclei (blue) of cells protruding into the basement membrane gaps (arrow in E). The cells protruding into the basement membrane gaps are positive for the neuronal marker Map2 (arrows). Labels as in Fig. 1. Scale bar: 29 µm in A,B; 35 µm in C-I.

View larger version (112K): [in this window] [in a new window]   Fig. 3. The developing cerebral cortex of the C3Ggt/gt mutant shows a failure of preplate splitting. (A,B) Confocal z-stack reconstruction of frozen sections of developing cerebral cortex of wild-type (A) and C3Ggt/gt mutant (B) mouse embryos treated with BrdU at E10.5 and recovered at E14.5. Note BrdU-positive cells on the pial side (arrows in A) and below (arrowheads in A) the early cortical plate in the wild type. By contrast, in the C3Ggt/gt developing cortex, BrdU-positive cells are almost exclusively found near the pial surface (arrows in B) and not in the area where the subplate would be expected (asterisk, B). (C,D) Calretinin staining (brown) of marginal zone cells (arrows) and subplate cells (arrowheads) in the wild-type (C) and C3Ggt/gt mutant (D) developing cortex. Note the absence in the mutant of subplate cells in a position below the marginal zone where the subplate would be expected (asterisk in D). (E,F) Developing cerebral cortex of wild-type (E) and C3Ggt/gt mutant (F) embryos treated with BrdU at E12.5 and recovered at E14.5. Note the position of the labelled wild-type cells of the future layer VI in the nascent cortical plate (arrows in E) in contrast to the disorganised position of the C3Ggt/gt mutant cells (arrows in F). Note that in this experiment some preplate cells were also labelled. Labels as in Fig. 1. Scale bar: 21 µm in A; 23 µm in B; 26 µm in C,D; 17 µm in E,F.

View larger version (126K): [in this window] [in a new window]   Fig. 4. Abnormal distribution of the basement membrane and of RC2-positive radial glial cells in the developing cortex of the C3Ggt/gt mutant. Epifluorescence (A,B,G,H) and confocal z-stack reconstructions (C-F) of frozen sections stained for laminin (red) and RC2 (green). Wild-type (A,C,E,G) and C3Ggt/gt mutant (B,D,F,H) mouse developing cerebral cortex at E12.5 to E14.5 as indicated. Wild-type radial glial processes (A,C,E,G, green, arrowheads in E) overlap (yellow, arrows in E) with basement membrane laminin (red). C3Ggt/gt mutant radial glial processes are entangled (B,D,F,H, long arrows in F) and show little overlap with laminin (short arrow in F). Asterisk, blood vessel; PS, pial surface; other labels as in Fig. 1. Scale bar: 25 µm in A,B; 249 µm in C; 242 µm in D; 137 µm in E; 141 µm in F; 11 µm in G,H.

View larger version (126K): [in this window] [in a new window]   Fig. 5. Abnormal distribution of reelin-expressing marginal zone cells in the developing cortex of the C3Ggt/gt mutant. (A,B) Confocal images of frozen sections stained for reelin (green) and counterstained with bisbenzimide (blue). (C,D) Differential interference contrast images of paraffin sections stained for calretinin (brown). (E-J) Epifluorescence images of frozen sections stained for reelin (green), laminin (red) and counterstained with bisbenzimide (blue). Wild-type (A,C,E,G,I) and C3Ggt/gt mutant (B,D,F,H,J) mouse developing cerebral cortex at E12.5 to E14.5 as indicated. Note that cells positive for reelin (A,B) and calretinin (C,D) are evenly distributed in the wild-type (arrowheads in A,C), but not in the C3Ggt/gt mutant (arrowheads in B,D) developing cortex. Also note that the gaps in reelin-positive cells coincide with gaps in laminin distribution (F,H,J, arrows). Scale bar: 25 µm in A-J.

View larger version (56K): [in this window] [in a new window]   Fig. 6. C3Ggt/gt mutant cortical neurons fail to migrate in ex vivo brain slice cultures. Confocal microscopy (A,B,E-K) and epifluorescence images (C,D) of wild-type (A,C,E,G) and C3Ggt/gt mutant (B,D,F,H-K) mouse forebrain slices. Brain slices were recovered at E12.5. Proliferating neural precursor cells were infected with eGFP-expressing retrovirus to mark (green) proliferating cells and their differentiating and migrating progeny. Brain slices were cultured for 48 hours and then visualised by fluorescence microscopy. (A,B) Frozen sections of brain slices counterstained with TRITC-phalloidin marking filamentous actin reveal the structure of the cortex primordium. Note that wild-type, but not C3Ggt/gt mutant, forebrain slices developed a cortical plate (A, versus arrows in B) and that wild-type (arrows in A), but not C3Ggt/gt mutant (arrowheads in B), neurons migrated away from the ventricle. Wild-type neurons exhibited long radial processes (arrowheads in E,G), whereas C3Ggt/gt mutant neurons had multiple short processes (arrowheads in D,F,H-K). For time-lapse, see Movies 1, 2 in the supplementary material. Dashed line, ventricular surface; CP, cortical plate; LV, lateral ventricle; PS, pial surface. Scale bar: 141 µm in A,B; 73 µm in C,D; 96 µm in E,F; 37 µm in G; 27 µm in H; 18 µm in I; 29 µm in J,K.

View larger version (79K): [in this window] [in a new window]   Fig. 7. C3Ggt/gt mutant cortical neuroepithelial cells fail to extend processes and fail to migrate on complex or defined matrix in vitro. (A-N) Cortical neuroepithelial explants of wild-type (A,C,E,G,I-K) or C3Ggt/gt mutant (B,D,F,H,L-N) E10.5 mouse embryos were plated on complex extracellular matrix (A-D,I-N) or recombinant laminin (E-H) and cultured; (A,B) day 3 of culture, (C-H) day 6, (I-N) day 3 to day 4. For time-lapse, see Movies 3, 4 in the supplementary material. (G,H) Cultures on laminin were stained for the neuronal marker type III β-tubulin (red) and counterstained with bisbenzimide (blue). Differential interference contrast images (A-F), epifluorescence images (G,H) and time-lapse phase-contrast images (I-N). (A,C,E,G,I) Processes and migrating neurons were seen in wild-type (arrowheads) but not in C3Ggt/gt mutant cultures. Dashed line, migration front. Scale bar: 72 µm in A-D; 37 µm in E-H; 70 µm in I-N.

View larger version (29K): [in this window] [in a new window]   Fig. 8. C3G activation in response to reelin. (A) Reelin is present in the supernatant of 293T cells transfected with a reelin expression construct, but not in the supernatant of cells transfected with an empty vector (mock). (B) Immunoblot probed for tyrosine phosphorylated protein (pC3G) and reprobed for total C3G (lower panel) in lysates of E16.5 wild-type cortical neurons treated with supernatant of reelin-expressing or mock transfected 293T cells for 15 minutes and precipitated with anti-C3G antibody. Numbers below lanes indicate relative densitometry readings corrected for total C3G. (C) Immunoblot probed for Rap1 in lysate of neural precursor cells treated with reelin or control supernatant (mock) for 24 hours, then subjected to RalGDS-Rap1-binding domain affinity purification of GTP-loaded Rap1. Numbers below lanes indicate relative densitometry readings.

View larger version (59K): [in this window] [in a new window]   Fig. 9. Comparison of the C3Ggt/gt mutant phenotype with the Reeler phenotype and the nervous-system-specific β1 integrin conditional mutant phenotype. (A) At E14.5, the developing wild-type mouse cortex consists of ventricular (VZ), subventricular (SVZ) and intermediate (IZ) zones, subplate (SP), the nascent cortical plate (CP), marginal zone (MZ) and has a continuous basement membrane (BM) at its pial surface. Cajal-Retzius (CR, green) cells expressing reelin are located in the marginal zone. Radial glial processes (RGP) extend from the ventricular zone to the basement membrane at the pial surface, where they are anchored with endfeet to the extracellular matrix protein lattice. Migrating bipolar neurons (biN) are guided by radial glial processes and may transiently become multipolar (muN). (B) β1 integrin deficiency in the brain leads to a disruption of the basement membrane, loss of parallel radial glial process orientation and loss of endfeet anchoring, as well as an undulating cortical plate (U-CP) with invasion of the marginal zone by cortical plate cells (Graus-Porta et al., 2001). (C) Loss of reelin leads to a lack of reelin expression in the Cajal-Retzius cells and to a failure of cortical plate cells to split the preplate (PP) into marginal zone and subplate, resulting in an unsplit preplate (US-PP) and the stacking of cortical plate cells (CPC) below the unsplit preplate (Sheppard and Pearlman, 1997). In addition, lack of reelin leads to oblique orientation of the radial glial processes. (D) The C3Ggt/gt mutant phenotype combines all elements of the Reeler phenotype and the β1 integrin mutant phenotype: lack of preplate splitting, the stacking of cortical plate cells below the unsplit preplate, disruption of the basement membrane, invasion of the marginal zone and pericerebral space by cortical plate cells, loss of radial glial process orientation and loss of endfeet anchoring. In addition, lack of C3G leads to an accumulation of migrating neurons in the multipolar stage.

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