(A–E) Quantification of the MMP activity revealed by in situ zymography performed with FITC-conjugated gelatin. (A) Cytoarchitectural organization of the E15 neocortex visualized by semi thin section of E15 neocortex stained with toluidin blue. (B, C, D) Representative microphotographs of in situ zymography performed with FITC-conjugated gelatin in control condition (B), in the presence of 1,10-phenantroline (general MMP inhibitor) (C), and in the presence of a selective inhibitor of MMP-2/-9 (D). (E) Quantification of the zymographic signals in the entire neocortex. (F–H) Co-localization of MMP-dependent gelatinolytic enzymatic activity (viewed by in situ zymography) with MAP2 in E15 neocortex. (F) The immunostaining of the dendrites with anti-MAP2, (G) the corresponding zymographic signal and (H) the overlay of the two images. (I) Higher magnification of the white selection box in the overlay. (H, I: white arrow indicates gelatinolytic activity surrounding growing dendrites). MZ: Marginal Zone; CP: Cortical Plate; SP: Subplate zone; IZ: Intermediate Zone; SVZ: Sub-Ventricular Zone; VZ: Ventricular Zone.

(A) RT-PCR analysis of MMP2 and MMP9 transcripts in E15 whole brain, E15 whole cortex and dissociated E15 cortical neurons after 2 day in culture. GAPDH housekeeping gene was used as internal control for each sample (bottom panel). B) RT-PCR analysis of Sema3A transcripts in cortical neurons. Molecular weight marker (GeneRuler™ 100 bp DNA Ladder, Fermentas) was used to check for correct transcript band (on left).

Expression pattern of MMP-2 and MMP-9 in the mouse neocortex at E15. (A–D) Microphotographs were obtained from confocal microscope analysis of the double staining (C) for MMP-2 (A), and MAP2 (as a dendritic marker) in dendrites growing in the cortical plate (B). (D) Co localization profile of MMP-2 and MAP2 signals demonstrating the presence of MMP-2 in the dendrites. (E–H) Confocal microscope images of the double staining (G) for MMP-9 (E) and MAP2 (F) in dendrites of the cortical plate. (H) Co localization profile of MMP-9 and MAP2. Here the non-superposed graphs prove the absence of co localization between MMP-9 and MAP2. White arrows point out representative line profiles allowing better visualization of double staining at the sub cellular level.

(A–B) Double staining on dissociated cortical neurons for neurofilament (in green, as an axonal marker) and MAP2 (in red, as a dendritic marker) illustrating the decrease of axonal length and the increase of dendritic length in presence (B) or not (A) of Sema3A. (C) Quantification of the dendritic growth promoting effect of Sema3A. Cortical neurons are grown without treatment or with Sema3A + a large spectrum MMP inhibitor GM6001, or selective inhibitors of MMP-2/9 or MMP-3. (* p<0.01 student's t test, ns not significant).

(A) Western Blotting analysis of MMP-2 and MMP-9 expression. The expression of MMP-2 increases in the presence of Sema3A, while no modification occurs for MMP-9. (B) Determination of MMP-2 and MMP-9 enzymatic activity by ELISA assay. MMP-2 activity increases in the presence of Sema3A without modification MMP-9 activity. (* p<0.01 student's t test/control, ns not significant).

(A) Dissociated neurons were grown without treatment or in the presence of Sema3A±blocking antibodies. The block of Neuropilin-1 (NRP1) suppressed Sema3A-induced dendritic growth. (B) Quantification of MMP-2 activity in culture media from dissociated neurons treated or not with Sema3A±anti-NRP1. The block of Neuropilin-1 (NRP1) abolished Sema3A-induced increase of MMP-2 activity. (* p<0.01 student's t test/control, ns not significant).

(A) Analysis of embryonic neurons dendritic length. Treatment of dissociated neurons with 100 ng/ml of Sema3A and/or with Gö 6976 increased dendritic length. (B) Analysis of MMP-2 activity by enzymatic ELISA assay. Sema3A treatment increased MMP-2 activity in culture media from cultured neurons. The inhibition of PKCα with 2.3 nM of Gö suppressed the increase of MMP-2 activity induced by Sema3A. (* p<0.01 student's t test/control, ns not significant).

The inhibition of PKCα blocks Sema3A-induced MMP-2 at the protein level but not at the transcriptional level. (A) RT-PCR analysis revealing no change of MMP-2 expression at the transcription level. (B) Western blot analysis revealing the lack of Sema3A-induced MMP-2 protein level augmentation in the presence of Gö 6976 (translational effect).