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Fig. 7. Inhibition of RA signaling alters SVZ cell morphology and impairs migration to the olfactory bulb. (A-D) Images of GFP (green) and calretinin (red) immunofluorescence double labeling after electroporating SVZ cells with EGFP (A), or EGFP plus dominant-negative (dn) RAR (B), dnRXR (C), or both dominant-negative constructs (D) as depicted in Fig. 3D. Explants were cultured for 4 DIV after electroporation and resectioned at 50 µm. After electroporation of EGFP (Con; A) or EGFP plus dnRXR (C), GFP-labeled cells appear in the SVZ and RMS and overlap with calretinin-expressing cells. Expression of EGFP with dnRAR (B) or dnRAR plus dnRXR (D) decreases the migration of GFP-positive cells; many appear in the septum and are calretinin-negative. Arrows in A-D outline the RMS. (E,F) Higher magnification images of GFP+ SVZ cells near the regions marked with arrowheads in A and B, respectively. GFP-labeled cells in the Con explant show a radial cell-like morphology (E), while those co-expressing dnRAR appear disorganized (F). (G) Quantification of migration distance from the SVZ to the most rostral GFP-positive cell in the distal RMS/olfactory bulb. Maximum migration distance was calculated by measuring the linear distance from the anterior border of the lateral ventricle to the most rostral GFP-positive cell (see inset and methods). GFP+ cells that express dnRAR or dnRAR/dnRXR migrate significantly shorter distances. (H) Quantification of GFP-positive cell number in the distal RMS and olfactory bulb (rostral to the line shown in the inset) reveals significantly fewer GFP+ cells migrating to the bulb in explants expressing dominant-negative retinoid receptors. For G and H, single asterisk denotes P<0.05, and double asterisk P<0.01. (I,J) P2 slices were electroporated with a dnRAR-IRES-GFP plasmid (J) or IRES-GFP control (I) and cultured for 4 DIV, fixed and resectioned at 50 µm. After electroporation of IRES-GFP vector alone (Vector), GFP-labeled cells migrated in the RMS toward the olfactory bulb (OB). SVZ cell expression of dnRAR-IRES-GFP (dnRAR) inhibits the rostral migration of GFP-positive cells. Arrows in I and J point to the RMS. Scale bar: in D, 200 µm for A-D; in E, 25 µm for E,F; in J, 200 µm for I,J.

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