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.