The Journal of Neuroscience, May 16, 2007, ():
Zic1 and Zic3 Regulate Medial Forebrain Development through Expansion of Neuronal Progenitors
J. Neurosci. Inoue et al.
27: 5461
Supplemental Data
Files in this Data Supplement:
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Fig. S1
Location of mouse Zic1, Zic2, and Zic3 mRNAs and Zic protein in the developing hippocampus. (A-I) In situ hybridization for Zic1 (A–C), Zic2 (D–F), and Zic3 (G–I) was performed on coronal sections at E12.5 (A, D, G) and E16.5 (B, C, E, F, H, I). (C), (F), and (I) are higher magnifications of rectangle (B) or the corresponding areas in (E and H), respectively. Arrows in (G) and (I) indicate the boundaries of Zic3 expression in the cortical hem. (A, D, G) are more caudal adjacent sections of the same brain shown in Fig. 1 (A, B, E, F, I, J). (B, E, H) are caudal-level sections of the same brain shown in Fig. 1 (C, G, K). (J–L) Immunofluorescent staining of Zic proteins on coronal sections at E12.5 (J) and E16.5 (K, L). (L) is a higher magnification of the hippocampus region in (K). III, third ventricle; CA1, hippocampal field CA1; CA3, hippocampal field CA3; cpe, choroid plexus; dg, dentate gyrus; fm, fimbria; he, cortical hem; hf, hippocampal fissure; hip, hippocampus; lge, lateral ganglionic eminence; mge, medial ganglionic eminence; ncx, neocortex (cerebral cortex); th, thalamus; st, striatum.
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Fig. S2
Molecular marker expression patterns in coronal sections through thalamus of Zic1/3 mutant mice. Sections of E14.5 brains from Zic1 -/- (A–E) and Zic1/3 (Zic1 -/- Zic3 Bn/Bn) (F–J). In situ hybridization for Hes5 (A, F), Tubb3 (B, G), Nkx2.1 (C, H), Dlx2 (D, I), and Lhx6 (E, J). Dorsomedial structures (hippocampus and thalamus) are hypoplastic and partly disorganized while ventral structures are less severely affected in these sections. Asterisks in (F) and (G) indicate hypoplastic thalamus. hip, hippocampus; lge, lateral ganglionic eminence; mge, medial ganglionic eminence; ncx, neocortex; th, thalamus.
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Fig. S3
Molecular marker expression patterns showing defective septal structures in Zic1/3 mutant. Coronal sections through septum of E15.5 wild-type (Zic1+/+ Zic3 +/Y) (A–G), and Zic1/3 mutant (Zic1 -/- Zic3 Bn/Y) (H–N). In situ hybridization for Dlx1 (A, H), Dlx2 (B, I), Dlx5 (C, J), Mash1 (D, K), ER81 (F, M), Vax1 (F, M) and GAD67 (G, N). lge, lateral ganglionic eminence; ncx, neocortex; se, septum. Asterisks in (H) indicate hypoplastic septum in Zic1/3 mutant. Closed arrowheads in (H–J, L–M) indicate the altered marker expression patterns reflecting medial expansion of the septal SVZ of Zic1/3 mutant. Open arrowheads in (K) indicate dispersed Mash1 signal that may occur concomitantly with the septal SVZ expansion in Zic1/3 mutant. Signals indicated by arrows in (G, N) may represent medial septal neurons that was decreased in Zic1/3 mutant.
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Fig. S4
Proliferation and differentiation of neural progenitors in the hippocampal primordium in the Zic1/3 mutant. (A–D) Proliferation and differentiation in the hippocampal primordium in Zic1 -/- (A, C) and in Zic1 -/- Zic3 Bn/Y (B, D) embryos after 1 h pulse labeling of BrdU at E15.5. Immunofluorescence staining was performed for BrdU (A, B), and class III beta tubulin (C, D). The outlines of the hippocampus are indicated by dashed lines in (A) and (B). Regions used for quantification of BrdU-positive cells in (I) are indicated by arrows. dg, dentate gyrus; fm, fimbria; hvz, hippocampal ventricular zone. (E–H) Neuronal differentiation and cell cycle exit fraction analysis. Animals were exposed to a single-pulse of BrdU at E13.5. 24 h later (at E14.5), the animals were sacrificed. Neighboring sections were subjected to immunofluorescence staining for class III beta-tubulin (E, F), and BrdU (green) and Ki67 (red) (G, H). Class III beta tubulin stained layer was slightly thicker in Zic1/3 than in Zic1-/- (arrows in E, F). The boxed areas in (E, F) corresponds to (G, H), respectively. (I) Quantification of cell proliferation at E15.5. The numbers of BrdU-positive cells in the region of hippocampal ventricular zone (hvz) and fimbria (fm) were determined in wild-type, Zic1 -/-, Zic3 Bn/Y, and Zic1 -/- Zic3 Bn/Y (or Zic1 -/- Zic3 Bn/Y). The results are shown as mean number of multiple comparable sections with SDs. **, P < 0.01; *; P<0.05 by t-test. (J) Quantification of cell fractions exiting cell cycle. The results represented by (G, H) were quantified and used to calculate cell fractions exiting the cell cycle in the hippocampal ventricular zone. The ratio of the number of cells labeled only with BrdU (BrdU+/Ki67-, no longer dividing) to the number of double-labeled BrdU+/Ki67+ cells (yellow, re-entered cell cycle) was compared. Zic1/3 showed a significantly higher ratio than did the wild type, Zic1-/-, or Zic3 Bn/Y (or Bn/Bn). The results represent the mean ratios of the numbers of cells no longer dividing to the numbers of cells that had re-entered the cycle in the hippocampal primordium. *, P < 0.05 by t-test.
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Fig. S5
Effects of Zic3 misexpression in cerebral cortex examined at a later stage. A Zic3 expression plasmid vector, pCAG-myc-tagged-Zic3-IRES-EGFP (CAG-Zic3-IE) (D–F) or its control vector, pCAG-IRES-EGFP (CAG-IE) (A–C) was transfected by in utero electroporation into the dorsolateral cerebral cortex, where Zic3 is not expressed. Electroporation was performed at E14.5, and the fates of the transfected cells were examined at P4 by examining the GFP signals. Transfected cells were detected with immunofluorescence staining for anti-GFP (A–F), the neuronal marker MAP2 (A, D) and NeuN (B, E), and stem cell and astrocyte marker GFAP (C, F). A higher magnification of the indicated region is shown in the inset in (A), (B), and (F). While most CAG-EGFP-transfected cells (EGFP+) migrated out of VZ and were located in the CP (A–C), many of CAG-Zic3-transfected cells (EGFP+) remained in the VZ (D–F). CAG-Zic3-transfected cells in the VZ were positive for GFAP expression (inset in F). (G) Quantification of the percentage of MAP2, NeuN and GFAP-expressing cells in the transfected (EGFP+) cells. Means of three independent experiments are shown with SDs.
