Fig. 3. Effects of Grg4 misexpression on tectal laminar formation. (A-G) Tecta at E12. (A) Low-power micrograph of an RCAS-AP-misexpressing tectum, which served as the control. Immunohistochemistry is with anti-gag antibody. The transfection sites are indicated by brackets. (B,B') RCAS-Grg4-misexpressing tectum. In situ hybridization for Grg4 (B) and DAPI staining (B'). (C,D) DAPI staining of the control (C) and Grg4-misexpressing (D) tecta. (E) Higher magnification of Grg4 expressing site in E. (B,B',D,E,G) are from adjacent sections of the same tectum. On B and B', the site that corresponds to D and E is indicated by arrows. (F,G) Immunohistochemistry with anti-neurofilament antibody on the control (F) and Grg4-misexpressing tecta (G). (H-T) E14 tecta. Immunohistochemistry with anti-PV (H,I), anti-Glutamate (K,L) and anti-NgCAM antibodies (N,O). In situ hybridization for ER81 (Q,R) and for Lim1 (S,T). Merged figures of Grg4 and ER81 (R') and Grg4 and Lim1 (T') at the site indicated by brackets on R and T, respectively. Grg4 misexpression was assessed immunohistochemically with antibody against the HA tag (J,M,P,R',T'). Control (H,K,N,Q,S) and Grg4-misexpressing site (I,J,L,M,O,P,R,T). L and M are the same field of the same section. Grg4 expressing site is indicated in L. In Grg4-misexpressing regions, lamina g seemed to be reduced (D), but lamina i/j was increased (D,G). Arrowheads in G indicate radial neuronal fibers. Expression of PV (H) indicates that lamina g is specifically disrupted by Grg4 misexpression (I). Cells that express glutamate in SGC and SAC are reduced at the Grg4-misexxpressing site (right hand side of L, where Grg4 misexpressing site is indicated as Grg4). ER81 and Lim1, which are normally expressed in lamina i/j, are expressed in deeper layers in the Grg4-misexpressing region (Q-T). Scale bars: 100 µm for A-T; 20 µm for G'.

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