Fig. 5. Grg4 instructs the tectal postmitotic cells to take a late migratory pathway. (A) Control E8 tecta in which RCAS-AP was electroporated at E2. (B) Grg4 misexpression by RCAS-Grg4. (C) Application of morpholino antisense oligonucleotide for Grg4 at E5. (D) Grg4-5' misexpression by RCAS-Grg4-5'. (E) En2 misexpression by RCAS-En2 infection. (F) Relative cell numbers of Sox2- and Sox14-positive cells. In A-E: (a) DAPI staining, (b) in situ hybridization for Sox2, (c) in situ hybridization for Sox14, (d) in situ hybridization for ER81. (A, part e; B, part e) Immunostaining for PV. (B, part f) Low-power micrograph to show Grg4 misexpression. (C, part e) immunostaining for FITC to show FITC-conjugated morpholino. (D, part e) Immunostaining for HA Tag. Immunostaining for En2. In the control (A), Sox2 is expressed in lamina IIu and the ventricular layer (VL). Sox14 is expressed in laminae IV and IId. ER81 is expressed specifically in lamina IId. PV is expressed in laminae III-II at E8. In the Grg4-misexpressing region, reduction of lamina I, IIu and IV is assessed from the expression of Sox2, PV and Sox14 (B, part a-c; B, part e). Treatment with morpholino antisense oligonucleotide for Grg4 exerted reverse effects: expansion of lamina IIu (C, part b), reduction of lamina IId (C, parts c,d). Grg4-5' acted as a dominant-negative form for Grg4; expansion of lamina I and IIu (D, part b), reduction of lamina IId (D, parts c,d). En2 misexpression also resulted in the reverse effects to Grg4: expansion of lamina IIu (E, part b), reduction of lamina IId (E, parts c,d). For quantification, percent of Sox2- and Sox14-positive cells around the lamina II in total cells of mantle layer was calculated (F). The number of cells in a rectangle, one side of which is 100 µm and parallel to the ventricular surface, was counted on four samples from two embryos each. The graph shows that Grg4 forces tectal postmitotic cells to follow a late migratory pathway. Error bars represent s.e.m. Asterisks indicate significant differences from the control (single asterisk, P<0.05; double asterisks, P<0.005). AP, control AP-transfected tecta; Grg4, Grg4 misexpressing tecta; Mor, tecta treated with morpholino antisense oligonucleotide. Scale bars: 100 µm.

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