Fig. 1. Laminar formation of the tectum. (A) Laminar formation by early and late migratory cells. (B) Two hours after electroporation with lacZ vector at E5. (Upper panel) Immunohistochemistry with anti-lacZ. (Lower panel) Immunohistochemistry with anti-Hu-C/D. Hu-positive early neurons accumulate in the upper layer of the ventricular layer before migrating into their destination, so this layer could be distinguished as the early neuronal layer (*). lacZ staining is observed in the neuroepithelial layer, not in the early neuronal layer at 2 hours after electroporation. NE, neuroepithelial layer. (C) Horizontal section of an E6 tectum that was labeled at E3. DAPI staining (left panel) reveals laminae I and II and the ventricular layer. In the ventricular layer, the early neuronal layer could be distinguished (*). Cells labeled at E3 migrate out of the neuroepithelium and make up lamina II and I by E6. (D,H) DAPI staining of E8 (D) and E12 (H) tecta to show laminar formation. (E-G) Distribution of lacZ-positive cells at E8. Electroporation with pMiwZ was carried out at E3 (E), E6 (F) and E5 (G). Left panel shows lacZ staining, and right panel shows the number of lacZ-positive cells in each lamina. (I,J) Distribution of lacZ-positive cells at E12. Electroporation with pMiwZ was carried out at E3 (I) and E6 (J). Left panel shows lacZ staining, and right panel shows the number of lacZ-positive cells in each lamina. As the cells labeled at E6 migrated into lamina IId (F), these cells (late migratory cells) may have divided the laminae that had been formed by early migratory cells (C). Thus, the laminae of early migratory cells are split into two (E): upper laminae (laminae IV-IIu) and deeper lamina (lamina I). Distribution of late migratory cells at E8 and E12 indicates that lamina IId at E8 is remodeled to laminae h and i/j of the SGFS (F,J). E5 is the transitory stage and the ventricular layer may be composed of a mixture of the progenitors of early and late migratory cells. Thus, the cells labeled at E5 showed ubiquitous distribution (G). Scale bars: 100 µm.

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