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Figure 2


Fig. 2. Fgf8 electroporation expands the specific population posterior to ZLI in the early embryonic stage. (A) The selected angles of section and plane are illustrated, in sagittal view, with anterior towards the left. Brains were embedded and sectioned perpendicular to ZLI as indicated by Shh expression (pink). The section at the halfway point of the diencephalic tube along the D/V axis, which is also at the end of Spry2 expression (purple) is selected (arrow). The section contains p1 (yellow), p2 (orange) and p3 (green). (B) Scheme of the semi-coronal section used in this study at the position indicated in A. The dorsal (pallium) and ventral (subpallium) telencephalon are divided by the cortical hem (purple) and pallium/subpallium boundary (antihem, red). The diencephalon contains p1, p2 and p3. Fgf8 and EGFP constructs are electroporated close to ZLI at E10.5 (C,E,G,I), and sFgfr3 and EGFP constructs are electroporated at E9.5 (D,F,H,J). Brains were collected at E12.5, and only those exhibiting strong EGFP fluorescence were processed for in situ hybridization. The electroporated side is to the left. No shift is observed with p3 markers Dlx1 (C, blue) and Dlx2 (D, blue) and the ZLI marker Shh (brown) in Fgf8-overexpressing (C) or -inhibited brain (D). Two-color fluorescent in situ hybridization with Ngn2 (green) and Mash1 (red) reveals an alternating pattern of expression in the diencephalic VZ. The Mash1+ Rim VZ is expanded (red bracket), whereas Ngn2+ VZ is shrunken (green bracket) in Fgf8-overexpressing brain (E). The site of electroporation is shown by GFP expression (blue) by in situ hybridization (inset). In Fgf8-inhibited brain, Ngn2+ VZ (green bracket) is expanded and Mash1+ Rim VZ (red arrow) is shrunken (F). Nkx2.2 (brown), expressed in the region flanking ZLI (arrow), is expanded by Fgf8 electroporation, without expansion of Shh (blue) (G). A specific marker of the Rim, Sox14 (blue), is also expanded by Fgf8 electroporation (arrow) (I). Expression of Nkx2.2 and Sox14 is shrunken in Fgf8-inhibited brain (H,J, arrow). Scale bar in J: 500 µm.