DEV021618 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Fgf8 expression. Dorsal view at E10.5 (A, arrow). Smaller but similar expression of Fgf18 is observed in the diencephalon, like that of Fgf8, as revealed on dorsal (B, arrow) and lateral (C, arrow) views. Lateral view of the brain shows restricted expression of Fgfr1 in p3 (D) and strong expression of Fgfr2 and Fgfr3 in both p2 and p3 (E,F). Scale bar in F: 500 µm.

• Supplemental Figure S2 -

  Fig. S2. Brains are electroporated with Fgf8 and Egfp at E10.5 and harvested at E13.5. Anterior is towards the left. Ectopic expression of Fgf8 in p2 increases cell growth and causes malformation (A, arrow), whereas expression close to ZLI is associated with no gross increase in size (B). When brains receive excess Fgf8 signal in p2 (left side of brain) (C), ectopic En2 expression is induced in p2 at E12.5 (D). TUNEL assay of Fgf8-electroporated brain reveals no increase of cell death (E,F, arrow). Pulse labeling of cells with BrdU at E12.5 for 24 hours in Fgf8-electroporated brains reveals no significant difference of cell growth (G,H). Strong upregulation of Spry2 is observed in Fgf8-electroporated brain at E11.5 (I, arrow). Complete loss of Spry2 expression is observed in sFgfr3-electroporated brain (J, arrow). Scale bar in D: 50 µm for C-H; 200 µm for I,J.

• Supplemental Figure S3 -

  Fig. S3. Shh results. Expression of Shh (A,B) and Shh activity demonstrated by expression of Ptc (C,D) at E11.5 is not altered by Fgf8 electroporation, as demonstrated by whole-mount in situ hybridization. Anterior is towards the left. Dorsal view of the site of electroporation reveals Egfp expression prior to in situ hybridization for brains in A and B (E,F). Wnt3a expression in ZLI is not altered by Fgf8 (G,H, arrows). Scale bar in H: 500 µm for A-F; 50 µm for G,H.

• Supplemental Figure S4 -

  Fig. S4. Gene expression shifts. Egfp construct is electroporated close to ZLI at E10.5 (A), and no shift is observed in the Rim marker Nkx2.2 (brown) or the ZLI marker Shh (blue) (B). Fgf8 overexpression causes a shift in gene expression in the postmitotic region labeled by Lhx9 (C, arrow). Fgf8 overexpression downregulates Otx2 expression in the p2 progenitor region (D, arrows). Diencephalic p2 tissue responds differently to Fgf8 and Shh electroporations. Fgf8 electroporation expands expression of the Rim marker Gad67 (blue) and shrinks expression of the p2 marker Gbx2 (brown, bracket) (E). Expression plasmid carrying chick Shh (Agarwala et al., 2001) is electroporated at E10.5. Overexpression of Shh causes expansion of Gbx2 expression, which extends to the p2/p3 border (F, bracket). Scale bar in D: 50 µm in A-F.

• Supplemental Figure S5 -

  Fig. S5. Rim marker expression. Expression pattern of the Rim markers at E10.5 (A-C) and E11.5 (D-I). The Rim is already detectable in sectioned brain at E10.5 by the gap between Ngn2 and Zli (A, arrow). Both Rim markers Nkx2.2 (B, arrow) and Mash1 (C, arrow) are also detectable. FGF activity is restricted to the dorsal part of the diencephalon (F,I), although the Rim markers are also expressed in ventral diencephalon (D,E,G,H). Scale bar in I: 200 µm for D-I; 100 µm for A-C.