Supp1: Fig.S1 Assessment of recombination in NesCre/+ and Olig3Cre/+ mice
(A–D) Double immunofluorescence for Olig3 and EYFP in NesCre/+; R26-stop-EFYP/+ embryos at E10.0 (A,B) and E11.5 (C,D). A and C are for Olig3, showing the thalamus. B and D are for EYFP, the readout of Cre-mediated recombination. Arrows define the boundaries of the thalamus. At E10.0, a scattered population of Olig3-expressing thalamic progenitor cells express EFYP. At E11.5, however, most of the diencephalon has undergone recombination, including the entire thalamus (C,D). (E–H) Triple immunofluorescence for Mash1, Ngn2 and EYFP in NesCre/+; R26SmoM2+ embryos at E11.5. R/C indicates the mixed zone pTH-R/C, where both Mash1 and Ngn2 are expressed in a non-overlapping manner (G). The mixed zone pTH-R/C has undergone homogeneous recombination and thus has homogeneous SmoM2 expression at this stage (H). (I,J) Double immunofluorescence for Olig3 and EYFP in Olig3Cre/+; R26SmoM2+ embryos at E10.5. EYFP is expressed in the entire thalamus that expresses Olig3. Scale bar, 100 µm for A,B,E-H; 200 µm for C,D,I,J.
Supp2: Fig.S2 Analysis of cell proliferation in SmoM2-expressing and Shh knockout embryos
Triple immunoflourescense for BrdU, PH3, and TuJ1 in E11.5 Olig3Cre/+; R26SmoM2+ and control littermate (A,B), or E12.5 NesCre/+; ShhC/C and control litter mate (C,D) showing pattern of cell proliferation and differentiation are normal. Analysis of the number of PH3-labelled cells within the thalamic ventricular zone (region between ZLI and arrowhead) showed that the number of cells in M-phase was not significantly different in Olig3Cre/+; R26SmoM2+, but was reduced by about 50% in NesCre/+; ShhC/C brains. Arrowheads mark the caudo-dorsal thalamic boundary indicated by Olig3 expression. Scale bar, 200 µm.
Supp3: Fig.S3 Early postmitotic markers in SmoM2-expressing embryos
In situ hybridization of Gbx2 (A,B,F,G), RORα (C,H), Irx2 (D,I), BHLHB4 (E,J) on sagittal (A,G) and frontal (B–E,G–J) sections of E12.5 NesCre/+; R26SmoM2/+ and Cre(−) control embryos. Rostral is to the left in A,G, and midline is to the left in other panels. (A,F) Sagittal sections of the forebrain and midbrain showing Gbx2 expression in the postmitotic thalamus. In NesCre/+; R26SmoM2/+ embryos, Gbx2 expression is extended caudo-dorsally, and the pretectum appears to be significantly compressed. (B,G) Gbx2 expression is extended caudo-dorsally, covering almost the entire diencephalon in NesCre/+; R26SmoM2/+ brains. (C,H) Similarly, RORα expression is expanded (compare arrowheads). (D,I) Expression of Irx2, which is normally found in the pretectum and the caudo-dorsal tip of the postmitotic thalamus, is reduced in NesCre/+; R26SmoM2/+ embryos (I, region between arrows). (E,J) BHLHB4, a rostral pretectal marker, is also reduced in NesCre/+; R26SmoM2/+ embryos (J, arrow). Arrowheads in A–H show the caudo-dorsal border of the thalamic mantle zone. PT, pretectum; TH, thalamus; ZLI; zona limitans intrathalamica; PTh, prethalamus; MB, midbrain. Scale bar, 500 µm for A and F, 200 µm for other panels.
Supp4: Fig.S4 Postmitotic lineage of Olig2-expressing progenitor cells and induction of Sox2 in Olig2-electroporated thalamus
(A–C) Frontal sections of E11.5 (A) and E14.5 (B,C) embryos that are Olig2Cre/+ ; R26stop-EGFP/+. Midline is to the left. A. EGFP immunoreactivity in rostro-ventral part of the thalamic progenitor zone (double arrows). Note that there is no gap in EGFP distribution from pTH-C to the ZLI (arrowhead), implicating that Olig2 is transiently expressed in pTH-R progenitor cells as well. (B,C) EGFP immunoreactivity at E14.5 heavily overlaps with Sox2, a marker for the rostro-ventral thalamic nuclei. Putative VP and MGv nuclei are particularly strong for EGFP expression, whereas dLG is not as strong (B). (D,E,F) Frontal sections of E14.5 mouse brain electroporated with Olig2 and EGFP plasmids at E10.5. Midline is to the left. D is a control side, whereas E is the electroporated side of the same brain. F is a higher magnification image of the boxed region in E, and shows that only on the electroporated side Sox2 is ectopically induced. Scale bar, 50 µm for F, 200 µm for all the other panels.
Supp5: Fig.S5 Expression of Fgf8, Spry1 and Spry2 in SmoM2-expressing and Shh cko embryos
Frontal sections of E12.5 control (A–D), NesCre/+; R26SmoM2/+ (E–H) and NesCre/+; Shhc/c (I–L) forebrains. In situ hybridization for Shh (A,E,I), Fgf8 (B,F,J), Spry1 (C,G,K) and Spry2 (D,H,L) is shown. (A–D) In control embryos, Fgf8 is expressed near the dorsal midline of the caudal diencephalon (B, double arrow) as well as immediately rostral to the dorsal part of the ZLI (B, arrow). The Shh expression in the ZLI is shown as a reference (A, arrowhead). Spry1 and Spry2 are both expressed near the Fgf8-expressing regions (C,D, double arrows and arrow). (E–H) In NesCre/+; R26SmoM2/+ embryos, Fgf8, Spry1 and Spry2 are still expressed in similar locations, without obvious expansion or an increased intensity. (I–L) Fgf8, Spry1 and Spry2 are expressed near the rostral part of the ZLI in NesCre/+; Shhc/c mice, similar to the control (J,K,L, arrow). Scale bar, 200 µm.