DEV015891 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. The Shh immunoreactivtity in ventral telencephalon is sustained in Shh-CKO mutants. Shh protein was localized in the dorsal pallium (A,A′) and in the floor plate of ventral telencephalon at E13.5 (A, arrow) and E10.5 (C, arrow). The Shh-immunoreactive cells were scattered in the dorsal pallium in wild type (A, arrowheads); however, these cells were not observed in Shh-CKO embryos (B). In Shh-CKO mutants, Shh-immunoreactivity was decreased in dorsal telencephalon (B,B′), while Shh expression in floor plate of the ventral telencephalon was not affected (B, arrow). The immunoreactivity of Cre recombinase was restricted in the dorsal telencephalon of Shh-CKO embryos at E13.5 (D). The staining of the ventricular surface of the ventral telencephalon was non-specific because it was not in the nucleus (the Cre had a nuclear localizing signal) Scale bar in D: 100 µm for A,B,D.

• Supplemental Figure S2 -

  Fig. S2. Neuronal differentiation is reduced in the absence of Shh signaling in the dorsal pallium. Immunostaining on parasagittal sections of E15.5 and E18.5 dorsal pallium with anti-Neuronal class III β-tubulin antibody (Tuj1). The total thickness of the dorsal pallium (from ventricle to pial surface) and the stained regions by Tuj1 antibody were measured. (A) Both Shh-CKO and Smo-CKO embryos showed the significantly decreased Tuj1-positive region and total thickness of the dorsal pallium at E15.5 (Tuj1: Shh, 202.0±11.79 µm; n=3, P<0.05; Smo, 183.2±8.68 µm; n=3, P<0.01; total: Shh, 314.26±7.05 µm; n=3 P<0.05; Smo, 297.78±12.10 µm; n=3, P<0.05) compared with wild type (Tuj1: 235.7±13.38 µm; total: 365.56±26.08 µm; n=3). (B) At E18.5, Smo-CKO showed significant decrease in thickness of both of them (Tuj1: 349.81±25.12 µm; n=3, P<0.05; Total: 420.56±28.92 µm, n=3, P<0.05), whereas Shh-CKO did not show significant decrease (Tuj1: 388±76.86 µm; total: 464.15±88.12 µm, n=3) when compared with wild-type embryos (Tuj1: 446.42±47.05 µm; total: 535.80±49.42 µm; n=3).

• Supplemental Figure S3 -

  Fig. S3. Basal progenitors in SVZ are more severely affected in the absence of Shh signalling. Neural stem cells in VZ were distinguished by PAX6 immunoreactivity (red) and all proliferative cells were detected by Ki67 immunoreactivity (green) in SVZ and VZ at E15.5 (A-C). The Pax6-negative/Ki67-positive cells were considered as basal progenitors in SVZ. Parasagittal sections were counterstained with DAPI. Examples of anti-Pax6 and -Ki67 staining of the dorsal pallium of wild type (A), Shh-CKO (B), and Smo-CKO mutants (C) are shown. (D) There was no significant difference in Pax6-positive cell number among wild type (175.5±11.13; n=3), Shh-CKO (162.3±7.48; n=3) and Smo-CKO (163.1±2.77; n=3) embryos. (E) Pax6-negative/Ki67-positive cells were not significantly affected at E15.5 in Shh-CKO and Smo-CKO (20.5±4.11 and 18.8±4.52, respectively; n=3) compared with wild type (27.2±4.44; n=3). Immunostaining of parasagittal sections were performed with anti-PAX6 (red) and anti-PHH3 (green) antibodies. Mitotic cells in M-phase were labeled with an anti-PHH3 antibody in the dorsal pallium at E15.5 wild type (F), Shh-CKO (G) and Smo-CKO (H) embryos. Neural stem cells in M-phase were not significantly affected (I). The basal progenitors in M-phase (PAX6-negative/PHH3 positive) of Smo-CKO showed significant reduction (J, 1.84±0.01; n=3, P<0.05) compared with wild type (J, 3.08±0.54; n=3). Cells were counted in 100 µm sampling boxes from the ventricular surface to the pial surface (A-C) and on the entire sections (F-H). Scale bar: 100 µm.

• Supplemental Figure S4 -

  Fig. S4. Basal progenitors in SVZ/VZ of the dorsal pallium is reduced in the absence of Shh signalling. In wild type (A), Shh-CKO (B) and Smo-CKO (C), basal progenitors in SVZ/VZ of the dorsal pallium were detected by anti-TBR2 antibody at E18.5. (D) In wild type, many TBR2-positive cells were present in SVZ/VZ (69.1±2.38; n=3), while significantly fewer TBR2-positive cells were observed in Shh-CKO (36.2±3.42; n=3, P<0.01) and Smo-CKO (29.8±1.38; n=3, P<0.01). Cells were counted in 100 µm sampling boxes (white box, A-C) from the ventricular surface to the pial surface. Scale bar: 50 µm.

• Supplemental Figure S5 -

  Fig. S5. Shh protein is expressed in Cajal-Retzius neurons and GABAergic interneurons in the dorsal telencephalon. Double-immnostaining on parasagittal section of E13.5 and E15.5 dorsal pallium with anti-Shh antibody (A-C) and several neuronal markers. At E13.5, reelin (A′) and calretinin (B′), which are CR neurons markers, were detected in some Shh-positive cells in MZ of the dorsal pallium (A′′,B′′). At E15.5, Shh protein was co-expressed in GABAergic interneurons in MZ of the dorsal pallium (C′,C′′). Arrows indicate double-stained cells. Scale bar: 50 µm