DEV021535 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Enhancement of neocortical Cajal-Retzius cell formation in Hes1;Hes3;Hes5 cKO mice. (A-F) Reelin- (A,C,D,F) and p73-expressing cells (B,E) increased in number in Hes1;Hes3;Hes5 cKO mice, compared with the control at both E12.5 and E14.5. Boxed regions in A,C,D,F were enlarged in A′,C′,D′,F′. (G) Quantification of the number of reelin protein-expressing cells at the dorsomedial telencephalon at E14.5. Cajal-Retzius cells significantly increased in number in Hes1;Hes3;Hes5 cKO mice. **P<0.001, t-test. Scale bars: 200 µm.

• Supplemental Figure S2 -

  Fig. S2. Normal formation of the cortical layers and the hippocampus in Hes1;Hes3;Hes5 cKO mice. (A-L) Cortical layers were normally formed in Hes1;Hes3;Hes5 cKO mice at E18.5. (M-V) Coronal sections of the hippocampal region. All hippocampal field-specific markers were normally expressed in Hes1;Hes3;Hes5 cKO mice at E18.5. Scale bars: 100 µm in A-L; 200 µm in M-V.

• Supplemental Figure S3 -

  Fig. S3. Increased expression of Hey1 in the dorsal telencephalon of Hes1;Hes3;Hes5 cKO mice. Coronal sections of the control (A,C) and Hes1;Hes3;Hes5 cKO mice (B,D) at E12.5 were hybridized with RNA probes indicated at the bottom of each panel. Expression of Hey1, but not Hey2, was upregulated in the dorsal telencephalon of Hes1;Hes3;Hes5 cKO mice. Scale bars: 200 µm.

• Supplemental Figure S4 -

  Fig. S4. Hypoplastic hindbrain-choroid plexus in Hes1;Hes5 double KO mice. Side-view (A,C) and rear view (B,D) of E10.5 control embryos (A,B) and Hes1;Hes5 double KO embryos (C,D), which were hybridized with Ttr RNA probe. Hes1;Hes5 double KO embryos showed hypoplastic development of the choroid plexus in the hindbrain. Scale bars: 1 mm.

• Supplemental Figure S5 -

  Fig. S5. No significant increase of Cajal-Retzius cell differentiation in the pallial-subpallial boundary of Hes1;Hes3;Hes5 cKO mice. Coronal sections of the control (A-D,I) and Hes1;Hes3;Hes5 cKO mice (E-H,J) at E11.5 (A-H) and E12.5 (I,J) were hybridized with RNA probes as indicated at the bottom of each panel. Dbx1⁺ pallial-subpallial boundary progenitor cells were not changed in number in Hes1;Hes3;Hes5 cKO mice (E,J), compared with the control (A,I). (B-D,F-H) The expression of Ngn1, Ngn2 and Mash1 was not significantly affected in Hes1;Hes3;Hes5 cKO mice. Scale bars: 100 µm.

• Supplemental Figure S6 -

  Fig. S6. No significant increase of Cajal-Retzius cells in the piriform cortex of Nes-CreER^T2;Hes1;Hes3;Hes5 cKO mice. (A,B) The Nes-CreER^T2 line5-1 mice were crossed with the Rosa26-lacZ reporter mice, and 6 mg tamoxifen was administered at E9.5. X-gal staining was carried out with coronal sections of E12.5 embryos. No X-gal-positive cells were observed in the choroid plexus epithelium. (C-F′) Hes1 was expressed throughout the telencephalon of the control mice at E12.5 (C-D′). By contrast, tamoxifen treatment efficiently downregulated Hes1 expression throughout the neuroepithelium of Nes-CreER^T2;Hes1;Hes3;Hes5 cKO mice at E12.5 (E-F′). In these mice, Hes1 expression was significantly reduced in the hem but was maintained in the prospective choroid plexus region (E-F′). (G-L) The hem (Wnt3a⁺) and the choroid plexus epithelium (Msx1⁺, Ttr⁺) were formed normally in Nes-CreER^T2;Hes1;Hes3;Hes5 cKO mice (J-L), as in the control (G-I). (M-P′) Reelin-expressing cells were not increased in number in Nes-CreER^T2;Hes1;Hes3;Hes5 cKO mice (O-P′), compared with the control (M-N′) at E12.5. Boxed regions in C-F and M-P are enlarged in C′-F′ and M′-P′. Scale bars: 500 µm in A-F,M-P; 100 µm in G-L.

• Supplemental Figure S7 -

  Fig. S7. Normal response to Bmp signaling, cell death and cell proliferation in Hes1;Hes3;Hes5 cKO mice. (A,B) Bmpr1a expression on coronal sections of E11.5 embryos. Expression of Bmpr1a was normal in the telencephalon of Hes1;Hes3;Hes5 cKO mice. (C-F) E10.5 telencephalic explants of the control (C,E) and Hes1;Hes3;Hes5 cKO embryos (D,F) were cultured for 72 hours in the absence (C,D) or the presence of BMP4 (E,F). Telencephalic explants of Hes1;Hes3;Hes5 cKO mice ectopically expressed Msx1 by BMP4 treatment, like control explants. (G-J) TUNEL assay on coronal sections of the dorsal telencephalic midline of the control and Hes1;Hes3;Hes5 cKO mice at E11.5. The number of cells positive for TUNEL was similar between the control (G) and Hes1;Hes3;Hes5 cKO mice (I). (K,L) The number of BrdU incorporated cells (1.5-hour exposure) was similar between the control (K) and Hes1;Hes3;Hes5 cKO (L) mice at E10.5. Scale bars: 100 µm in A,B,G-L; 1 mm in C-F.

• Supplemental Figure S8 -

  Fig. S8. Regulation of Cajal-Retzius cell formation by Ngn2. (A-F) Reelin- (A,C,D,F) and p73-expressing cells (B,E) decreased in number in Ngn2 KO mice, compared with the control at both E12.5 and E14.5. Boxed regions in A,C,D,F are enlarged in A′,C′,D′,F′). (G) Quantification of the reelin protein-expressing cell number in the dorsomedial telencephalon at E14.5. The number of Cajal-Retzius cells significantly decreased in Ngn2 KO mice. **P<0.001, t-test. Scale bars: 200 µm.

• Supplemental Figure S9 -

  Fig. S9. Normal patterning of the dorsal telencephalic midline in Ngn2-null mice. Coronal sections of the wild-type (A-C,G-I) and Ngn2-null mice (D-F,J-L) at E11.5 (A-F) and E12.5 (G-L) were hybridized with RNA probes as indicated at the bottom of each panel. Patterning of the dorsal telencephalic midline region occurred normally in Ngn2-null mice as in the wild-type mice. Scale bars: 200 µm.

• Supplemental Figure S10 -

  Fig. S10. Normal generation of the choroid plexus epithelial cells in Rbpj cKO embryos. Coronal sections of the control (A-H,Q) and Rbp-j cKO (I-P,R) at E12.5 (A-P) and E14.5 (Q,R) were hybridized with RNA probes as indicated at the bottom of each panel. Expression of Hes5 was severely downregulated in Rbp-j cKO mice (L). By contrast, Hes1 was still expressed in Rbp-j cKO embryos (K). Ttr and Msx1 were normally expressed in the telecephalon of Rbp-j cKO embryos (M,N). Although the morphology of the choroid plexus was altered, the epithelial cells were formed normally in the absence of Rbp-j, suggesting that the Notch-Rbp-j pathway is not involved in cell fate specification of the choroid plexus epithelial cells. Scale bars: 200 µm.