Supplemental Data. Regulation of Cerebral Cortex Size and Folding by Expansion. of Basal Progenitors

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1 Supplemental Data Regulation of Cerebral Cortex Size and Folding by Expansion of Basal Progenitors Miki Nonaka-Kinoshita, Isabel Reillo, Benedetta Artegiani, Maria Ángeles Martínez- Martínez, Mark Nelson, Víctor Borrell & Federico Calegari

2 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Animals Tetbi 4D mice were screened using forward 5 -CCTGTGGAGATGTAGAAGAAAACC-3 and reverse 3 -TCAAGTAGTCGGGGATGTCG-5 PCR-primers to amplify a 291-bp fragment of luciferase. Primary embryonic fibroblast from 3 tetbi 4D founders were screened for doxycycline-dependent induction of the 4D cassette by luciferase assays after transfection with rtta expression vectors as previously described (Schonig and Bujard, 2003). The founder line whose fibroblasts revealed the best induction relative to leakiness was then crossbred for more than three generations to the transactivator nestin rtta line (Mitsuhashi et al., 2001) (Jackson Laboratory). Genotypes were identified by RFP expression and genomic PCR with 4D / serving as internal negative control. Pigmented ferrets (Mustela putorius furo) were obtained from Marshall Bioresources (North Rose, NY) and kept on a 16:8 hours light:dark cycle. For both species, the morning of the vaginal plug was defined as embryonic day (E) 0.5. Animals were treated according to local regulations of the German, Spanish and EU authorities and experimental protocols approved by the Regierungspräsidium Dresden or the Universidad Miguel Hernández IACUC as appropriate. Viral preparation and surgery For HIV viral preparation, p6nst90-gfp and p6nst90-4dg plasmids were used to produce high titer stocks ( cfu/ml) lentiviruses encoding for ubiquitin-driven GFP nls or Cdk4-T2A-cyclin D1-T2A-GFP nls, as previously described (Artegiani et al., 2

3 2011). Approximately 1 µl of the viral suspension was injected intraventricularly in E13.5 embryos under similar conditions as described for in utero electroporation (Lange et al., 2009). Both viral preparation and in utero manipulation were recently described (Artegiani et al., 2012). Briefly, timed-pregnant C57/Bl6 females were isoflurane anesthetized, their uterine horns exposed, and virus solution injected through the uterine walls in the lateral ventricles. For viral injections in ferret, stocks of retroviral particles (5x cfu/ml) encoding CAG-GFP, or CAG-Cdk4-T2A-cyclin D1-T2A-RFP were prepared as previously described (Tashiro et al., 2006). Ferret kits aged P1 were anesthetized with 1.5% isoflurane and 4 µl of retroviral suspension injected into the OSVZ by means of a stereotaxic frame. Alternatively, progenitor cells in the VZ of the occipital cerebral cortex of P1 ferrets were electroporated, either with DNA plasmids encoding EF-driven GFP or a mixture of plasmids encoding EF-GFP, CMV-Cdk4 and CMV-cyclin D1. Both viral injection and electroporation in ferrets were previously described (Borrell, 2010; Reillo et al., 2011). Immunohistochemistry and in situ hybridization Immunohistochemistry was performed on 50 µm thick sections by 30 min permeabilization with 0.3% Triton X-100 in PBS, 30 min quenching with 10 mm NH 4 Cl in PBS, 1 hour blocking with 0.2% gelatin, 300 mm NaCl, and 0.3% Triton X-100 in PBS (IHC), followed by overnight incubation at 4 C with primary antibodies in IHC buffer, washing and 2 hours incubation with secondary antibodies at room temperature. Eventually, treatment with 2 M HCl between quenching and blocking was used for BrdU detection. Antigen retrieval by microwave boiling for 1 min in citrate buffer ph 6 (Dako) 3

4 was performed before blocking for Tbr2 immunodetection. Conditions for primary and secondary antibodies and DAPI counterstaining are summarized in Supplemental Table 1. Alternatively, sections were incubated with appropriate biotinylated secondary antibodies with ABC complex (1:100, Vector) and incubated with fluorescentlyconjugated avidin-biotin reagents or developed with nickel enhancement as described elsewhere (Borrell et al., 1999). When necessary, sections were counterstained with Nissl. In situ hybridization was performed on 10 µm thick cryostat sections mounted on Superfrost adhesive slides, dried at room temperature and hybridized overnight at 68 C with 500 ng/ml DIG-labeled probe in hybridization solution, washed at 68 C in posthybridization solution (50% formamide, 2x SSC, 0.1% Tween) and maleic acid (100 mm maleic acid ph 7.5, 150 mm NaCl, 0.1% Tween) at room temperature. Alkaline phosphatase reaction was obtained by anti-dig conjugated antibody (1:2000) in maleic acid plus 10% goat serum overnight at 4 C followed by 1-2 days incubation in NBT- BCIP (Sigma). Antisense probes were prepared using the DIG RNA Labeling Kit SP6/T7 (Roche) after cloning into pcrii-topo vector (Invitrogen). 4

5 SUPPLEMENTAL REFERENCES Artegiani, B., Lange, C., and Calegari, F. (2012). Expansion of embryonic and adult neural stem cells by in utero electroporation or viral stereotaxic injection. Journal of visualized experiments : JoVE. Artegiani, B., Lindemann, D., and Calegari, F. (2011). Overexpression of cdk4 and cyclind1 triggers greater expansion of neural stem cells in the adult mouse brain. J Exp Med 208, Borrell, V. (2010). In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation. Journal of neuroscience methods 186, Borrell, V., Del Rio, J.A., Alcantara, S., Derer, M., Martinez, A., D'Arcangelo, G., Nakajima, K., Mikoshiba, K., Derer, P., Curran, T., et al. (1999). Reelin regulates the development and synaptogenesis of the layer-specific entorhino-hippocampal connections. J Neurosci 19, Lange, C., Huttner, W.B., and Calegari, F. (2009). Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors. Cell Stem Cell 5, Mitsuhashi, T., Aoki, Y., Eksioglu, Y.Z., Takahashi, T., Bhide, P.G., Reeves, S.A., and Caviness, V.S., Jr. (2001). Overexpression of p27kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells. Proc Natl Acad Sci U S A 98, Reillo, I., de Juan Romero, C., Garcia-Cabezas, M.A., and Borrell, V. (2011). A role for intermediate radial glia in the tangential expansion of the mammalian cerebral cortex. Cereb Cortex 21, Schonig, K., and Bujard, H. (2003). Generating conditional mouse mutants via tetracycline-controlled gene expression. Methods Mol Biol 209, Tashiro, A., Zhao, C., and Gage, F.H. (2006). Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo. Nat Protoc 1, Zhu, P., Aller, M.I., Baron, U., Cambridge, S., Bausen, M., Herb, J., Sawinski, J., Cetin, A., Osten, P., Nelson, M.L., et al. (2007). Silencing and un-silencing of tetracyclinecontrolled genes in neurons. PloS one 2, e533. 5

6 SUPPLEMENTAL TABLE 1 ANTIGEN COMPANY CAT.NR. DILUTION SPECIES REQUIRES BrdU Abcam ab6326 1:200 m HCl Ctip2 Abcam ab :200 m Cux1 Santa Cruz sc :250 f GFP Aves Labs GFP :1000 n.a. Ki67 Novocastra NCL-Ki67p 1:200 m/f Olig2 Millipore AB9610 1:1000 m Pax6 Covance PRB-278P 1:200 m PH3 Upstate :1000 f PH3 Millipore :500 m antigen retrival RFP Rockland :2000 m/f S100b Abcam ab868 1:1000 m Satb2 Abcam ab :200 m Tbr2 Abcam ab :400 m/f antigen retrival primary Jackson :1000 antibody (Cy-labeled) Table 1. Antibodies used in this study. List of antibodies, distributors, working dilutions, their us in mouse (m) or ferret (f) and special requirement are indicated (left to right). 6

7 SUPPLEMENTAL FIGURE AND FIGURE LEGENDS Figure S1. Overexpression of 4D does not influence the proliferation of radial glial cells (A-C) Fluorescence pictures (A, left and B) and quantifications (A, right and C) of cells labeled for Pax6 (A) or Pax6/Tbr2 (B-C) in cortices of E15.5 CT or 4D mice treated with 9TB-dox starting at E11.5. (D) Radial thickness of cortical layers of mice treated as in (A-B). Graphs represent absolute numbers of cells in a cortical column of 100 µm width. Scale bar in A = 100 µm. Data are mean ± S.E.M.; *P < 0.05; ***P < 0.001; n > 3 pairs of embryos from different litters. 7

8 Figure S2. Overexpression of 4D increases cortical thickness at E18.5 (A) Whole mount pictures of E18.5 CT or 4D embryos treated with 9TB-dox form E11.5 to E15.5. Yellow and red lines outline the boundaries of one hemisphere and are reproduced in the right panel to appreciate size differences. (B) Fluorescence pictures of DAPI-stained brain sections and stereological quantifications of the whole brain of embryos treated as in (A). Yellow and red lines outline the boundaries of the ventricular and cortical surface area, respectively. Scale bar, 500 µm. Data are mean ± S.E.M.; *P < 0.05; n > 3 pairs of embryos from different litters. 8

9 Figure S3. 4D overexpression increases cortical surface area at P21 (A) Phase contrast pictures of 150 µm thick coronal sections from the anterior to the posterior (from top to bottom) boundaries of the P21 brain of mice injected with GFP (right) or 4DG (left) viruses at E13.5. (B) Nuclear density (left) and total number of cells (right) in the CP of P21 mice injected with GFP or 4DG viruses at E13.5. Total numbers are calculated by considering volume (Figure 4A) and density (D). (C) Fluorescence pictures of the cortical plate of CT or 4DG mice treated as in (A) followed by immunohistochemistry for the astrocyte and oligodendrocyte markers S100b or Olig2, respectively, (green) and DAPI counterstaining (blue). Scale bar, 25 µm (C). Data are mean ± S.E.M.; *P < 0.05; **P < 0.001; n > 3 pairs of embryos from different litters. 9