Supplementary Figure S1. Monolayer differentiation of mouse ESCs into telencephalic neural precursors. (a) Schematic representation of the protocols

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1 Supplementary Figure S1. Monolayer differentiation of mouse ESCs into telencephalic neural precursors. (a) Schematic representation of the protocols used to differentiate mouse ESCs. (b) Representative images and quantification of Sox1-GFP ESCs differentiated for 5 days as a monolayer. On the 5th day 70.5±6% of the cells were Sox1 positive (c) qrt-pcr analysis showing the decrease in the expression of pluripotency associated genes and the upregulation in the expression of telencephalic markers during the first 4 days of monolayer differentiation. (d) The bar chart indicates the downregulation of the non-neural ectoderm marker cytokeratin14, of the endoderm marker Gata6 and of the mesoderm marker T at day4 of the neural differentiation process. (e) qrt-pcr analysis showing how specifically ventral telencephalic markers are upregulated during the neuronal differentiation by monolayer of ESCs in the absence of Activin. (f) Immunofluorescent microscopy images and quantification of Nestin/Foxg1 double + cells (54.2±4.3), Otx2 (14.69±1.42%), Pax2 (11.62±1.72%) and Pax5 positive cells (3.58±0.13%) after 7 days of monolayer differentiation. (D) For all experiments the average fold change from three independent experiments (±SEM) is shown. Scale bar=25µm (b) and 50µm (f).

2 Supplementary Figure S2. TGF-ß1 and TGF-ß3 are unable to induce neuronal precursor differentiation. (a) Nestin and β-iii-tubulin double immunostaining and (b) quantifications of cultures treated with control conditions, TGF-ß1 (10ng/ml) or TGF-ß3 (10ng/ml) for 3 days. In these cultures we observe that neither TGF-ß1 nor TGF-ß3 can induce differentiation of ESC derived neural precursors (nestin + / β-iii-tubulin + cells: control 78.3±3.7%/ 19.8±0.9%; TGF-ß1 75.6±4.2%/ 21.6±1.4% and TGF-ß3 treated 76.4±2.6%/ 22.1±0.4%; n=3, mean±sem). ESCs were differentiated for 5 days as a monolayer, then re-plated into poly-d-lysine/laminin coated dishes and cultured in NBB27 media (controls), TGF-ß1 (10ng/ml) or TGF-ß3 (10ng/ml). Scale bar=25µm.

3 Supplementary Figure S3. Gli3 defective neuronal precursors present a hyperactivation of the Shh pathway and don t respond to Activin. (a) The levels of differentiation were analyzed in wild-type (wt) and Gli3 defective cells (Xt) by Nestin and β-iii-tubulin immunostaining after 3 days of control or Activin treatment and (b) the proportion of Nestin + / β-iii-tubulin + wiltypecells plotted (wild-type cells: control 70.5±3.5%/ 22.4±1.91% and Activin treated 42.8%±2.4%/ 57.2±3.63%/; Xt cells: control 84±4.2%/ 15.3±1.53% and Activin treated 76.4%±3.9%/ 20.3±2.18%). In these experiments we observe that the Gli3 defective cells are unable to differentiate in response to Activin. (c) Analysis of the normalized mrna levels of Shh target genes in wild-type and Gli3 -/- cells after 7 days of differentiation. An increase in the expression of Shh target genes is observed in Gli3 defective cells. (d) qrt-pcr analysis of Shh target gene expression in wt and Gli3 -/- cells after 3 days of control or Activin treatment. Activin has only minor effects on the levels of expression of these genes in Gli3 -/- cells compared to controls. For all experiments n=3, mean±sem. ESCs were differentiated for 5 days as a monolayer, then replated into poly-d-lysine/laminin coated dishes and cultured in NBB27 media (controls) or NBB27+10ng/mlActivin for 3 days (a-b and d) or 7 days (c). Scale bar=50µm.

4 Supplementary Figure S4. Combined effects of Cyclopamine and high RA concentrations can mimic Activin induced differentiation. (a) Nestin and β-iii-tubulin immunostaining and (b) quantification of the proportion of positive cells for control, Activin, cyclopamine, RA or RA plus cyclopamine treatments. This data indicates that cyclopamine and RA cooperate to induce neuronal differentiation and the levels of differentiation they induce are similar to those induced by Activin (nestin + /β-iii-tubulin + cells: control 75.1±5.6%/ 22.4±2.3%; Activin 37.8±5.4%/ 58.6±4.3%; 10µM RA 81.34±6.4%/ 15.3±1.2%; cyclopamine 60.8±3.6%/ 32.5±3.2% and cyclopamine +10µM RA treated cells 47.6±2.32%/ 46.7±3.8). ESCs were differentiated for 5 days as a monolayer, then re-plated into poly-d-lysine/laminin coated dishes and cultured in NBB27 media (controls), NBB27+10ng/mlActivin, NBB27+10µM Cyclopamine, NBB27+10µM RA, NBB27+10µM Cyclopamine+5µM RA or NBB27+10µM Cyclopamine+10µM RA. Scale bar=25µm.

5 Supplementary Figure S5. Activin treatment increases the proportion of Dlx2 + interneurons. Immunostaining and quantification for (a) CoupTFII/Nestin (Control 31.48±5.3%; Activin 55.16±4.6%) and CoupTFII/β-III-tubulin (Control 24.3±4.32% and Activin 58.6%±5.17%) indicating that the proportion of CoupTFII positive cells increases upon Activin treatment; (b) β-iii-tubulin/dlx2 (control 36.3±7.1% and Activin 52±5.2%) showing an increase in the proportion of β-iii-tubulin/dlx2 double positive cells upon Activin treatment; (c) calretinin/gaba (76.34%±6.8%) indicating that the majority of calterinin positive cells also stain for GABA and for calretinin/th illustrating how these two markers are very rarely coexpressed (<5%); (c ) high magnification image of (c); (d) calretinin/couptfii indicating that the majority of calretinin expressing neurons are also positive for CoupTFII (95.7±5%) and (e-f) for calretinin/reelin indicating that Activin treatment rarely generates neurons that co-express these markers only (Control 50±3.68% Activin16±1.44%) and that the proportion of reelin positive cells increases upon Activin treatment (Control 6±0.5%, Activin 14.38±1.2%). n=3, mean±sem. ESCs were differentiated as in Fig.S4 but cultured in NBB27 media ±Activin for 5 days (d), 3 days (a and b), 6 days (c) or 7 days (e). For (e) BDNF (10ng/ml) was added from day 2 after re-plating. Scale bar=50µm (a and b), 25µm (c, d and e, except for TH-calretinin where the scale bar=50µm and (c ) where it is 14µm).

6 Supplementary Figure S6. Activin treated telencephalic neural precursors derived from mouse ESCs integrate into the cortex of adult mice. (a) Low magnification composite of images of the injection sites (arrows) in the cortex of animals injected with GFP labelled, Activin treated, telencephalic neural precursors derived from mouse ESCs. The pial surface and white matter (WM) are indicated. (b) Immunostaining for Calretinin to visualize those cells in the cortex that co-express GFP (arrows). (c) 2-photon imaging of a transplanted GFP cell one month after injection into the adult cortex. (c ) High magnification of the area marked by a rectangle in c showing dendritic spines (arrows). Scale bar = 400μm (a), 25μm (b) and 10μm (c-c ).

7 Supplementary Figure S7. A conserved role for Activin in human ESCs. (a) Schematic representation of the protocol used to differentiate human ESCs. (b) qrt-pcr analysis showing the decrease in the expression of pluripotency associated genes and the upregulation in the expression of telencephalic markers during the first 18 days of monolayer differentiation. (c) Representative images of cells expressing Oct3/4, Sox1 and Pax6 during this process and quntification of the percentage of positive cells at each time-point. (d) Representative images of cells expressing calretinin/β-iii-tubulin for the H1 human ESCs line and the 2F8 and 4FHuang ipscs and quatinfication of double positive cells in control conditions and after Activin treatment (H1: control 5.4±1.5%, Activin 70.3±8.8%; 2F8: control 4.9±2.2%, Activin 63.2±15.8%; 4FHuang: control 4.4±1.8%, Activin 58.7±10.9%. (e) Double immunostaining for calretinin and GABA in Activin treated hescs (94.73±2.07% double positive). For all experiments the average fold change from three independent experiments (±SEM) is shown. hescs were differentiated as in Fig.7. Scale bar=50µm (c), 75µm (d) and 14µm (e).

8 Supplementary Figure S8. Activin inhibits Shh signalling and requires RA signalling during the differentiation of human ESCs. (a) qrt-pcr analysis indicating how the levels of Gli1 and Ptch1 change after 24h exposure to the indicated conditions and (b) how the expression of the Aldh1 family of genes are promoted by Activin signaling whilst Cyp26b1 is repressed. (c) Immunohistochemistry and (d) quantification of the proportion of neural precursors and neurons indicates that inhibition of RAR blocks the neural differentiation induced by Activin in hescs. Nestin+/β-III-tubulin + cells: Control=72.1±2.9%/14.6±1.9%; -RA+Act=62.6±5.5%/23.1±2.6%; +RA=64.0±5.5%/16.0±0.9%; +RA+Actv=35.9±5.5%/46.6±3.9%; +RA+Actv+RXRinh=32.0±5.8%/42.5±8.2%; +RA+Actv+RAR inh= 57.1±8.1%/ 31.6±7.1%; +RA+RAR inh= 69.1± 3.6%/ 17.5± 2.1%. For all experiments n=3, mean±sem Student s t test. **p<0.005 for the comparison between +RA+Act versus RA+Act and *p<0.05 for the comparison between +RA+Act versus +RA+Act+RAR inh. hescs were cultured as in Fig.7 until they were re-plated into poly-l-lysine/laminin coated dishes and at this stage they were cultured in N2B27 (controls), N2B27+10ng/ml Activin, N2B27+10µM RXR inhibitor, N2B27+10µM RXR inhibitor+10ng/mlactivin, N2B27+10µM RAR inhibitor or N2B27+10µM RAR inhibitor+10ng/mlactivin for 6 days (H1/iPSCs) or 7 days (H7). The NBB27 media was either VitA deficient or supplemented with VitA for these experiments. Scale bar=50µm.

9 Supplementary Table S1 Primers used for qrt-pcr Primers used for mouse cdnas Forward Reverse Nkx2.1 GGACACCATGCGGAACAGCG CCATGCCACTCATATTCATGCC Gsx2 GGACACCCGCAGCACCACG CCTTTTGCCATTGGGTACCTGG Pax6 CCATCTTTGCTTGGGAAATC GCTTCATCCGAGTCTTCTCC Gli1 CCCAATACATGCTGGTGGTGC CCGTGTGCGACCGAAGGTGC Patched1 CGAGACCAACGTGGAGGAGC GGAGTCTGTATCATGAGTTGAGG GAD1 GGCATCTTCCACTCCTTCGC CACAGATCTTCAGGCCCAGTTTTCT Foxg1 GACCCGTCGAGCGACGACG GGACAGGAAGGGCGACATGG Six3 GGTTTAAGAACCGGCGACAG TACCGAGAGGATCGAAGTGC Sox1 CCTCGGATCTCTGGTCAAGT TACAGAGCCGGCAGTCATAC Oct4 GGTTTAAGAACCGGCGACAG TACCGAGAGGATCGAAGTGC Nano GGTTTAAGAACCGGCGACAG TACCGAGAGGATCGAAGTGC Rex1 CGAGTGGCAGTTTCTTCTTGG GACTCACTTCCAGGGGGCAC Aldh1a1 GCCTGCACAACCTGCAGTCC GCCGCTCACTGAATTGTGCC Aldh1a2 GGTTTAAGAACCGGCGACAG TACCGAGAGGATCGAAGTGC Aldh1a3 GGTTTAAGAACCGGCGACA TACCGAGAGGATCGAAGTGC Cyp26b1 GCTCCATGGGATTCCCGCTC CGACGACTGGAAGCCGGAAC STRA6 GGTGACAGATGACTACAGCAGC GGTGCGGTGAGCTGGCAAAG Dlx2 CTACACCGCCGCGTACACCTCCTAC CGTGGTTTCCGGACTTTCTTTGGCTTCCCG Dlx6 CCTGGCCCTTCCCGAGAGAG GGGTCACTCTCGTGTGGGTTAC Dlx5 CCTCGCCCTGCCAGAACGC GGCATCTCCCCGTTTTTCATGA Er81 GGTCTGCTTGCAGTCAAGAG GGAGTGCTGGATGGTGTCGG Lhx6 CGAGTGCTCCGTGTGTCGCA CCAGTCACTGGCATAGATTTGTC Lhx2 CCCTACTACAACGGCGTGGG GGTAGGGCTGGTCACGATCC Crabp1 GCACACAGACACTTCTTGAGGG CGGACATAAATTCTTGTGCACACC Zcchc12 CCTGGAGGCAGGCTTTCTCAC CGCTTTATCTTCAGCAGTGCAGC β-actin GGTGACAGATGACTACAGCAGC GGTGCGGTGAGCTGGCAAAG Primers used for human cdnas Forward Reverse GSX2 CGCACCTGTCTGCACCGCC CCAGCTCCAGGAGTTGCGTG DLX2 ACTACCCCTGGTACCACCAGAC TCTGCTCTCAGTCTCTGGCGAGTTCTC GAD67 CGTCTTCGACCCCATCTTCGT CGCAGATCTTGAGCCCAGTT Calretinin CCTTACCTGCACCTGGCCGA CCAGAGCCTTTCCTTGCCTTC β-actin TCACCACCACGGCCGAGCG TCTCCTTCTGCATCCTGTCG