Supplemental Data

Xinwei Cao, Samuel L. Pfaff, Fred H. Gage

FIGURE LEGENDS

Figure S1. (A) Specificity of the TEAD1 antibody. 293T cells were transfected with

Myc-tagged TEAD1, 3, and 4 overexpression constructs. Western blot shows that the

TEAD1 antibody could only detect TEAD1 but not TEAD3 and 4. The weak bands in

TEAD3, 4, and mock samples are likely to be the endogenous TEAD1 of 293T cells. (B)

Comparison of the expression patterns of TEAD1 and pYAP. ( C to E) At thoracic levels,

TEAD1 is also expressed in a group of cells located at the ventral-lateral region of the neural tube. Some of these cells express the motor neuron marker Isl1/2. Regions in (D) and (E) correspond to that within the dashed square in (C). ( F) In situ hybridization shows that TEAD4 is expressed in the ventricular zone of the chick neural tube. ( G) Co- immunoprecipitation from HH27-28 forebrain and midbrain lysates shows interaction of endogenous YAP and TEAD in neural progenitor cells.

Figure S2. In situ hybridization (ISH) shows that misexpressing YAP ∆60-89 (A) and

TEA R59K -VP16 (B) did not induce cyclin D1 expression. YAP ∆60-89 and TEA R59K -VP16

cDNAs are followed by an IRES-GFP cassette to monitor their expression. The image

next to the ISH photograph shows GFP expression in an adjacent section.

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Figure S3. (A) Testing YAP shRNAs by Western blot. Chick embryonic fibroblasts were transfected with HA-YAP and one of the shRNA constructs. HA-mRFP (monomeric red fluorescent ) was cotransfected as the control. shYAP#1 and #2 specifically inhibited YAP expression but had no effect on mRFP expression. The target sequence of shGFP is: GGCACAAGCTGGAGTACAACTA. (B to I) Inhibiting YAP and TEAD

279 with YAP N and TEA, respectively, led to increased levels of cleaved caspase 3 (B and

C, arrows), but did not affect BrdU incorporation (D and E) or cyclin D1 mRNA levels (F

to I). In panel B, the transfected side is indicated by cotransfected GFP. In panels C to E,

279 YAP N and TEA were detected with antibodies against YAP and , respectively. In panels F to I, the image next to the ISH photograph shows the expression of cotransfected

GFP in an adjacent section.

Figure S4. ( A) Domain structures of YAP 279 ∆-EnR and TEA R59K -EnR constructs. (B to

D) Misexpressing YAP 279 ∆-EnR did not significantly affect proliferation (B) or neuronal

differentiation (C and D). ( E to G) Misexpressing TEA R59K -EnR did not significantly affect proliferation (E) or neuronal differentiation (F and G).

Figure S5. Testing the shLats1 construct. HA-Lats1 KD was cotransfected with the shRNA cloning vector pRFPRNAi (A to C) or shLats1 (D to F). The dsRED protein expressed from pRFPRNAi was used to monitor transfection efficiency (A and D). Lats1 KD was detected by an HA antibody. To obtain a quantitative comparison, the laser power, gain, and iris for each color channel were kept constant during the acquisition of these two sets of images. The HA immunofluorescence signal obtained from the cotransfection of

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shLats1 and Lats1 KD was lower than that from the cotransfection of pRFPRNAi and

Lats1 KD (cf. B and E), indicating that shLats1 reduced the expression level of Lats1 KD .

The reason that the Lats1 KD instead of the Lats1 construct was used in this experiment was that the former had a higher expression level.

3 Table S1. PCR Primers Used for Cloning and Mutagenesis

Gene name and purpose Sequence cTEAD1 forward w/ Cla I, in frame with pMIW3-N-Myc AAGGA ATCGAT AGCGATTCTGCAGATAAACCTATTG cTEAD1 reverse w/ Xba I CATA TCTAGA TTAGTCCTTTACAAGCCTGTAGATATG cTEAD1 DBD reverse w/ EcoR V, in frame with pMIW3-N-Myc AATG GATATC GGGGAATTCCTGGCAGGCCCAGC TEA domain R59K mutation, sense CTTAGAACAGGGAAGACA AA GACCAGGAAGCAGGTGTC TEA domain R59K mutation, anti-sense GACACCTGCTTCCTGGTC TT TGTCTTCCCTGTTCTAAG cTEAD3 forward w/ EcoR V, in frame w/ pMIW3-N-Myc GCCG GATATC CCATAGCGTCCAACAGCTGGAACGCCAGC cTEAD3 reverse w/ Xba I CATA TCTAGA AGCCCCTAGTCCTTCACCAACTTGTAGAC cTEAD4 forward w/ Cla I, in frame w/ pMIW3-N-Myc GCCG ATCGAT TTGGAGCTTCTAGCTGGCACCATTACCTCC cTEAD4 reverse w/ Xba I CATA TCTAGA TAGTCTCTAGTCCTTCACCAGCCGGTAG cYAP forward w/ EcoR V, in frame with pMIW3.1-N-HA GCCG GATATC TGGATCCCGGGCAGCCTCAGCCGCAGC cYAP reverse w/ Xba I GTCT TCTAGA GGCCCCTATAACCAAGTAAGAAAACTC cYAP90 forward w/ EcoR V GCCG GATATC TGCCGGACTCCTTCTTCAAGCCG cYAP279 reverse w/ EcoR V, in frame w/ pMIW3-N-Myc-EnR CATA GATATC TCAGCCTTTCCTTCTCCATCTG cYAP S126A mutation, sense CAGCATGTTCGTGCTCAT GCCTCTCCAGCATCACTGC cYAP S126A mutation, antisense GCAGTGATGCTGGAGAGG CATGAGCACGAACATGCTG mouse YAP forward w/ EcoR V and Bgl II CATA GATATCAGATCT GCCATGGAGCCCGCGCAACAGCCGCCGCCC mouse YAP reverse w/ Not I CACG GCGGCCGC TGGCTCCCTGCAGCTCTATAACCACGTGAG cMST2 forward w/ EcoR V, in frame w/ pMIW3.1-N-HA GCCG GATATC TGGAGCAGGCAGCGCCCAAGAGCAAACTG cMST2 reverse w/ Xba I CATA TCTAGA GTTACCAGTGTCCACACTGACTCATAGC cMST2 kinase-dead mutation K55R, sense GACAAGTTGTAGCAATTA GGCAGGTCCCTGTGGAGTC cMST2 kinase-dead mutation K55R, antisense GACTCCACAGGGACCTGC CTAATTGCTACAACTTGTC cLats1 forward w/ Not I, in frame w/ pMIW3.1-N-HA CTGT GCGGCCGC TGAAGAGAAGTGAGAAGCCAGAAGGTTATAG cLats1 internal reverse w/ Hind III CCTCTTTGCT AAGCTT GGCTCCAGTCTCG cLats1 internal forward w/ Hind III CGAGACTGGAGCC AAGCTT AGCAAAGAGG cLats1 reverse w/ Xba I CACG TCTAGA GTGTTTACTCCTCTACTAAACATAAACTAGGTCACG cLats1 kinase-dead mutation DA, sense GATGGTCATATTAAATTGACTG CCTTCGGACTCTGTACAGG cLats1 kinase-dead mutation DA, antisense CCTGTACAGAGTCCGAAG GCAGTCAATTTAATATGACCATC cLats2 forward w/ Not I, in frame w/ pMIW3.1-N-HA CTGT GCGGCCGC TGAGGCCAAAGACTTTTCCTGCTACGACATATTC cLats2 internal reverse w/ Bsa I CTGAGGCTCA GGTCTC ATCACTCGCATGC cLats2 internal forward w/ Bsa I GCATGCGAGTGAT GAGACC TGAGCCTCAG cLats2 reverse w/ Nhe I CACG GCTAGC TTACACATATACAGGTTGACAAGCTCCAGTC 4 cLats2 kinase-dead mutation DA, sense GATGGGCATATCAAACTGACTG CCTTCGGACTATGTACGGG cLats2 kinase-dead mutation DA, antisense CCCGTACATAGTCCGAAG GCAGTCAGTTTGATATGCCCATC chick Cyclin D1 forward w/ EcoR I, into pBSSK CATA GAATTC GAGAGAGCGAGCGAGAGACTGACTCAGTCC chick Cyclin D1 reverse w/ Kpn I, into pBSSK CACG GGTACC TCTTAAATGTTCACATCTCGCACATCAG chick Cyclin D1 forward w/ EcoR V, in frame w/ pMIW vectors GCCG GATATC TCGAGACGAAGGAGGCAGACATGG chick Cyclin D1 reverse w/ Xba I CATA TCTAGA TTAAATGTTCACATCTCGCACATCAG chick Cyclin D2 forward w/ EcoR I, into pBSSK CATA GAATTC ATGGAGTTGCTGTGCTGCGAGGTGGATCC chick Cyclin D2 reverse w/ Kpn I, into pBSSK CACG GGTACC TCACAGGTTGATATCTCTCACATCTGTGG chick c-Myc forward w/ EcoR I, into pBSSK CATA GAATTC ATGCCGCTCAGCGCCAGCCTCCCCAGCAAG chick c-Myc reverse w/ Kpn I, into pBSSK CACG GGTACC TATGCACGAGAGTTCCTTAGCTGCTCAAG chick N-Myc forward w/ EcoR I, into pBSSK CATA GAATTC ATGCCGGGAATGATCAGCAAGAACCCGGAC chick N-Myc reverse w/ Kpn I, into pBSSK CACG GGTACC TTCTGCTCCTCTGCCTGAAGGGAATGGAC

5 Table S2. Plasmid Concentrations Used for In Ovo Electroporation

Plasmid Name Concentration ( µg/ µl) * YAP, YAP ∆60-89 0.4 - 0.8 TEA-VP16, TEA R59K -VP16 0.4 - 0.8 TEAD1 0.2 - 0.8 YAP-IRES-GFP, YAP ∆60-89 -IRES-GFP 0.6 TEA-VP16-IRES-GFP, TEA R59K -VP16-IRES-GFP 0.6 Cyclin D1-IRES-GFP 1.5 shYAPs (for 45 hpe embryos) 0.9 shYAPs (for 22 hpe embryos) 0.4 mYAP 0.4 279 279 ∆ YAP N , YAP N 1.5 R59K R59K TEA, TEA , TEA N 0.4 d4EGFP N 0.8 TEA-EnR, TEA R59K -EnR 0.2 YAP 279 -EnR, YAP 279 ∆-EnR 0.2 – 0.4 Mst2 KD 3 Lats2 KD 3 shLats1 0.6 Lats1 KD 1.2 pRFPRNAi 0.6 Mst2 0.8 YAP S126A 0.2 GFP 0.3

* For electroporation, 9 l of DNA and 1 l of 1% Fast Green were combined. The concentrations listed here are the final concentration in 10 l of solution.

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Table S3. Antibodies Used for Immunostaining, Western Blot (WB), and Immunoprecipitation (IP)

Antibody Name Source Host Dilution YAP Cell Signaling rabbit 1:150 WB: 1:500 YAP (H125) Santa Cruz Biotechnology rabbit IP: 4 g/ml lysate 1:150 phospho-YAP Ser127 Cell Signaling rabbit WB: 1:1,000 1:500 TEF-1/TEAD1 BD Biosciences mouse WB: 1:1,000 – 2,500 Chemicon rabbit 1:2,000 Sox2 (Y17) Santa Cruz Biotechnology goat 1:500 BrdU Accurate rat 1:200 Tuj1 Covance mouse 1:2,000 PKC ζ/aPKC Santa Cruz Biotechnology rabbit 1:1,000 phospho-Histone H3 Ser10 Chemicon Upstate rabbit 1:1,000 cleaved caspase 3 Cell Signaling rabbit 1:200 1:50 Myc (9E10) Santa Cruz Biotechnology mouse WB: 1:500 1:200 Myc Sigma rabbit IP: 6 g/ml lysate 1:200 HA (HA.11) Covance mouse WB: 1:1,000 1:200 HA Novus rabbit IP: 10 g/ml lysate GFP Invitrogen rabbit 1:1,000 NF (3A10) mouse 1:50 Lim1/2 (4F2) Developmental Studies mouse 1:50 Hybridoma Bank Lhx3 (67.4E12) (supernatant form) mouse 1:50 Isl1/2 (39.4D5) mouse 1:50 Ngn2 Jonas Muhr rabbit 1:200

7 Figure S1 Cao et. al.

A

TEAD1TEAD3TEAD4Mock

anti-TEAD1

anti-Myc

TEAD1 pYAP TEAD1 pYAP B HH27-28

TEAD1 Lim1/2 TEAD1 Isl1/2 TEAD1 Lhx3 C D E HH27-28, thoracic

TEAD4 mRNA G F AP

Input IP: anti-YIP: anti-Myc

Blot: IgG

HH27-28 anti-TEAD1 TEAD1 Figure S2 Cao et. al.

Cyclin D1 GFP DAPI A 60-89 ∆ , 22 hpe YAP

B R59K TEA -VP16 TEA Figure S3 Cao et. al.

A HA-mRFP HA-YAP

shYAP#1shYAP#2shGFP HA-YAP Blot: HA HA-mRFP

Caspase 3 GFP BrdU YAP B D 279 N , 22 hpe YAP

Caspase 3 Myc BrdU Myc C E TEA, 22 hpe

Cyclin D1 GFP DAPI Cyclin D1 GFP DAPI F 8 hpe G 12 hpe 279 N

YAP

H 8 hpe I 12 hpe TEA Figure S4 Cao et. al.

A YAP 279 ∆ -EnR WW EnR

R59K TEAR59K -EnR TEA* EnR

BrdU YAP NF YAP Lim1/2 YAP B CD ∆ 279 -EnR, 22 hpe YAP BrdU Myc NF Myc Lim1/2 Myc EF G R59K TEA -EnR, 22 hpe TEA Figure S5 Cao et. al.

dsRED HA Merge AB C KD Lats1 + pRFPRNAi D EF KD Lats1 + shLats1