1

Visvanathan

Supplementary figure legends

Figure S1. Immunohistochemical analysis of chick neural tube electroporated with SCP1 (A) and SCP1 plus REST (B). Ectopic BrdU+-cells in the lateral zone do not express neuronal marker NF and postmitotic marker p27kip1, whereas they maintain expression of progenitor markers Nkx6.1 and Pax6 (double positive cells in arrows). A ventral quadrant of electroporated side of neural tube is shown and the outline of neural tube is marked in yellow dotted line. The medial (M) to lateral (L) orientation is as indicated.

Figure S2. (A) Electronic overlays of adjacent sections of E11.5 mouse embryo processed for in situ hybridization with miR-124 (purple) and SCP1 (blue). (B) In situ hybridization of miR-124 in E12.5 mouse embryo. (C) Quantification of GFP::SCP1-3'UTR and NF fluorescence intensity in the neural tube reveals the down-regulation of GFP in NF+-neuronal domain. On the X-axis, values 1 and 330 correspond to the most medial and lateral sides of the neural tube, respectively. The average pixel intensity of GFP and NF fluorescence in unsaturated images was determined using Axiovision software (Zeiss). The average pixel intensity from four readings performed across medial to lateral neural tube is as shown. Error bars indicate the standard deviations.

Figure S3. BrdU-staining analyses of chick neural tube electroporated with SCP1 (A), SCP1 plus miR-124 (B), miR-124-sensitive SCP1::SCP1-3'UTR (C), and miR-124 plus SCP1::SCP1-3'UTR (D). SCP1 expression, both with and without miR-124, results in laterally positioned BrdU+-cells. In contrast, miR-124-sensitive SCP1-excpression vector (SCP1::SCP1-3'UTR) , both with and without miR-124, has no such effect.

Figure S4. Immunohistochemical analysis of neuronal differentiation (TuJ+-cells) in transfected P19 cells. (A) miR-124, but neither miR-124-mt nor miR-128, triggers TuJ-expression. miR-124-mediated neurogenesis is attenuated by miR-124-resistant SCP1. (B)Neurite outgrowth upon miR-124 expression in P19 cells is as indicated in arrows.

Supplementary materials and methods

DNA constructs

CMV-miR-124 and CMV-miR-128 vectors were generated by cloning ~320-nt miR-124-2and ~320-nt miR-128 genomic regionsinto pAd-Track-CMV. GL3::SCP1-3'UTR and GFP::SCP1-3'UTR reporters were created by cloning ~1.6 kb mouse SCP1-3'UTR into pGL3 (Promega) and pEGFP-C2 (Clontech). SCP1-ires-GFP, SCP1-pi-ires-GFP, and REST expression vectors have been previously described (Yeo et al. 2005). miR-124-resistant SCP1 was generated by cloning SCP1-ORF into pCS2 vector. For miR-124-sensitive SCP1 construct, ~1.6 kb SCP1-3'UTR was inserted into downstream of SCP1-ORF. Chick SCP1 cDNA was purchased from ARK Genomics.

Sequences of oligonucleotides used in this study

Antisense / Sense
miR-124 / 5’-GCAUUCACCGCGUGCCUUAAU / 5’-UAAGGCACGCGGUGAAUGCCA
miR-124-mt / 5’-GCAUUCACCGCGUCGCUUAAU / 5’-UAAGCGACGCGGUGAAUGCCA
miR-128 / 5’-AAGAGACCGGUUCACUGUGAGA / 5’-UCACAGUGAACCGGUCUCUUUU
miR-1 / 5’-ACAUACUUCUUUACAUUCAAUA / 5’-UGGAAUGUAAAGAAGUAUGUAA
2'-OMe miR-124 / 5’-UGGCAUUCACCGCGUGCCUUAA
2'-OMe scrambled miR-124 / 5’-GAUCCCUCCCUCGGUAAGUAG U

RT-PCR primers

Gene / Forward / Reverse
ms Ngn2 / 5’-AACTCCACGTCCCCATACAG / 5’-AGGTGAGGCGCATAACGATG
ms NeuroD / 5’-CCTGATCTGGTCTCCTTCGT / 5’-AAGAAAGTCCGAGGGTTGAG
ms TuJ / 5’-GTCTCTAGCCGCGTGAAGTC / 5’-CCTGGGCACATACTTGTGAG
ms CyclophilinA / 5’-GTCTCCTTCGAGCTGTTTGC / 5’-GATGCCAGGACCTGTATGCT
GFP / 5’-TACGGCAAGCTGACCCTGAAGTTC / 5’-CTTCAGCTCGATGCGGTTCACCAG
ms SCP1 / 5’-CCAGTCCAGTACCTGCTTCC / 5’-CCCATTCGCTGTAGGAACTC

Antibodies used in this study

We used rat anti-BrdU(Harlan), mouse anti-NF (3A10, DSHB), mouse anti-p27kip1 (BD Transduction Laboratories), mouse anti-TuJ (Babco), mouse anti-NeuN (Chemicon), rabbit-tubulin (Santa Cruz), rabbit anti-GFP (Invitrogen), rabbit anti-cyclinA (Abcam), rabbit anti-LacZ (Sigma), rabbit anti-Nkx6.1 (Briscoe et al. 2000) and mouse anti-Pax6 (DSHB) antibodies.

TUNEL assays

TUNEL assays were performed using In Situ Cell Death Detection Kit (Roche).

References

Briscoe, J., Pierani, A., Jessell, T.M., and Ericson, J. 2000. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell101(4): 435-445.

Yeo, M., Lee, S.K., Lee, B., Ruiz, E.C., Pfaff, S.L., and Gill, G.N. 2005. Small CTD phosphatases function in silencing neuronal gene expression. Science307(5709): 596-600.