Supplemental data

Isolation of miR-9

An Otx2 forebrain-midbrain (FM) that directs Otx2 expression in the forebrain and midbrain does not

have its activity in the presumptive telencephalic region at E9.0 (Kurokawa et al., 2004). To identify that

function in development of the telencephalic primordium, a microarray analysis was conducted to compare

expression profiles between FM-active and inactive regions. Head was dissected from E9.0 transgenic embryos

harboring a lacZ gene under the control of Otx2 FM enhancer, and vitally stained with chromogenic solution for

βGal activity as described (Kimura et al., 1997). After staining, rostral brain was dissected into the

βGal-negative telencephalic region and βGal-positive di-mesencephalic region, and total RNAs were isolated from each region. Five μg of total RNAs was subjected to the One-Cycle Target Labelling method for synthesis

of biotin-labelled cRNA probes. The cRNA probes were subsequently fragmented and hybridized to the

GeneChip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA), according to the manufacturer’s

instruction. The microarray image data were created with GeneChip Scanner3000 (Affymetrix), and subsequently analyzed with GeneChip Operating Software (Affymetrix). Signal intensities of each transcript on the chip were compared between βGal-negative and -positive regions, and among transcripts of which expression levels were higher than 1.5 fold in the FM-inactive region, cDNA transcripts of 1432 bp (BC096523) and 3408 bp (AK032343) in length were found to have no open reading frame and to encode miR-9s; they correspond to miR-9-2 and miR-9-3, respectively.

Plasmid construction

To express miR-9, miR-9-1, miR-9-2 and miR-9-3 were isolated as approximately 200 bp genomic fragments with

23 mature miR-9 nucleotides in the middle by genomic PCR. Primer sets used are: forward

(5’-ataagaatgcggccgcCCGCTTGGTTTCAGCCTAGATT-3’; small letters indicate linker sequences with the

underline at NotI restriction site) and reverse (5’-atagtttagcggccgcCATTTTGACCAGGCCTCCAGAG-3’) for miR-9-1; forward (5’-ataagaatgcggccgcAAAGCCAAAGAGGATCGAGA-3’) and reverse

(5’-atagtttagcggccgcCTGGTTTTTACTGTCTCTTG-3’) for miR-9-2; forward

(5’-ataagaatgcggccgcAACTGGGGATGGAGAAATCGCGAG-3’) and reverse

(5’-atagtttagcggccgcCCCAAAATCCTAGTCTGCAGCTCA-3’) for miR-9-3, respectively. To express these in

P19 cells, the 200 bp fragments were inserted into pEF-1α vector (Invitrogen, Carlsbad, CA) at the NotI site to produce miR-9-1WT, miR-9-2WT and miR-9-3WT. To express miR-9 in mouse telencephalon, the 200 bp miR-9-2

fragment was inserted into pCMS-EGFP vector (Clontech, Tokyo, Japan) at the NotI site to produce

CMV-miR9-2WT-SV-EGFP.

To generate luciferase reporter constructs, wild type and mutant DNA fragments harboring putative miR-9 target sequences of Foxg1, Id4, Lhx5 and Nr2E1 3’ UTRs were synthesized as DNA oligonucleotides. Their sizes were 48 bp, and target sequences were placed in the middle of each fragment: top strand

(5’-gTTCTTTCCTTTTGTTTACTTTTAGACCAAAGATTGGATTCTAGCAAA-3’) and bottom strand

(5’-cTTTGCTAGAATCCAATCTTTGGTCTAAAAGTAAACAAAAGGAAAGAA-3’) for luc-Mm-Foxg1WT;

top strand (5’-gTTCTTTCCTTTTGTTTACTTTTAGACGTTTGATTGGATTCTAGCAAA-3’) and bottom

strand (5’-cTTTGCTAGAATCCAATCAAACGTCTAAAAGTAAACAAAAGGAAAGAA-3’) for

luc-Mm-Foxg1MT; top strand

(5’-gCACGCGCCTGCGCAATTTCTCGGGACCAAAGTCAATATTCTGAAGGG-3’) and bottom strand:

(5’-cCCCTTCAGAATATTGACTTTGGTCCCGAGAAATTGCGCAGGCGCGTG-3’) for luc-Mm-Lhx5WT; top

strand (5’-gGAAAAATGTTCTCTGCTTGCTACCAAAGGACAAACTCTTGGAAACGG-3’) and bottom strand (5’-cCCGTTTCCAAGAGTTTGTCCTTTGGTAGCAAGCAGAGAACATTTTTC-3’) for luc-Mm-Id4WT; top strand

(5’-gGTGTCCCGTCATCAAAGTGCTGAAACCAAAGGCCATAGGGTCAGCGA-3’) and bottom strand

(5’-cTCGCTGACCCTATGGCCTTTGGTTTCAGCACTTTGATGACGGGACAC-3’) for luc-Mm-Nr2E1WT;

top strand (5’-gGTTTGTTTGTTTGTTTACTTTTAGACCAAAGATTGGGTTTTAGAAAA -3’) and bottom strand (5’-c TTTTCTAAAACCCAATCTTTGGTCTAAAAGTAAACAAACAAACAAAC-3’) for luc-Gg-Foxg1WT; top strand

(5’-gGCTGGGTTTTTTGTTTACTTTTAGACCAAAGATTGGGTTGTAGATCA-3’) and bottom strand

(5’-cTGATCTACAACCCAATCTTTGGTCTAAAAGTAAACAAAAAACCCAGC-3’) for luc-Xt-Foxg1WT; top strand (5’- gATTAACAATACAGTTTCTTTGTAGGCCAAAGAATGGGTATCCAGCAA -3’) and bottom

strand (5’-c TTGCTGGATACCCATTCTTTGGCCTACAAAGAAACTGTATTGTTAAT-3’) for luc-Dr-Foxg1WT. Letters underlined indicate sequences predicted to make base pairing with a seed sequence of miR-9; letters in bold indicate four nucleotides substituted; small letters indicate additional nucleotides for concatemerization. After annealing and concatemerization of each oligonucleotide, the concatemers of four copies were isolated and subcloned into pGL3-TK vector (Promega, Madison, WI) at the XbaI site to place them at 3’ downstream of firefly luciferase gene directed by TK and to generate luc-Mm-Foxg1WT, luc-Mm-Foxg1MT, luc-Mm-Lhx5WT, luc-Mm-Id4WT or luc-Mm-Nr2e1WT, respectively.

Probes for in situ hybridization and antibodies for immunohistochemistry and immunoblot

Sources of probes used for RNA in situ hybridization are: TTR (Wakasugi et al., 1985), Wnt3a (Roelink and

Nusse, 1991), Foxg1 (Tao and Lai, 1992), Reelin (D'Arcangelo et al., 1995), NeuroD1 (Schwab et al., 1998),

Ngn2 (Sommer et al., 1996), p21 (Siegenthaler and Miller, 2008) and (Meyer et al., 2002; Cabrera-Socorro et al., 2007). An EGFP PCR product, which possesses T7 RNA polymerase site at the 3’ end, was obtained from pCMS-EGFP vector (Clontech) and used as the template for EGFP RNA probe. BM Purple or INT/BCIP (Roche,

Basal, Switzerland) was used for chromogenic reactions. In situ hybridization to detect miR-9 expression was carried out with the antisense oligonucleotide of the 23 mature nucleotides. NeuroD1, Ngn2, p73, p21, and

Foxg1 cDNA clones were obtained from the Functional Annotation of Mouse-3 (FANTOM3) library (Carninci

et al., 2005). Primary antibodies used for immunohistochemistry were anti-Foxg1 (1:500; Watanabe et al., 2005) , anti-Reelin

(1:200; Chemicon, Temecula, CA), anti-p73 (1:200; LabVision, Fremont, CA), anti-p21 (1:2000; BD

Biosciences, San Diego, CA), anti-GFP-Alexa488 (1:200; Invitrogen, Eugene, CA), anti-GFP (1:200; Nakalai

Tesque, Kyoto, Japan) and anti-Tuj1 (1:500, Covance, Berkeley, CA); secondary antibodies were conjugated with Alexa 350, 488 or 594 (1:200; Invitrogen). Immunoblot was conducted with primary antibodies against

Nr2E1 (1:500; PPMX, Tokyo, Japan), Foxg1 (1:1000; Watanabe et al., 2005) and GAPDH3 (1:5000, Everest

Biotech, Upper Heyford, UK), and with secondary antibodies conjugated with horseradish peroxidase (1: 2000;

Upstate, Temecula, CA, or Cell Signaling, Beverly, MA).

RT-PCR primer sets

Primers to detect the expression of each gene were designed in a single exon encoding 3’ UTR: forward

(5’-TATTGGCCATTTATTGTTTTGTCCT-3’) and reverse (5’-TACCAAGTGCATTTGCTAGAATCC-3’) for

Foxg1; forward (5’-CTGCACACACATCCACCGAG-3’) and reverse (5’-GCGTCACTGCTCTGATTCCC-3’)

for Emx2; forward (5’-CAATGTCCCAGGCTCATTCA-3’) and reverse

(5’-TCAGTGCCAACTACCTGTTGGT-3’) for Otx2; forward (5’-CCTTGGTGACCCTCCTCAAG-3’) and

reverse (5’-TTCCTGGGCAGCTCTAAACTG-3’) for Hoxb1; forward (5’-ACAGGCTGAGCCAGGTTCTG-3’) and reverse (5’-AATGATTCAGCGGGATCTTCC-3’) for Nr2E1; forward

(5’-GTGATGTGAAGTTCCCCATAAGG-3’) and reverse (5’-CTACTGAACTGCTGGTGGGTCA-3’) for

TATA binding , respectively. Primers to detect each miR-9 pri-miRNA were designed at 3’ downstream of the loop structure: forward (5’-AAAATAACCCCATACACTGCGC-3’) and reverse

(5’-TCGGCCACTACCAGCGTT-3’) for miR-9-1; forward (5’-AAAACTCCTTCAAGGTCACCGA-3’) and reverse (5’-GGTTGCAGTCTTTCTTTCCGG-3’) for miR-9-2; forward (5’-ATGACTCTCACAACTTCTGC-3’) and reverse (5’-TTCTCTGGGCTCCTCTGGCT-3’) for miR-9-3.

Cell culture, transfection and Luciferase assay

Mouse P19 cells were cultured in Dulbecco’s minimal essential medium (DMEM; Invitrogen) supplemented

with 10% fetal bovine serum (Equitech-Bio, Kerrville, TX). In the Luciferase assay, routinely, 9 x103 cells were

plated onto 96-well plates and transfected the next day with a firefly luciferase reporter, a renilla luciferase vector (Promega) and a miR-9 expression vector (1 μg DNA each); renilla luciferase vector served to normalize

transfection efficiency. Transfection was performed using Lipofectamine according to the manufacturer’s

protocol (Invitrogen, Carlsbad, CA). Cells were harvested 48 hours after the transfection and processed with the

Dual-Glo Luciferase Assay System (Promega); Luciferase activity was measured with 1420ARVOsx-1

luminometer (PerkinElmer, Waltham, MA). Firefly luciferase mRNA was detected with the primers: forward

(5’-CGCTGGAGAGCAACTGCATA-3’) and reverse (5’- CCAGGAACCAGGGCGTATCT-3’). It was

normalized by the amount of the amplification of renilla luciferase cotransfected; the primers used were forward

(5’-GAGAACGCCGTGATTTTTCTG-3’) and reverse (5’-CCACAGGTAGCTGGAGGCAG-3’). In examining

miR-9 effects on Foxg1 expression in neuralized P19 cells, 1.5 x105 cells were plated onto gelatin-coated 6-well

plates and transfected the next day with a miR-9 expression vector (1μg DNA) or with LNA antisense

oligonucleotides against miR-9, or miR-159 as a control, (40 nM). One day after the transfection or two days

after plating the cells were treated with 1 nM all-trans RA (Sigma, St. Louis, MO) for two days in serum-free

medium (DMEM Nutrient Mixture F-12 HAM, Sigma) supplemented with ITS-X supplement (Invitrogen) and

0.05 mM 2-mercaptoethanol (Sigma). The cells were further cultured for indicated days without RA before the

assay. All experiments were repeated at least three times.

Supplementary Figure 1:

Quantitative RT-PCR for miR-9-1, miR-9-2 and miR-9-3 expression in whole embryos at indicated stages (left

panel) and in whole brain, telencephalon and mesencephalon at E10.5 (right panel). The ordinate gives the copy

number of each transcript.

Supplementary Figure 2:

miR-9 targets. (A) Plausible base pairing to the putative binding sites of the 3’ UTRs of mouse Lhx5, Nr2e1, Id4

and Foxg1 mRNAs. (B) Luciferase assay for miR-9 targeting of Lhx5, Nr2e1, Id4 and Foxg1 3’UTRs.

Supplementary Figure 3:

Effects of miR-9 on Foxg1 protein expression in neuralized P19 cells. (A) Pattern of P19 neural differentiation.

The expression of brain and spinal cord markers (Foxg1, Emx2, Otx2, Hoxb1) is compared between P19 cells

treated with or without 1 nM RA for two days after the second day of plating by quantitative RT-PCR. Cells were

cultured another two days after removal of RA. (B) Foxg1 and miR-9-2 expression with 1nM RA treatment. After

removal of RA, the cells were further cultured for indicated days. (C) The effect of miR-9-2 over-expression on

Foxg1 and Nr2e1 protein expression in RA-treated P19 cells. GAPDH3 served as loading control. Experiments

were conducted three times; Foxg1 protein levels quantified were 26%, 23% and 35% of the control by miR-9-2

WT; 83%, 97%, and 73% by miR-9-2MT, respectively. Nr2e1 protein levels were 84%, 97%, and 110% of the control by miR-9-2WT; 97%, 83%, and 102% by miR-9-2MT, respectively. (D) The effects of miR-9-2 over-expression on Foxg1 and Nr2e1 expression at mRNA level in the P19 cells. (E) The effects of miR-9 knock-down on Foxg1 protein expression in the P19 cells. Experiments were conducted two times; Foxg1 protein levels quantified were 1.6, and 1.4 fold of the control by LNA-miR-9AS, while they were 0.8, and 1.1 fold by

LNA-miR-159AS as a control.

Supplementary Figure 4:

Effects of miR-9 on Cajal-Retzius cell differentiation in vivo. CMV-miR-9-2WT-SV40-EGFP or SV40-EGFP was electroporated at E11.5 (A) or at E14.5 (B). Twenty four hours later, the neocortices were immunostained for p73

(Aa), p21 (Ab), Reelin (Ac, B), and EGFP. Scale bars: (A) 20 μm; (B) 100 μm.

Supplementary References

Cabrera-Socorro A, Hernandez-Acosta NC, Gonzalez-Gomez M, Meyer G (2007) Comparative aspects of p73

and Reelin expression in Cajal-Retzius cells and the cortical hem in lizard, mouse and human. Brain Res

1132:59-70.

Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, Oyama R, Ravasi T, Lenhard B, Wells C, et

al. (2005) The transcriptional landscape of the mammalian genome. Science 309:1559-1563.

D'Arcangelo G, Miao GG, Chen SC, Soares HD, Morgan JI, Curran T (1995) A protein related to extracellular

matrix deleted in the mouse mutant reeler. Nature 374:719-723.

Kimura C, Takeda N, Suzuki M, Oshimura M, Aizawa S, Matsuo I (1997) Cis-acting elements conserved between

mouse and pufferfish Otx2 genes govern the expression in mesencephalic cells. Development

124:3929-3941.

Kurokawa D, Kiyonari H, Nakayama R, Kimura-Yoshida C, Matsuo I, Aizawa S (2004) Regulation of Otx2

expression and its functions in mouse forebrain and midbrain. Development 131:3319-3331.

Meyer G, Perez-Garcia CG, Abraham H, Caput D (2002) Expression of p73 and Reelin in the developing human

cortex. J Neurosci 22:4973-4986.

Roelink H, Nusse R (1991) Expression of two members of the Wnt family during mouse development--restricted

temporal and spatial patterns in the developing neural tube. Genes Dev 5:381-388.

Schwab MH, Druffel-Augustin S, Gass P, Jung M, Klugmann M, Bartholomae A, Rossner MJ, Nave KA (1998)

Neuronal basic helix-loop-helix proteins (NEX, neuroD, NDRF): spatiotemporal expression and targeted

disruption of the NEX gene in transgenic mice. J Neurosci 18:1408-1418.

Siegenthaler JA, Miller MW (2008) Generation of Cajal-Retzius neurons in mouse forebrain is regulated by

transforming growth factor beta-Fox signaling pathways. Dev Biol 313:35-46.

Sommer L, Ma Q, Anderson DJ (1996) neurogenins, a novel family of atonal-related bHLH factors, are putative mammalian neuronal determination genes that reveal progenitor cell heterogeneity in the

developing CNS and PNS. Mol Cell Neurosci 8:221-241.

Tao W, Lai E (1992) Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene

family, in the developing rat brain. Neuron 8:957-966.

Wakasugi S, Maeda S, Shimada K, Nakashima H, Migita S (1985) Structural comparisons between mouse and

human prealbumin. J Biochem (Tokyo) 98:1707-1714.

Watanabe K, Kamiya D, Nishiyama A, Katayama T, Nozaki S, Kawasaki H, Watanabe Y, Mizuseki K, Sasai Y

(2005) Directed differentiation of telencephalic precursors from embryonic stem cells. Nat Neurosci

8:288-296.