SUPPLEMENTARY INFORMATION

Transcription

1 doi: /nature06403 Supplementary Information: Nanog safeguards pluripotency and mediates germline development Supplementary Tables SUPPLEMENTARY INFORMATION mrna Forward primer Reverse primer Annealing Product Temp ( o C) size (bp) Nanog ATGAAGTGCAAGCGGTGGCAGAAA CCTGGTGGAGTCACAGAGTAGTTC Oct4 GGCGTTCTCTTTGGAAAGGTGTTC CTCGAACCACATCCTTCTCT Sox2 GGCGGCAACCAGAAGAACAG GCTTGGCCTCGTCGATGAAC Dnmt3L ACATCTGCCTCTGCTGTGGAACTC TCCCTCTTGATCATGGAAGGCCTT Dppa2 CACCATGTCATACTTCGGCCTGGAGACT ACTCTACTCTTTTCTCCTTTGGCACCC Dppa4 TTCTGGATGAGAAAGGCACC TGCCCCAAGTGTGTTCATAA Ecat1 TGTGGGGCCCTGAAAGGCGAGCTGAGAT ATGGGCCGCCATACGACGACGCTCAACT Ecat8 CTGGTGCAGGGCTCTGATTAAGTC TTTACACAGCCGTTCTTCTCGTCA Ecat16 GACCGGGCTGGCTTCCTGTCACCTAGT TTTACCATTTTCGGTGGCAAGGCTTCC Eras CAAAGATGCTGGCAGGCAGCTACC GACAAGCAGGGCAAAGGCTTCCTC Esg1 ATAAGCTTGATCTCGTCTTCC CTTGCTAGGATGTAACAAAGC Fbx15 TTCCTGTGTAGCACCTTCCA TCCTACGCTGTCCATGTACT Fgf4 CGAGGGACAGTCTTCTGGAG TCTTACTGAGGGCCATGAAC Fgf5 CTTCAGTCTGTACTTCACTGG AAAGTCAATGGCTCCCACGAA Gdf3 AGTTTCTGGGATTAGAGAAAGC GGGCCATGGTCAACTTTGCCT Nr0b1 TCCAGGCCATCAAGAGTTTC ATCTGCTGGGTTCTCCACTG Rex1 GACATCATGAATGAACAAAAAATG CCTTCAGCATTTCTTCCCTG Sall1 CACCATGTCACGGAGGAAGCAAGCGAAGC TTACAAGGGGTTGGCAGATGTTCGTAAA Sall4 ATCCGAGCACAGCCCACCTTTGTC ACGCTGGTGTACTGGTTCCAGGAG Sox15 CACCATGGCGCTGACCAGCTCCTCACAA TTAAAGGTGGGTTACTGGCAT Stella CACCATGGAGGAACCATCAGAGAAAGTC CTAATTCTTCCCGATTTTCGCATTCT T GTGACTGCCTACCAGAATGA ATTGTCCGCATAGGTTGGAG Tcl1 CACCATGGCTACCCAGCGGGCACACA TTATTCATCGTTGGACTCCGAGTCTATCAG Utf1 GGACCCTTCGATAACCAGATCC CAGGTTCGTCATTTTCCGCA Zfp296 AAGCACCCAGATCTGTTGACCT GAGCCTCTGGGGTATCTAGG GAPDH CCCACTAACATCAAATGGGG CCTTCCACAATGCCAAAGTT Supplementary Table 1. Oligonucleotides used for RT-PCR. 1 DMSO included at 5%. 1

2 doi: /nature06403 SUPPLEMENTARY INFORMATION mrna Forward primer Reverse primer UPL probe # Product size (bp) Oct4 GTTGGAGAAGGTGGAACCAA CTCCTTCTGCAGGGCTTTC Rex1 CAGCTCCTGCACACAGAAGA ACTGATCCGCAAACACCTG GATA6 GGTCTCTACAGCAAGATGAATGG TGGCACAGGACAGTCCAAG GATA4 GGAAGACACCCCAATCTCG CATGGCCCCACAATTGAC TBP GGGGAGCTGTGATGTGAAGT CCAGGAAATAATTCTGGCTCA Supplementary Table 2. Oligonucleotides used for Q-PCR. 2

3 doi: /nature06403 SUPPLEMENTARY INFORMATION Cell line 1 Embryo number % Germ cell chimaerism 2 (n) RCNβ(t) (+/-) (1); >70 (1) RCNβH-B(t) (-/-) 3 10 (1); >70(2) Supplementary Table 3. Summary of contributions to germ cell chimaerism of genital ridges at E11.5 in chimaeras with >70% somatic chimaerism. 1 Cell lines used in morula aggregations with MF1 morulae are indicated, as are the Nanog genotypes (brackets) 2 DAPI staining in confocal sections allowed counting of total cell number. Percentage somatic chimaerism was calculated as (GFP + /DAPI + ) x 100 in the area of the genital ridge after excluding Oct4 + or Mvh + cells from the GFP + population. Percentage germ cell chimaerism is (GFP + /Mvh + Oct4 + ) x

4 doi: /nature06403 SUPPLEMENTARY INFORMATION Cell line 1 % somatic Number of % Germ cell chimaerism 2 (n) chimaerism 2 embryos RCNβ(t) (+/-) < (1); <10 (2); (1) (1); <10 (3); (1) NA >70 3 <10 (1); (1); >70 (1) RCNβH B(t) (-/-) < > RCNβH B(t) < Rescue 0 (+/-) NA >70 3 <10 (1); (1); >70 (1) RCNβH B(t) < Rescue 5 (+/-) (1); <10 (1); (1) NA >70 0 NA RCNβH B(t) < (3) Rescue 10 (+/-) NA (1) >70 1 <10 (1) Supplementary Table 4. Summary of contributions to chimaerism in somatic and germ cells within the genital ridge at E Cell lines used in morula aggregations with MF1 morulae are indicated, as are the Nanog genotypes (brackets) 2 DAPI staining in confocal sections allowed counting of total cell number. Percentage somatic chimaerism was calculated as (GFP + /DAPI + ) x 100 in the area of the genital ridge after excluding Oct4 + or Mvh + cells from the GFP + population. Percentage germ cell chimaerism is (GFP + /Mvh + Oct4 + ) x

5 doi: /nature06403 SUPPLEMENTARY INFORMATION Supplementary Figure 1. Origin of cell lines used in this study. The three distinct strands of genetic manipulations applied in this study are outlined. *, RC cells are transfected with a CAG directed Nanog transgene in which Nanog is constitutively expressed. Following activation of Cre recombinase by Tamoxifen, Nanog is excised and GFP comes under CAG control (see Figure 2 for diagrammatic detail). Cells in which GFP is expressed from the CAG cassette are indicated in green font. The Nanog locus genotypes are indicated in brackets. In (c), cells targeted with the conditional vector are indicated Nanog Cond and Nanog Δ before and after excision of the loxp flanked Nanog gene. Supplementary Figure 2. Genetic construction of TNG cells. a, The region of the Nanog gene around exon 1 is shown at the top. Coding regions of exons are in orange, the 5 UTR is in blue and the homology arms used for construction of the targeting vector are in red. The targeting construct is shown in the middle; egfp was inserted between the homology arms precisely at the Nanog AUG codon. GFP expression is linked through an IRES to puromycin resistance encoded by the pac gene and followed by a polya site (ippa). The positions of the flanking probes used for Southern analysis and the fragement sizes produced by Sex AI digestion are indicated. b, Southern analysis of Sex AI digested DNA from targeted cells; T, E14Tg2a; A, B & C are from puromycin resistant clones that had (A, B) or had not undergone homologous recombination. 5

6 doi: /nature06403 SUPPLEMENTARY INFORMATION Supplementary Figure 3. Individual SSEA1 +, GFP - TNG cells give rise to GFP + TNG cells. a, TNG cells were analysed for SSEA1 and GFP expression (left) and cells within the indicated gates were purified by two sequential sorts. The purity of the sorted populations was determined by FACS analysis of 1000 cells for both SSEA1 +, GFP - (middle) and SSEA1 +, GFP + populations (right). b, Individual sorted cells from the middle and right hand panels in (a) were deposited in separate wells of a 96 well plate. After 7 days incubation, the number of wells containing GFP + cells was determined for plates seeded with the GFP + or GFP - fractions (left). After 21 days, the number of wells containing alkaline phosphatase positive undifferentiated colonies was determined for plates seeded with the GFP + or GFP - fractions (right). The x-axes indicate data obtained from plates seeded with cells from either the GFP + fraction or the GFP - fraction. Numbers are the average and standard deviations from 96 well plates for GFP + (n=3) and GFP - (n=5) fractions. c, Examples of 96 well plates seeded with GFP - or GFP + fractions from TNG cells and stained for alkaline phosphatase activity (shown in red) after 21 days. Supplementary Figure 4. GFP - TNG cells are more prone to differentiate than GFP + TNG cells. TNG cells were sorted into SSEA1 +, GFP + and SSEA1 +, GFP - populations of >99.5% purity and cultured for 3 days. SSEA1, GFP profiles were determined at daily intervals. Supplementary Figure 5. Simple replacement of Nanog alleles. a, The Nanog gene is diagrammed at the top, with exons shown as orange boxes. The extents of the 5 and 3 homology arms are indicated in red and the probes from 6

7 doi: /nature06403 SUPPLEMENTARY INFORMATION the regions flanking the homology arms are indicated in green. An example of a simple targeting vector is shown in the centre. Variant simple targeting vectors are identical except for the substitution of the hph gene for β-geo or hph-tk. A targeted locus is diagrammed at the bottom. The sizes of bands expected by Southern analysis using Stu I or Bam HI are indicated and can be contrasted with those expected from the wild type locus indicated at the top. b, Southern analysis of DNA from RCN cells (+/+), RCNβ cells (+/-) and two independent -/- clones (RCNβH-A; RCNβH-B) was digested with Stu I or Bam HI and hybridised with the 5 and 3 probes indicated in a. Supplementary Figure 6. Nanog deletion by Tamoxifen. a, Quantitative immunoblot analysis of Nanog overexpression in RCN cells. The indicated volumes (μl) of lysates from RCN cells and RCN(t) cells [labeled (t)] were analysed by probing with anti-nanog and anti-tubulin antibodies. b, Northern analysis of RNA from the indicated cell lines using probes from the Nanog ORF and GAPDH at the indicated times following induction of Cre activity by the addition of Tamoxifen. c, Emergence of Nanog -/- cells. RCNβH-B cells were plated at 8x10 4 cells/cm 2, treated with 1μM 4OH-Tamoxifen and photographed at the indicated times following the start of Tamoxifen treatment. Cells were passaged by re-plating at 8x10 4 cells/cm 2 after photography on d3 and d7. Note that following Nanog deletion, egfp becomes ubiquitously expressed. d, Analysis of expression of Nanog related transcripts in Nanog -/- cells. RT-PCR analyses were performed using primers that would detect Nanog, NanogPc, NanogPd 7

8 doi: /nature06403 SUPPLEMENTARY INFORMATION as well as Dppa4. 35 PCR cycles were performed on cdna derived from 10ng RNA from 2 separate cultures of RCNβΗ Β(t) and RCNβHTK(t). Supplementary Figure 7. Colony forming capacity of Nanog +/+, Nanog +/- and Nanog -/- ES cells. Cells were plated in the indicated LIF concentrations (units/ml), incubated for 6 days and stained for alkaline phosphatase activity. Differentiated colonies with a primitive endodermal morphology were counted separately from other differentiated colonies whether they were purely differentiated or of mixed morphology. Examples of colonies of each category are shown. Cell lines were RCN(t), +/+; RCNβ(t), +/-; RCNβH-B(t), -/-. Representative data from a single experiment performed in triplicate is shown; error bars are standard deviations (n=3). Supplementary Figure 8. Targeting with a conditional vector in the absence of Nanog overexpression. a The structure of the Nanog alleles in a Nanog +/- line is indicated on the top two lines. The third line diagrams the conditional targeting vector used to disrupt the wild type Nanog allele resulting in the allele shown on line four. The positions of probes flanking the homology arms used for identification of targeted cells according to the indicated restriction fragment length polymorphisms are indicated. b, Southern analysis to demonstrate homologous recombination. The cell lines used for DNA preparation include E14Tg2a before or after targeting with the simple β-geo targeting vector, E14Tg2a (Nanog +/βgeo ), Tβ and seven homologous recombinants, E14Tg2a (Nanog βgeo/conditional ), TβC. 8

9 doi: /nature06403 SUPPLEMENTARY INFORMATION c, PCR analysis indicates that resolution of homologous recombination had occurred 5 to the 5 loxp site in 5/7 putative conditionally targeted clones. d, RT-PCR analysis of conditionally targeted lines for expression of Nanog and GAPDH. Lysates were from the indicated cell lines. TβC44cre and TβC55cre are derived from TβC44 and TβC55 following Cre transfection and FACS purification of GFP expressing cells. B(t), control lysate from RCNβH-B(t). e, Immunoblot analysis of Nanog protein expression in conditionally targeted lines and deletion derivatives; abbreviations as in (d). f, Representative clonal self-renewal assays of conditionally targeted TβC44 (Nanog β-geo/cond ) cells and the deletion derivative TβC44cre6 (Nanog β-geo/δ ). Error bars are standard deviations (n=3). g, Post-natal chimaera generated by injection of Nanog -/- cells [TβC44cre6 (Nanog β-geo/δ )] into C57Bl/6 blastocysts. Patches of sandy coat colour indicate ES cell contribution. Supplementary Figure 9. Differentiation under defined neural induction conditions. a. Q-PCR analysis during defined neural differentiation. mrna levels for Rex1, Oct4, GATA4 and GATA6 are given in arbitrary units following normalisation to the TBP mrna level after the indicated number of days. Error bars are standard deviations (n=3). b Immunofluorescent detection of β-iii tubulin after 6 days in the defined neural induction protocol. 9

10 doi: /nature06403 SUPPLEMENTARY INFORMATION Supplementary Figure 10. Potency of Nanog -/- cells. a-f, contribution of Nanog -/- cells in teratomas. Panels show H&E sections obtained from teratomas obtained following injection of RCNβH-B(t) cells under the kidney capsule; a, keratinised epithelium; b, ciliated epithelium; c, chondrocytes; d, neurectoderm; e, glandular tissue; f, skeletal muscle. g, chimaera obtained from injection of Nanog -/- cells obtained by deletion of the Nanog transgene from the cell line RCNβHTK. Supplementary Figure 11. Nanog -/- cells have a defect in germ cell development. RGB versions of panels shown in Figure 4 are shown in the top row alongside panels from genital ridges of further chimaeras from Nanog -/- ES cells examined at E12.5. The bottom rows are the same sections showing separate staining for Mvh and Oct4. Supplementary Figure 12. Rescue of germ cell development in Nanog -/- cells by re-targeting of Nanog. a The structure of the two alleles in Nanog -/- cells is indicated in the top part of the panel. Below this is diagrammed the targeting vector used to rescue a functional Nanog allele by re-targeting and at the bottom is diagrammed a rescued allele. The positions of probes flanking the homology arms used for identification of targeted cells according to the indicated restriction fragment length polymorphisms is indicated. b, Southern analysis of Nanog in E14Tg2a, RCNβHTK(t); TK(t) and RCNβH- B(t); B(t) as well as nine RCNβHB(t) sub-clones in which homologous recombination of the rescuing DNA (R) had occurred. Separate hybridisation analyses indicate that in lines RH3 and RH9 replacement of the hph allele has occurred, whereas in the remaining rescue lines the geo allele has been replaced. 10

11 doi: /nature06403 SUPPLEMENTARY INFORMATION c, Immunoblot analysis of Nanog protein expression in cell lysates from E14Tg2a (+/+), RCNβHB(t) (-/-) and the indicated rescue cell lines. d, Comparative clonal self-renewal assays of Nanog -/- RCNβHB(t) cells and a representative rescue clone (RCNβH-B(t)R10). Error bars are standard deviations (n=3). 11

12 doi: /nature06403 SUPPLEMENTARY INFORMATION Supplementary Video Legends Supplementary Video 1. Bright field of GFP - TNG cells from 24-44h postplating. SSEA1 +, GFP - TNG cells were sorted by FACS to >99.5% purity, re-plated in GMEMβ/10%FCS/100u/ml LIF and incubated in 7% CO 2 at 37 o C. Time lapse imaging was initiated 24h after re-plating and continued for 20h with images being recorded at 7 min intervals. This video shows the series of bright field images. Supplementary Video 2. Fluorescent field of GFP - TNG cells from 24-44h postplating. As for Video 1, except this video shows the fluorescent images. Supplementary Videos 12

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