Supplementary Table S1. Primer sequences used in this study

Genotyping

Prdm14 wild-type allele

Prdm14 mutant allele

Prdm1 wild-type allele (floxed)

Prdm1 mutant allele

Bmp4 wild-type allele

Bmp4 mutant allele

Smad1 wild-type allele

Smad1 mutant allele

Prdm14-mVenus, Prdm1-mVenus transgenic allele

Q-PCR

Spike RNA (1000 copies)

Gapdh

Prdm1

Dppa3 (stella)

Hoxb1

Hoxa1

Snai1

Pou5f1 (Oct4)

Sox2

Nanog

Dnd1

Kit

5'-AAGGTTCTGGGAACTGGATGTC-3', 5'-CACAATATGCTGGCATGCGTTC-3'

5'-CCTCACTCAGCCAGCTCGGT-3', 5'-GGTCTCTGGCATCTTAGTTG-3'

5'-GTCGCCTGATCGTGCTCGAC-3', 5'-GCTGGATTCACGTAGCGCATC-3'

5'-GTCGCCTGATCGTGCTCGAC-3', 5'-GCACAGGCCTGTGATCCTAGC-3'

5'-CAAACTTGATCTTTCGGACCTG-3', 5'-TTCCCGGTCTCAGGTATCAAAC-3'

5'-CAAACTTGATCTTTCGGACCTG-3', 5'-TTCGCCAATGACAAGACGCTG-3'

5'-TCAGTGGTGCTTTGAACCCT-3', 5'-TTGTGAGGGAAGATGACTGG-3'

5'-TCAGTGGTGCTTTGAACCCT-3', 5'-TGGATGTGGAATGTGTGCGA-3'

5'-ACTCATCTCAGAAGAGGATCTG-3', 5'-CACAGTCGAGGCTGATCTCG-3'

　

5'-GCCATATCGGCTCGCAAATC-3', 5'-AACGAATGCCGAAACCTCCTC-3'

5'-ATGAATACGGCTACAGCAACAGG-3', 5'-CTCTTGCTCAGTGTCCTTGCTG-3'

5'-AGCATGACCTGACATTGACACC-3', 5'-CTCAACACTCTCATGTAAGAGGC-3'

5'-AGGCTCGAAGGAAATGAGTTTG-3', 5'-TCCTAATTCTTCCCGATTTTCG-3'

5'-GATCCTACAGGTCTTGGGACC-3', 5'-AGCTCAAAGGCACTGAACTGAG-3'

5'-GTGACTAGTCTTCTGCATGTCG-3', 5'-TCTGCTCTGGACCACATCACTC-3'

5'-AGCAGGGTGGTTACTGGACAC-3', 5'-CCATTATTCATGGTCCCTTCTG-3'

5'-GATGCTGTGAGCCAAGGCAAG-3', 5'-GGCTCCTGATCAACAGCATCAC-3'

5'-CATGAGAGCAAGTACTGGCAAG-3', 5'-CCAACGATATCAACCTGCATGG-3'

5'-CTTTCACCTATTAAGGTGCTTGC-3', 5'-TGGCATCGGTTCATCATGGTAC-3'

5'-CCCTAAATGGGTTAAGCAGAGC-3', 5'-GGCAAGGTTCCTCACAACTAAAG-3'

5'-GGCATTCCTATGTGATTATTGTGTC-3', 5'-TGCAAACTCCAATCTATGACTGAAC-3'

Critical function of Prdm14 for the establishment of the germ cell lineage in mice

Masashi Yamaji, Yoshiyuki Seki, Kazuki Kurimoto, Yukihiro Yabuta, Mihoko Yuasa, Mayo Shigeta, Kaori Yamanaka, Yasuhide Ohinata, and Mitinori Saitou

b

c

01234

Gapdh

01234

Prdm14

01234

Prdm1

01234

Dppa3

(stella)

01234

Pou5f1

(Oct4)

E6.75 E7.25

Prdm2 / Riz1Prdm16

Prdm9 / MeisetzPrdm10 / Tristanin

Prdm15

Prdm1 / Blimp1Prdm4

Prdm6 / PrismPrdm12

Prdm5Prdm13

Prdm8BAE39788

Suv4-20h2

mKIAA1936Smyd1Smyd2

Smyd3Smyd5

Suv39h1

Eu-HMTase1 / GLPEu-HMTase2 / G9a

EsetSetdb2

SetmarWhsc1

Ash1Ezh2Ezh1Mll2Mll

Mll5

Setd1bSetd1a

Mll3Setd8

Setd5Setd7 / Set7/9

Whsc1l1Nsd1

Suv39h2

Suv4-20h1

Prdm14

a

ES

B: BrainH: HeartS: SpleenLu: LungLi: LiverM: Sk. muscleK: KidneyT: TestisO: Ovary

8: EG(E8.5)12: EG(E12.5)

B H S Lu Li M K T O 8 12ES

4.8kb

1.9kb

Gapdh

M_musculusR_norvegicusH_sapiensP_troglodytesM_mulattaC_familialisB_taurusM_domesticaG_fallusX_tropicalisT_nigroviridisD_rerioC_elegans

100847070706766606555475026

% IdentitiyPR domain Zinc finger domain

d

Supplementary Figure S1

Supplementary Figure S1.a, Phylogenetic tree of SET/PR domain-containing proteins. Prdm1 (Blimp1) and Prdm14 are highlighted in orange and red, respectively. b, Conservation of Prdm14 in the indicated species. The sequences of Prdm14 authologs in the indicated species were retrieved from the Ensembl database. c, Northern blot analysis of Prdm14 expression in various adult tissues, ES cells, and two independent EG cell lines derived from E8.5 and E12.5 PGCs, respectively. A control blot for Gapdh is shown at the bottom. d, Expression of Prdm14 in Prdm1 and Pou5f1 (Oct4)-positive single-cell cDNAs from MS (E6.75) and EB (E7.25) stage embryos ref. The vertical axis represents estimated log10 expression levels of the indicated genes and each bar corresponds to a single cell. Single cells were sorted according to their Prdm14 expression levels.

A referenceYabuta, Y., Kurimoto, K., Ohinata, Y., Seki, Y. & Saitou, M. Gene expression dynamics during germline specification in mice identified by quantitative single-cell gene expression profiling. Biol Reprod 75, 705-16 (2006).

Prdm14-mVenusDAPIPrdm14-mVenus

Oct4Prdm14-mVenus

MergePrdm14-mVenus

Merge

Prdm14-mVenus

E8.5

i

Supplementary Figure S2

c

Mal

eFe

mal

e

E12.5 E14.5 E16.5

fMerge

Prdm14-mVenus

E5.5 E6.75E6.5

E9.5g h

magnified

a Morula

MergePrdm14-mVenus

E3.5

MergePrdm14-mVenus

b

d e

i

j Pou5f1 (Oct4) Prdm143

2

1

0

3

2

1

0

25/50 (50%)

Log 1

0 co

py n

umbe

r

Prdm14-mVenus Prdm14-mVenusDAPI stella

stellaE7.75

Supplementary Figure S2.a-i, Expression of Prdm14-mVenus in a morula (a), a blastocyst (b), embryos at E5.5 (c), E6.5 (d), E6.75 (e), E7.75 (f), E8.5 (g), and E9.5 (h), and male and female genital ridges from E12.5 to E16.5 (i). Merged images with DAPI (white) are also shown. In d and e, anterior is to the left. f shows a posterior view. A majority of Prdm14-mVenus-positive cells co-expressed stella (Dppa3) (red). The boxed regions in h (upper panels) are magnified in the lower panels. Prdm14-mVenus-positive cells in the hindgut at E9.5 merged completely with Oct4-positive cells (red). In i, phase contrast images are shown at the left of each panel. Bars in a-f, and i, 50 µm; in g, 75 µm; in the upper panel in h, 400 µm; in the lower panel in h, 100 µm. j, Single-cell expression analysis of Pou5f1(Oct4) and Prdm14 in E3.5 inner cell mass (ICM) cells. The vertical axis represents the estimated copy number of each gene at the log10 scale and each bar represents a single cell. Prdm14 is expressed in approximately half of the ICM cells corresponding to epiblast-like cells ref. Prdm14 is initially weakly expressed in blastocysts at E3.5 (b), but then is quickly down-regulated, and no cells express Prdm14 at E5.5 (c). Prdm14 expression re-starts in a few cells locating in the region most proximal to the epiblast/primitive streak at E6.5 (ES stage, arrowheads in d) and subsequently continues only in the germ cell lineage until about E14.5.

A referenceKurimoto, K. et al. An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis. Nucleic Acids Res 34, e42 (2006).

Tota

l num

ber o

f rep

orte

r-pos

itive

PG

Cs

0

10

20

30

40

50

60

70

80

90

100Cas3(+)LS-EBMB-LBEHF-LHF

Prdm14 genotype +/- -/- +/+

a

Male Female

Wild Hetero Null

Delivery11 21 10

(26%) (50%) (24%)

40 89 43Fetus(E7.5) (23%) (52%) (25%)

c

d

b

3.9kb

+/+ +/-

Neo probe( HindIII )

+/+ +/-

8.4kb

4.2kb

5' probe( Bgl II )

Supplementary Figure S3

DAPI DAPI,Cas3

e

Prdm14

+/+

Prdm14-/-

f

mag

nifie

dCas3

Cas3Prdm1-mVenus

3.9 kb

ATG

ATG

ATG

5'probe

8.4 kb

neo probe

4.2 kb

BglII BglII

BglII

BglII

BglII

PGKneo

PGKneo

HindIII

HindIII

BglII

BglII

BglII

HindIII

HindIII

HindIII

Wild

Targeting construct

Targeted 1

Targeted 2

Supplementary Figure S3.a, Schematic representation of the strategy for gene targeting of Prdm14. The wild-type locus for Prdm14, the targeting construct replacing exons 2-5 of Prdm14 with Pgk-Neo flanked by loxP sequences, the resultant targeted allele, and the targeted allele from which Pgk-Neo is removed are shown. Positions of the 5' probe and the Neo probe are also shown. b, Southern blot analysis of the targeting event. The wild-type ES cell genome digested by BglII and probed with the 5' probe yields a single 8.4 kb band, whereas a heterozygously targeted ES cell genome generates two bands of 8.4 kb and 4.2 kb in length (upper). The same Prdm14 heterozygous clone, when digested with HindIII and probed with the Neo probe, generates a single 3.9 kb band, demonstrating the single, targeted integration of the targeting vector. c, Gross appearance of Prdm14-deficient mice (a male on the left and a female on the right) of 67 weeks of age. d, The numbers and percentages (in parenthesis) of Prdm14+/+, +/-, and -/- mice (E7.5 and delivered) generated from crosses between heterozygous females and males. The percentage of each genotype is in accordance with the expected Mendelian ratio. e, Typical images of Prdm14+/+ (upper panels) and -/- (middle panels) embryos with Prdm1-mVenus reporter (green) stained by anti-activated (cleaved) Caspase3 antibody (red). In the lower panels, magnified views of activated Caspase3-positive cells (red) with DAPI staining (white) are shown. The bars in the middle and the lower panels correspond to 75 µm and 15 µm, respectively. f, The numbers of activated Caspase3-positive cells among Prdm1-mVenus-positive cells in Prdm14+/+, +/-, and -/- embryos. Total numbers of Prdm1-mVenus-positive cells in LS/EB (grey), M/LB (dark grey), and E/LHF (black) stage embryos of each Prdm14 genotype are shown with the number of activated Caspase3-positive cells (red).

Supplementary Figure S4

Prdm14+/+ Prdm14-/-P

011

wk

Supplementary Figure S4.Absence of MVH-positive germ cells in Prdm14-/- testes. Histological sections of wild-type+/+ (left panels) and Prdm14-/- (right panels) testes of newborn (upper panels, post-natal day 0, P0) and 11-week-old (lower panels, 11 wk) mice were stained for MVH followed by counterstaining with hematoxylin. Wild-type+/+ testes contained MVH-positive germ cells (brown, spermatogonia at P0 ref), whereas Prdm14-/- testes were completely devoid of germ cells and there only remained Sertoli cells in the seminiferous tubules. Bar, 20 µm.

A referenceRussell, L.D., Ettlin, R.A., Sinha Hikim, A.P., Clegg, E.D. Histological and Histopathological Evaluation of the Testis, (Cache River Press, Clearwater, 1990).

Supplementary Figure S5

**

Prdm1

Prdm14

Pou5f1 (Oct4)

Hoxb1

+/+ +/- -/-0

1

2

3

0

1

2

30

1

2

30

1

2

3

Log 1

0 tra

nscr

ipt c

opy

num

ber

Supplementary Figure S5.Expressions of Prdm14 and Hoxb1 in the Prdm1- and Pou5f1 (Oct4)-positive single cells of each Prdm14 genotype. The vertical axis represents the estimated copy numbers of each gene at the log10 scale and each bar represents a single cell. Note that Prdm14 is more consistently expressed in the Hoxb1-negative cells. *: knocked out.

Supplementary Figure S6

+/+

-/-

Oct4 GLP DAPI Merge

(1/42, 2.4%)

(7/7, 100%)

Supplementary Figure S6.Impaired down-regulation of GLP in Prdm14-/-, Oct4-positive PGC-like cells at E8.25. Arrowheads indicate Oct4-positive (green), wild-type migrating PGCs (upper panels) with repressed GLP (red), whereas arrows indicate Prdm14-/- PGC-like cells (lower panels) with failed GLP down-regulation. Merged images with DAPI (blue) are also shown. Proportions (%) of GLP strong-positive (++) cells among Oct4-positive PGCs or PGC-like cells are shown in the right panels. Bar, 10 µm.

Supplementary Figure S7

a

Prdm14 Merge

b

c

Blimp1 1 117 239 605 738 856

Prdm14 1 246 360 424 561

0

1.2

1.0

0.8

0.6

0.4

0.2

Rel

ativ

e lu

cife

rase

act

ivity

Prdm14 Prdm1 pUAS-TK-Luc

0.01 0.05 0.1 0.2 pUAS-TK-Luc

0.01 0.05 0.1 0.2

Supplementary Figure S7.a, Protein structures of Prdm14 and Blimp1. Red and orange boxes represent PR-domains and zinc fingers, respectively. Amino acid numbers from the initial methionine are shown. b, Sub-nuclear localization of Prdm14 (green) in a PGC at E7.5. Merged image with DAPI (blue) is shown on the right. Bar, 2 µm. c, Relative activity of 5XUAS-TK-Luc in cells transfected with the indicated amount (µg) of the plasmids that express GAL4DBD-Prdm14 or Blimp1.

GAL4DBD-Prdm14 GAL4DBD-Prdm1