ニマルキャップ細胞群を用いた胚全体構造の構築への挑戦　　　

SFC－SWP 2015－010

AUTUMN2015年度　秋学期

ア

研究会優秀論文

慶應義塾大学湘南藤沢学会

森本健太　環境情報学部 4年

先端生命科学研究会

2015

Attempt to create overall embryonic structure

by the combination of artificially induced blastula centers

2

(AC)

RNA-seq Chordin Sizzled

X mRNA

gain-of-functional

CRISPR/Cas

β-Catenin VegT Siamois Twin

AC

AC

BCNE Nieuwkoop

Wnt8 mRNA VegT mRNA AC

AC AC

Keywords: , , RNA-seq, CRISPR/Cas,

3

...........................................................................................................................2

...................................................................................................................................3

1 .......................................................................................................................5

1.1. ...............................................................................................5

1.2. ...............................................................7

1.3. .......................................................9

2 RNA-seq ......................................12

2.1. .....................................................................................................................12

2.2. .....................................................................................................................15

2.2.1. ..........................................................................15

2.2.2. RNA cDNA ................................................................................15

2.2.3. RNA-seq ........................................................15

2.2.4. pCS2 ..................................................16

2.2.5. mRNA ................................................................................................16

2.3. .....................................................................................................................16

2.3.1. RNA-seq ....................16

2.3.2. SP-X ......................................19

2.4. .....................................................................................................................21

2.4.1. RNA-seq ............................................21

2.4.2. SP-X .................................................................22

3 RNA CRISPR/Cas ....24

3.1. .....................................................................................................................24

3.2. .....................................................................................................................26

3.2.1. ..........................................................................26

3.2.2. gRNA PAMmers gRNA ..................................................27

4

3.2.3. Cas9-nonNLS mRNA ........................................................................29

3.3. .....................................................................................................................29

3.3.1. CRISPR/Cas .....................................29

3.2.2. CRISPR/Cas .................31

3.4. .....................................................................................................................32

3.4.1. CRISPR/Cas ..................32

3.4.2. CRISPR/Cas ..................................34

4 .................36

4.1. .....................................................................................................................36

4.2. .....................................................................................................................38

4.2.1. ..........................................................................38

4.2.2. B-N-AC .....................................................................39

4.2.3. HE ..........................................................................................................40

4.2.4. BDA .......................................................................................................41

4.3. .....................................................................................................................41

4.3.1. AC ..............................................41

4.3.2. HE BDA ...............................42

4.4. .....................................................................................................................45

4.4.1. ..................45

4.4.2. ..........................................46

5 .....................................................................................................................48

.................................................................................................................................50

.........................................................................................................................53

.................................................................................................................................58

5

1

2

19

17

2

( Cell)

17~18

1)

2) 3)

1.1.

6

Trim36 (tripartite motif containing 36) Dnd1 (DND microRNA-mediated

repression inhibitor 1)

(Cuykendall et al., 2009) Dnd1 RNA

Dnd1 trim36 mRNA 3’-UTR

Trim36

(Mei et al., 2013) Trim36 E3

Dnd1 Trim36

30

30

(Houliston and Elinson, 1991) 4

(Kikkawa et al., 1996)( 1) Wnt/β-Catenin

β-Catenin β-Catenin TCF/LEF

Siamois Twin

(Molenaar et al., 1996; Brannon et al., 1997) Wnt/β-Catenin

Wnt11 Wnt8

(Tao et

al., 2005)

7

1

30

1.2.

(SpO)

SpO

(Nieuwkoop, 1969)

8

Nieuwkoop

(Nieuwkoop, 1973) Nieuwkoop SpO

4 T VegT

β-Catenin Wnt/β-Catenin

(Zhang and King, 1996) VegT

Wnt/β-Catenin Nieuwkoop

(Clements et al., 1999) VegT

Wnt/β-Catenin

Wnt/β-Catenin Siamois

Twin Siamois Twin BMP Chordin Noggin

Nieuwkoop BCNE (blastula Chordin- and

Noggin-expressing) ( 2) BCNE

(Kuroda et al., 2004) Nieuwkoop

Nodal BCNE

SpO Nieuwkoop Nodal

BCNE

(Agius et al., 2000; De Robertis and Kuroda, 2004)

9

2

(Stage 8) β-Catenin VegTNieuwkoop β-Catenin

BCNE SpO (Stage 10) BCNENieuwkoop Nodal

1.3.

FGF(Fibroblast growth factor)

(AC) AC

AC FGF

(Slack et al., 1989) FGF

(Kinoshita and

Asashima, 1995)( 3) SpO

10

Nodal TGF-β Nodal

Nieuwkoop

(Agius et al., 2000; De Robertis and Kuroda, 2004)

AC

(Ariizumi and Asashima, 2001)

3

(Stage 9) ACAC

11

RNA-seq

Chordin Noggin

Vent1 Sizzled

X Trim29 3

X

CRISPR/Cas

β-Catenin VegT mRNA Siamois Twin

mRNA CRISPR/Cas

AC

AC BCNE Nieuwkoop

12

2 RNA-seq

2.1.�

4

(Kikkawa et al., 1996)

(SpO)

SpO BCNE (blastula Chordin- and Noggin-expressing)

Nieuwkoop Nodal

(Agius et al., 2000; De Robertis and Kuroda, 2004) BCNE

Siamois Twin

BMP Chordin Noggin

(De Robertis and Kuroda, 2004; Kuroda et al., 2004) Siamois Twin

BMP4 BMP

(Ishibashi et al., 2008) β-Catenin VegT mRNA

Nieuwkoop Nodal

( 4) 20

13

(De Robertis and Kuroda, 2004)

(Tompsett et

al., 2013; Rotman et al., 2013)

RNA-seq

( SP-X)

Xolloid

Xolloid BMP Chordin C1

C2 C3 C4 Chordin

BCNE Xolloid Chordin

BMP

(Larraín et al., 2001; Oelgeschläger et al., 2000) SP-X

xHtrA1

xHtrA1

Biglycan Syndecan-4 Glypican-4

FGF (Fibroblast growth factor) GAG

(glycosaminoglycan) FGF-GAG FGF

FGF (Hou et al., 2007)

SP-X

SP-X SP-X

gain-of-functional SP-X

pCS2 pCS2-SP-X

14

SP-X mRNA

4

(Stage 10)BMP BMP

Chordin Noggin

15

2.2.

2.2.1.

�

(HCG10000, )

600 U

0.1x

(0.1x SS: 1x SS [58 mM NaCl, 0.67 mM KCl, 3.4 mM CaNO3-4H2O, 0.83

mM MgSO4-7H2O, 0.1 g/l Kanamycin Sulfate, 5 mM Tris-HCl, pH 7.4] 10

) 22 ˚C 1%

(100 ml 0.1x 1 ml

[#208-01026, ] 5N NaOH 3 ml )

2.2.2. RNA cDNA

3 15

RNA RNA ( , pH 5 76 ml Guanidine

Thiocyanate 23.6 g Ammonium Thiocyanate 15.2 g 3 M Sodium Acetate, pH 5.3

33.4 ml (#072-04945, ) 10 ml Nuclease-free

water (#129114, QIAGEN) 50 ml

RNA 15 µl Nuclease-free water

RNA (#48190-011, life

technology) RNase inhibitor (#SIN-201, ) dNTPs (#201913, QIAGEN)

M-MLV, RNaseH+ (#187-01286, Wako) 30 ˚C 10

42 ˚C 60 72 ˚C 10 cDNA

2.2.3. RNA-seq

Stage 10 (n=15)

RNA illumina HiSeq 100 bp Paired end

16

RNA-Seq Xenbase

(http://www.xenbase.org) Xenopus laevis (JGI 7.0)

TopHat ver. 2.0.9 (Trapnell et al., 2009)

Cufflinks ver.2.1.1 (Trapnell et al., 2010)

2.2.4. pCS2

pUC57-SP-X EcoRI XhoI

SP-X DNA

pCS2 EcoRI XhoI Antarctic

Phosphatase (#M0289L, NEB) pCS2

SP-X TaKaRa Ligation Kit Ver. 2 (#6022, TaKaRa)

2.2.5. mRNA

pCS2 SP-X pCS2-SP-X 5 ng Asp718

15 µl Nuclease-free water mRNA

6 µl SP6 mMessage mMachine Kit (#AM1340, Ambion)

37 ˚C 2 DNase 37 ˚C 15

mRNA

20 µl Nuclease-free water 500 ng/µl

2.3.

2.3.1. RNA-seq

Stage 10

RNA-seq

RNA-seq

45,336,894 42,035,856

17

TopHat

ver. 2.0.9 Xenopus laevis (JGI 7.0)

86.75%

86.95% Cufflinks ver.2.1.1 cuffdiff

2

Stage 10 19896

143

43

SP-X Trim29 3 (USP75AA USP107AA

USP276AA )

5

18

5

(A) X YFPKM(fragments per kilobase of exon per million mapped fragments)cuffdiff (B)

FPKM

BMP

19

2.3.2. SP-X

SP-X RNA-seq

SP-X SP-X gain-of-functional

SP-X

SP-X mRNA 4 2 4 nl 800

pg ( 6A) SP-X mRNA

28.8% (13/45) 84.4% (38/45)

( 6D, E, H, I, J, K)

(

6B) 13.3% (6/45) 15.6% (7/45)

( 6I, K)

( 1, 2) SP-X mRNA

20

6 SP-X mRNA

(A) 4 SP-X mRNA 4 24 nl mRNA 800 pg (B)

(C-E) Stage 29 C D ED 2 28.8% (13/45)E 2 84.4% (38/45)

(F-K) Stage 46 F G H-K H I 213.3% (6/45) J K 2

84.4% (38/45)1 mm

21

2.4.

RNA-seq

SP-X

1) RNA-seq

2) SP-X

2.4.1. RNA-seq

Stage 10 RNA RNA-seq

Chordin Noggin ADMP

Sizzled Bambi Wnt8

BMP4

BMP Stage 10

BMP4 Stage 8 Stage 12

Stage 10

X

Trim29

(tripartite motif containing 29) Trim29 MYST

22

Tip60 p53

(Sho et al., 2011; Yuan et al., 2010)

p53 Trim29

(Cordenonsi et al., 2003)

75

107 276 3

32 ELABELA

(Chng et al., 2013)

3

2.4.2. SP-X

SP-X

4

SP-X mRNA

( ; 13/45 ; 38/45 6D, E, H, I, J, K)

Chordin Noggin Follistatin BMP

BMP

(Piccolo et al., 1996; Zimmerman et al., 1997) BMP

SP-X mRNA

mRNA

MO

SP-X

( 4H, J) Pax6

23

Otx2 (Kenyon et al., 1999)

Pax6

(Chiang et al., 1996; Macdonald et al., 1995)

(Strähle et

al., 1993) SP-X mRNA Pax6

(Tran et al., 2007; Hoff et al., 2013; Horb et al., 1999) SP-X

mRNA

SP-X

24

3 RNA CRISPR/Cas

3.1.

20

gain- or loss-of-functional

(MO)

Zinc Finger TALEN CRISPR/Cas

F0 (Kim et al.,

1996; Cermak et al., 2011; Garneau et al., 2010) 4

Cas9 RNA

gRNA RNA PAM-presenting

oligonucleotides (PAMmer) 3 RNA

(O’Connell et al., 2014) ( 7)

CRISPR/Cas RNA

MO

MO

25

CRISPR/Cas siRNA

RNA

mRNA

CRISPR/Cas

CRISPR/Cas

β-Catenin VegT 2

Siamois Twin

β-Catenin

Wnt/β-Catenin

T-box VegT

β-Catenin VegT Nieuwkoop

β-Catenin VegT

BCNE (blastula Chordin- and Noggin-expressing)

BCNE

Siamois Twin BMP Chordin

Noggin (De Robertis and Kuroda, 2004; Kuroda et al., 2004)

Siamois Twin BMP4 BMP

(Ishibashi et al.,

2008) Nieuwkoop Nodal

MO

β-Catenin 2

(Heasman et al.,

2000) VegT Nieuwkoop

VegT (Heasman et al., 2001)

Siamois Twin BMP

26

(Ishibashi et al., 2008)

CRISPR/Cas β-Catenin

VegT Siamois Twin gRNA PAMmers

Cas9-nonNLS mRNA gRNA PAMmer 3

7 CRISPR/Cas

(A) CRISPR/Cas PAMgRNA Cas9 ( )

(B) CRISPR/Cas RNA mRNAPAM DNA PAMmer gRNA

Cas9 RNA

3.2.

3.2.1.

�

(HCG10000, )

600 U

0.1x

(0.1x SS: 1x SS [58 mM NaCl, 0.67 mM KCl, 3.4 mM CaNO3-4H2O, 0.83

mM MgSO4-7H2O, 0.1 g/l Kanamycin Sulfate, 5 mM Tris-HCl, pH 7.4] 10

) 22 ˚C 1%

(100 ml 0.1x 1 ml

27

[#208-01026, ] 5N NaOH 3 ml )

3.2.2. gRNA PAMmers gRNA

gRNA 20

PAMmer 19 NCBI RefSeq

4 PAMmer

( 8A) gRNA 2

gRNA DNA RNA polymerase

in vitro T7

gRNA FW (60 b) Cas9

gRNA RV (76 b) 2 gRNA

(100 µM each) 1 µl 10x PCR buffer for KOD-Plus-Neo 10 µl dNTPs (2

mM each) 1 µl 25 mM MgSO4 3 µl KOD-Plus-Neo (#436300, ) 1 µl

32 µl 94 ˚C 30 55 ˚C 1 68 ˚C 30

gRNA DNA gRNA DNA

500 ng DNA T7 RNA

polymerase (#10881767001, Roche) 37 ˚C 1

37 ˚C 30 DNase

gRNA Nuclease-free water

PAMmer 8B Integrated DNA

Technologies (IDT )

28

8 gRNA PAMmer

(A) gRNA PAMmer (B) gRNA-FWgRNA-RV PAMmer

29

3.2.3. Cas9-nonNLS mRNA

pCS2 NLS Cas9

(Cas9-nonNLS) pCS2-Cas9-nonNLS 5 ng Asp718

15 µl Nuclease-free water mRNA

6 µl mMESSAGE mMACHINE SP6 Transcription

Kit (#AM1340, Ambion) 37 ˚C 2 DNase

37 ˚C 15

mRNA 20 µl Nuclease-free water

1000 ng/µl

3.3.

3.3.1. CRISPR/Cas

CRISPR/Cas

β-Catenin VegT 2 1 β-Catenin gRNA

100 pg β-Catenin PAMmer 20 pg Cas9-nonNLS mRNA 1000 pg

β-Catenin

10.3% (9/87) 37.9% (33/87)

20.7% (18/87)

( 9E-G) CRISPR/Cas

Cas9 β-Catenin gRNA 100 pg β-Catenin PAMmer 20 pg

10.5% (6/57) 19.2%

(11/57) ( 9C,

D, G)

30

9 CRISPR/Cas β-Catenin

(A) 1 (B) C-F(C, D) β-Catenin gRNA 100 pg β-Catenin PAMmer 20 pg C

(36/57) D (11/57) (E, F) Cas9-nonNLS mRNA 1000 pg β-Catenin gRNA 100 pg β-Catenin PAMmer 20 pg

(KD) E (9/87) F(33/87) (G) β-Catenin

68.9% Typical Mildexo 1 mm

VegT VegT gRNA 100 pg VegT PAMmer 20 pg

Cas9-nonNLS mRNA 1000 pg 1 15.4%

(12/78) 15.4% (12/78)

14.1% (11/78) β-Catenin

( 10E-G) VegT gRNA 100

pg VegT PAMmer 20 pg 14.3% (9/63)

19.0% (12/63) ( 10C, D,

G)

31

10 CRISPR/Cas VegT

(A) 1 (B) C-F(C, D) VegT gRNA 100 pg VegT PAMmer 20 pg C

(33/63) D (12/63) (E, F) Cas9-nonNLS mRNA1000 pg VegT gRNA 100 pg VegT PAMmer 20 pg (KD)

E (12/78) F (12/78) (G) VegT 46.2%

Typical Mild exo1 mm

3.3.2. CRISPR/Cas

Siamois Twin

CRISPR/Cas 8

1 Siamois gRNA 50 pg Twin gRNA 50 pg Siamois PAMmer

10 pg Twin PAMmer 10 pg Cas9-nonNLS mRNA 1000 pg

9.7% (6/62) 45.2%

(28/62) 12.9% (8/62)

( 11E-G) Siamois gRNA 50 pg Twin gRNA 50pg Siamois

PAMmer 10 pg Twin PAMmer 10 pg 15.4% (8/52)

7.7% (4/52) (

11C, D, G)

32

11 CRISPR/Cas Siamois/Twin

(A) 8 2 (B) C-F(C, D) Siamois gRNA 50 pg Twin gRNA 50 pg Siamois PAMmer 10 pg

Twin PAMmer 10 pg C (36/52) D(4/52) (E, F) Cas9-nonNLS mRNA 1000 pg Siamois gRNA

50 pg Twin gRNA 50 pg Siamois PAMmer 10 pg Twin PAMmer 10 pg(KD) E (6/62) F

(28/62) (G) Siamois/Twin 67.7%Typical Mild

exo 1 mm

3.4.

CRISPR/Cas

β-Catenin VegT

Siamois/Twin β-Catenin VegT

Siamois/Twin MO

1) CRISPR/Cas

2)

3.4.1. CRISPR/Cas

CRISPR/Cas β-Catenin 10.3% (9/87)

VegT 15.4% (12/78)

33

Siamois/Twin 9.7% (6/62)

CRISPR/Cas

MO 70~80%

DNA PAMmer

PAMmer DNA

locked nucleic acids 2'-OMe

phosphorothioate

CRISPR/Cas RNA

(O’Connell et al., 2014)

phosphorothioate DNA DNA

PAMmer phosphorothioate

(Woolf et al., 1990; Hulstrand et al., 2010)

10 DNA 5' 3' 5 2'-OMe

RNA 20 DNA/RNA (ASOs: antisence oligo

nucleotides)

(Pauli et al., 2015) RNA ASOs RNase H

DNA/RNA RNA ( 12A)

ASOs

β-Catenin ASOs

( 12C) CRISPR/Cas

ASOs

gRNA

CRISPR/Cas gRNA

34

gRNA

3’ GG 2 3 PAM

(Farboud and

Mayer 2015) gRNA RNA

12 DNA/RNA (ASOs) β-Catenin

(A) ASOs ASOs 5' 5 2'-OMe RNA 10 DNA3' 5 2'-OMe RNA(mN ) phosphorothioate (*

) RNA ASOs DNA/RNA RNase(B) Stage 36 (C) 1~2 β-Catenin-ASOs 300 pg

76.6%(92/120)

3.4.2. CRISPR/Cas

CRISPR/Cas RNA

MO

miRNA non-coding RNA

35

(Michiue and Asashima 2005) Cas9

gRNA PAMmer

Cas9

dCas9 GFP mRNA /

RNA- 1

mRNA (Chen et al., 2013)

36

4

4.1.

1

ES (embryonic stem cell) iPS (induced

pluripotent stem cell) (Yamanaka, 2008; Okita et

al., 2008)

(Gokhale and Andrews, 2008)

37

(AC)

ES

iPS AC

AC

AC AC

(Ariizumi and

Asashima, 2001) AC canonical Wnt

(Harland, 2000)

AC

AC

(SpO) . SpO

SpO SpO

(Spemann and Mangold, 1924) SpO BCNE

(blastula Chordin- and Noggin-expressing) Nieuwkoop

Nodal (Agius et

al., 2000; De Robertis and Kuroda, 2004)

SpO SpO BCNE Nieuwkoop����

�

(��� ) AC

AC

β-Catenin Wnt8/β-Catenin

(Christian and Moon,

1993) Wnt8 mRNA BCNE AC

38

Wnt8 mRNA VegT mRNA Nieuwkoop

AC (Ishibashi et al., 2008) 8 1

Wnt8 mRNA Wnt8 mRNA VegT

mRNA AC BCNE Nieuwkoop

AC

B-N-AC (BCNE and Nieuwkoop centers-contained AC)

( 13)

13

AC BCNE NieuwkoopAC

4.2.

4.2.1.

�

(HCG10000, )

600 U

0.1x

39

(0.1x SS: 1x SS [58 mM NaCl, 0.67 mM KCl, 3.4 mM CaNO3-4H2O, 0.83

mM MgSO4-7H2O, 0.1 g/l Kanamycin Sulfate, 5 mM Tris-HCl, pH 7.4] 10

) 22 ˚C 1%

(100 ml 0.1x 1 ml

[#208-01026, ] 5N NaOH 3 ml )

4.2.2. B-N-AC

8 1 Wnt8 mRNA 1 pg 1

VegT mRNA 160 pg ( 14A) 1x

1 0.1x Stage

9 Stage 9 1x

0.3 mm AC BCNE Nieuwkoop

B-N-AC B-N-AC

AC AC

( 14B) poly-HEMA

1x 22 ̊C poly-HEMA

40 mm (#1-8549-01, ) 100 %

EtOH 4% poly-HEMA (12% of 2-hydroxyethyl methacrylate

#18894-100, Polysciences) 500 µl 4% poly-HEMA

40

14 AC

(A) 8 1 Wnt8 mRNAWnt8 mRNA VegT mRNA (Stage 9) AC

(B) A

4.2.3. HE

PBSFA (10x PBS

1:1:8 ) 70% EtOH

100% EtOH

8 µm

100%

EtOH 70% EtOH 1

41

1 70% EtOH 100% EtOH

(038-01045, ) 1/10

4.2.4. BDA

HE 100% EtOH 70%

EtOH 1x wash buffer

(1 M Tris-HCl 1.5 M NaCl pH 7.5 10 ) Streptavidin-AP

solution (1x wash buffer 150 ml, Streptavidin-AP 7.5 µl) 3

1x wash buffer AP buffer (1 M Tris-HCl 50 ml, 0.5 M MgCl2 50

ml, DW 400 ml) 5 7 ml BM purple 140 ml AP buffer

4˚C 1

4.3.

4.3.1. AC

BCNE Nieuwkoop

B-N-AC

8 1 Wnt8 mRNA 1 pg

Wnt8 mRNA 1 pg VegT mRNA 160 pg

B-N-AC AC

(n = 138)

2 AC ( 15A, n = 30)

29.7% (41/138)

70.2% (97/138) 20

( 15B) 72

( 15C, D, 3)

42

15 B-N-AC AC

(A) AC (B) B-N-ACAC 32

(C, D) B-N-AC AC 72 CD ( ) cg, cement gland 1

mm

4.3.2. HE BDA

72 HE

Wnt8

mRNA Wnt8 mRNA VegT mRNA

BDA (biotin dextran-amine)

BCNE

Nieuwkoop

HE

( 16B-F, H-L)

( 16B) BDA

Nieuwkoop Wnt8 mRNA VegT mRNA

43

( 16B’)

Wnt8 mRNA

Wnt8 mRNA VegT mRNA

( 16D’, F’, H’, K’, L’)

Wnt8 mRNA (

16C’-E’)

44

16 72

(A, G) B-N-AC AC A Wnt8 mRNABDA G Wnt8 mRNA VegT mRNA

BDA (B-F, B’-F’) A HEBDA HE BDA

(H-L, H’-L’) G HEBDA HE BDA

si, small intestine; mu, muscle; no, notochord; mt, mesothelium; me, mesenchyme 1 mm

45

4.4.

BCNE

Nieuwkoop AC

1)

2)

4.4.1.

B-N-AC AC

BDA Nieuwkoop

BCNE ( 15F’

H’, K’) FGF(Fibroblast growth factor) AC

(Kimelman, 1991;

Faas et al., 2013) Nieuwkoop Nodal

FGF

Nieuwkoop

BCNE ( 15C’

)

BCNE

BCNE

( 15C’-E’) BCNE

BCNE (Kuroda

46

et al., 2004) AC Nodal

BCNE ( 17)

17

B-N-AC ACBCNE ( )

Nieuwkoop ( )BCNE Nieuwkoop

4.4.2.

Wnt8 mRNA BCNE Wnt8

mRNA VegT mRNA Nieuwkoop

Wnt8 mRNA 8~32

(Christian and Moon, 1993)

Wnt8 Wnt8/β-Catenin β-Catenin

BMP Chordin Noggin

(De Robertis and Kuroda, 2004) Wnt8

Wnt8/β-Catenin (Hoppler and

Moon, 1998; Ramel and Lekven, 2004) Wnt8

Wnt8 BCNE

47

Wnt8 mRNA

dnGSK3 β-Catenin Wnt8/β-Catenin

BCNE

48

5 RNA-seq

(Stage 10)

SP-X gain-of-function

RNA CRISPR/Cas

β-Catenin VegT

β-Catenin VegT

Siamois Twin

gRNA PAMmer

AC

BCNE

Nieuwkoop BCNE

iPS

49

(Kuroda et al., 2015)

(Takebe et al.,

2013)

iPS

50

4

2

2

51

1

( :

)

4

52

DeNA

1 4

TA

SFC

!

53

Agius, E., Oelgeschläger, M., Wessely, O., Kemp, C., De Robertis, E.M. (2000).

Endodermal Nodal-related signals and mesoderm induction in Xenopus. Development 127, 1173–1183. [PMID: 10683171]

Ariizumi, T., and Asashima, M. (2001). In vitro induction systems for analyses of

amphibian organogenesis and body patterning. Int. J. Dev. Biol. 45, 273–279. [PMID: 11291857]

Brannon, M., Gomperts, M., Sumoy, L., Moon, R.T., and Kimelman, D. (1997). A

beta-catenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus. Genes Dev. 11, 2359–2370. [PMID: 9308964]

Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A.,

Somia, N. V, Bogdanove, A.J., and Voytas, D.F. (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82. [PMID: 21493687]

Chen, B., Gilbert, L. a, Cimini, B. a, Schnitzbauer, J., Zhang, W., Li, G.W., Park, J.,

Blackburn, E.H., Weissman, J.S., Qi, L.S., et al. (2013). Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491. [PMID: 24360272]

Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, H., and

Beachy, P. A (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407–413. [PMID: 8837770]

Chng, S.C., Ho, L., Tian, J., and Reversade, B. (2013). ELABELA: a hormone essential

for heart development signals via the apelin receptor. Dev. Cell 27, 672–680. [PMID: 24316148]

Christian, J.L., and Moon, R.T. (1993). Interactions between Xwnt-8 and Spemann

organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes Dev. 7, 13–28. [PMID: 8422982]

Clements, D., Friday, R.V., and Woodland, H.R. (1999). Mode of action of VegT in

mesoderm and endoderm formation. Development 126, 4903–4911. [PMID: 10518506]

Cordenonsi, M., Dupont, S., Maretto, S., Insinga, A., Imbriano, C., and Piccolo, S.

(2003). Links between tumor suppressors: p53 is required for TGF-beta gene responses by cooperating with Smads. Cell 113, 301–314. [PMID: 12732139]

Cuykendall, T.N., and Houston, D.W. (2009). Vegetally localized Xenopus trim36

regulates cortical rotation and dorsal axis formation. Development 136, 3057–3065. [PMID: 19675128]

De Robertis, E.M., and Kuroda, H. (2004) Dorsal-ventral patterning and neural

induction in Xenopus embryos. Annu. Rev. Cell Dev. Biol. 20, 295-308. [PMID: 15473842]

54

Faas, L., Warrander, F.C., Maguire, R., Ramsbottom, S. a, Quinn, D., Genever, P., and Isaacs, H. V (2013). Lin28 proteins are required for germ layer specification in Xenopus. Development 140, 976–986. [PMID: 23344711]

Farboud, B., and Meyer, B.J. (2015). Dramatic Enhancement of Genome Editing by

CRISPR/Cas9 Through Improved Guide RNA Design. Genetics 199, 959–971. [PMID: 25695951]

Garneau, J.E., Dupuis, M.È., Villion, M., Romero, D. a, Barrangou, R., Boyaval, P.,

Fremaux, C., Horvath, P., Magadán, A.H., and Moineau, S. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71. [PMID: 21048762]

Gokhale, P.J., and Andrews, P.W. (2008). New insights into the control of stem cell

pluripotency. Cell Stem Cell 2, 4–5. [PMID: 18371412] Harland, R. (2000). Neural induction. Curr. Opin. Genet. Dev. 10, 357–362. [PMID:

10889069] Heasman, J., Kofron, M., and Wylie, C. (2000). Beta-catenin signaling activity

dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222, 124–134. [PMID: 11784070]

Heasman, J., Wessely, O., Langland, R., Craig, E.J., and Kessler, D.S. (2001). Vegetal

localization of maternal mRNAs is disrupted by VegT depletion. Dev. Biol. 240, 377–386. [PMID: 10885751]

Hoff, S., Halbritter, J., Epting, D., Frank, V., Nguyen, T.M., van Reeuwijk, J., Boehlke,

C., Schell, C., Yasunaga, T., Helmstädter, M., et al. (2013). ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3. Nat. Genet. 45, 951–956. [PMID: 23793029]

Hoppler, S., and Moon, R.T. (1998). BMP-2/-4 and Wnt-8 cooperatively pattern the

Xenopus mesoderm. Mech. Dev. 71, 119–129. [PMID: 9507084] Horb, M.E., and Thomsen, G.H. (1999). Tbx5 is essential for heart development.

Development 126, 1739–1751. [PMID: 10079235] Hou, S., Maccarana, M., Min, T.H., Strate, I., and Pera, E.M. (2007). The secreted

serine protease xHtrA1 stimulates long-range FGF signaling in the early Xenopus embryo. Dev. Cell 13, 226–241. [PMID: 17681134]

Houliston, E., and Elinson, R.P. (1991). Patterns of microtubule polymerization relating

to cortical rotation in Xenopus laevis eggs. Development 112, 107–117. [PMID: 1769322]

Hulstrand, A.M., Schneider, P.N., and Houston, D.W. (2010). The use of antisense

oligonucleotides in Xenopus oocytes. Methods 51, 75–81. [PMID: 20045732] Ishibashi, H., Matsumura, N., Hanafusa, H., Matsumoto, K., De Robertis, E.M., and

Kuroda, H. (2008). Expression of Siamois and Twin in the blastula Chordin/Noggin signaling center is required for brain formation in Xenopus laevis embryos. Mech. Dev. 125, 58–66. [PMID: 18036787]

55

Kenyon, K.L., Moody, S.A., and Jamrich, M. (1999). A novel fork head gene mediates early steps during Xenopus lens formation. Development 126, 5107–5116. [PMID: 10529427]

Kikkawa, M., Takano, K., and Shinagawa, A. (1996). Location and behavior of dorsal

determinants during first cell cycle in Xenopus eggs. Development 122, 3687–3696. [PMID: 9012490]

Kim, Y.G., Cha, J., and Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc

finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160. [PMID: 8577732]

Kinoshita, K., and Asashima, M. (1995). Effect of activin and lithium on isolated

Xenopus animal blastomeres and response alteration at the midblastula transition. Development 121, 1581–1589. [PMID: 7600976]

Kuroda, H., Iwamiya, T., and Morimoto, K. (2015). The Experimental Procedures to

Create Life and Transplantable Heart. SFC J. 15, 262–282. Kuroda, H., Wessely, O., and De Robertis, E.M. (2004). Neural induction in Xenopus:

requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus. PLoS Biol. 2, E92. [PMID: 15138495]

Larraín, J., Oelgeschläger, M., Ketpura, N.I., Reversade, B., Zakin, L., and De Robertis,

E.M. (2001). Proteolytic cleavage of Chordin as a switch for the dual activities of Twisted gastrulation in BMP signaling. Development 128, 4439–4447. [PMID: 11714670]

Macdonald, R., Barth, K. a, Xu, Q., Holder, N., Mikkola, I., and Wilson, S.W. (1995).

Midline signalling is required for Pax gene regulation and patterning of the eyes. Development 121, 3267–3278. [PMID: 7588061]

Mei, W., Jin, Z., Lai, F., Schwend, T., Houston, D.W., King, M. Lou, and Yang, J.

(2013). Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification. Development 140, 2334–2344. [PMID: 23615278]

Michiue, T., and Asashima, M. (2005). Temporal and spatial manipulation of gene

expression in Xenopus embryos by injection of heat shock promoter-containing plasmids. Dev. Dyn. 232, 369–376. [PMID: 15614780]

Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S.,

Korinek, V., Roose, J., Destrée, O., and Clevers, H. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391–399. [PMID: 8756721]

Nieuwkoop, P.D. (1969). The formation of the mesoderm in Urodelean amphibians. I.

Induction by the endoderm. Roux’ Arch. f. Entw. Mech. 162, 34–373. Nieuwkoop, P.D. (1973). The organization center of the amphibian embryo: its origin,

spatial organization, and morphogenetic action. Adv. Morphog. 10, 1–39. [PMID: 4581327]

56

O’Connell, M.R., Oakes, B.L., Sternberg, S.H., East-Seletsky, A., Kaplan, M., and Doudna, J.A. (2014). Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266. [PMID: 25274302]

Oelgeschläger, M., Larraín, J., Geissert, D., and De Robertis, E.M. (2000). The

evolutionarily conserved BMP-binding protein Twisted gastrulation promotes BMP signalling. Nature 405, 757–763. [PMID: 10866189]

Okita, K., Hong, H., Takahashi, K., and Yamanaka, S. (2010). Generation of

mouse-induced pluripotent stem cells with plasmid vectors. Nat. Protoc. 5, 418–428. [PMID: 20203661]

Pauli, A., Montague, T.G., Lennox, K.A., Behlke, M.A., and Schier, A.F. (2015).

Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish. PLoS One 10, e0139504. [PMID: 26436892]

Piccolo, S., Sasai, Y., Lu, B., and De Robertis, E.M. (1996). Dorsoventral patterning in

Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86, 589–598. [PMID: 8752213]

Ramel, M.C., and Lekven, A.C. (2004). Repression of the vertebrate organizer by Wnt8

is mediated by Vent and Vox. Development 131, 3991–4000. [PMID: 15269175] Rotman, N., Guex, N., Gouranton, E., and Wahli, W. (2013). PPARβ interprets a

chromatin signature of pluripotency to promote embryonic differentiation at gastrulation. PLoS One. 8, e83300. [PMID: 24367589]

Sho, T., Tsukiyama, T., Sato, T., Kondo, T., Cheng, J., Saku, T., Asaka, M., and

Hatakeyama, S. (2011). TRIM29 negatively regulates p53 via inhibition of Tip60. Biochim. Biophys. Acta 1813, 1245–1253. [PMID: 21463657]

Slack, J.M., Darlington, B.G., Gillespie, L.L., Godsave, S.F., Isaacs, H.V., and Paterno,

G.D. (1989). The role of fibroblast growth factor in early Xenopus development. Development 107 Suppl., 141–148. [PMID: 2636135]

Spemann, H. and Mangold, H. (1924). Induction of embryonic primordia by

implantation of organizers from a different species. Roux’ Arch. Entw. Mech., 100, 599-638. [PMID: 11291841]

Strähle, U., Blader, P., Henrique, D., and Ingham, P.W. (1993). Axial, a zebrafish gene

expressed along the developing body axis, shows altered expression in cyclops mutant embryos. Genes Dev. 7, 1436–1446. [PMID: 7687227]

Takebe, T., Sekine, K., Enomura, M., Koike, H., Kimura, M., Ogaeri, T., Zhang, R.R.,

Ueno, Y., Zheng, Y.W., Koike, N., et al. (2013). Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484. [PMID: 23823721]

Tao, Q., Yokota, C., Puck, H., Kofron, M., Birsoy, B., Yan, D., Asashima, M., Wylie,

C.C., Lin, X., and Heasman, J. (2005). Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos. Cell 120, 857–871. [PMID: 15797385]

Tompsett, A.R., Wiseman, S., Higley, E., Giesy, J.P., and Hecker, M. (2013). Effects of

57

exposure to 17α-ethynylestradiol during sexual differentiation on the transcriptome of the African clawed frog (Xenopus laevis). Environ. Sci. Technol. 47, 4822–4828. [PMID: 23550701]

Tran, U., Pickney, L.M., Ozpolat, B.D., and Wessely, O. (2007). Xenopus Bicaudal-C is

required for the differentiation of the amphibian pronephros. Dev. Biol. 307, 152–164. [PMID: 17521625]

Woolf, T.M., Jennings, C.G., Rebagliati, M., and Melton, D.A. (1990). The stability,

toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos. Nucleic Acids Res. 18, 1763–1769. [PMID: 1692405]

Yamanaka, S. (2008). Induction of pluripotent stem cells from mouse fibroblasts by

four transcription factors. Cell Prolif. 41 Suppl. 1, 51–56. [PMID: 18181945] Yuan, Z., Villagra, A., Peng, L., Coppola, D., Glozak, M., Sotomayor, E.M., Chen, J.,

Lane, W.S., and Seto, E. (2010). The ATDC (TRIM29) protein binds p53 and antagonizes p53-mediated functions. Mol. Cell. Biol. 30, 3004–3015. [PMID: 20368352]

Zhang, J., and King, M.L. (1996). Xenopus VegT RNA is localized to the vegetal cortex

during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning. Development 122, 4119–4129. [PMID: 9012531]

Zimmerman, L.B., De Jesús-Escobar, J.M., and Harland, R.M. (1996). The Spemann

organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599–606. [PMID: 8752214]

58

1

https://youtu.be/MFa9TYQOZ1Q

2 SP-X mRNA

https://youtu.be/DdT9KFP1xCE

3

https://youtu.be/fVDPxN7A_ok

https://youtu.be/qiSvVLJpzwo

https://youtu.be/f6gRvLkJuJI

https://youtu.be/ITifNryVnn4

Printed in Japan　　印刷・製本　　ワキプリントピア

2016年 3 月31日　　　初版発行

SFC-SWP 2015-010

著者　森本健太

監修　先端生命科学研究会

発行　慶應義塾大学 湘南藤沢学会　　　〒252-0816　神奈川県藤沢市遠藤5322

　　　TEL：0466-49-3437

アニマルキャップ細胞群を用いた胚全体構造の構築への挑戦

■

本論文は研究会において優秀と認められ、出版されたものです。