Ikk2 regulates cytokinesis during vertebrate

Ikk2 regulates cytokinesis during vertebrate development

Hongyuan Shen1, Eun Myoung Shin1,5, Serene Lee2, Sinnakaruppan Mathavan2, Hiromi Koh3, Motomi Osato5, Hyungwon Choi1,3, Vinay Tergaonkar1,6,7 and Vladimir Korzh1,4

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Institute of Molecular and Cell Biology, Singapore; Genome Institute of Singapore, Singapore; Saw Swee Hock School of Public Health, National University of Singapore International Institute of Molecular and Cell Biology, Warsaw, Poland Cancer Science Institute, NUS, Singapore Department of Biochemistry, NUS, Singapore Center for Cancer Biology, UniSA, Adelaide, Australia

A

K44

M.musculus R.norvegicus H.sapiens G.gallus X.tropicalis D.rerio

1 1 1 1 1 1


- - - - MT - -

- - - - HQ - -

- - - - - - - - DAS - - -

- - - - PG - -

- MS WS P - MS WS P - MS WS P - MS R P P R AG WA P - MS R P P

P -

128 128 128 128 139 128

D D D D D D

AS AS AS AS S S S S

AL AL AL AL AL AL

R R R R R T

Y Y Y Y Y Y

L L L L L L

HE NR HE NR HE NR HE NR HE NR HG M R

HR HR HR HR HR HR


266 266 266 266 277 266

NS NS NS NS NS NK

V V V V K L

AE AE AE AG AG AG

R R R K K E

L L L L L L

E E E E E E

K WL K WL K WL K WL HWL R WL


375 375 375 371 386 404

K K K K T S


513 513 513 509 524 542

AWR AWR AWR AWK AWR V WR


649 649 649 645 662 676

K K K K K K

B

I I I I I I

L L L L L L

T N E G L T L DM D L T N E G L T L DM D L L N E G H T L DM D L V NDAV V ADT DL V N DG R Q G E G D L L MDNR R A DE P L E E E E E E

RQK RQK RQK RQK RQQ RQK

ME ME ME ME T E ME

Q Q Q Q Q Q

E E E E E E

WN L WN L WN L WN L WN L WN L

L L L L L L

AVE AVE AVE AVE T VE T AL L L L L L L

I I I I I I

I I I I I I

S L PTQ S L PTQ S L T TQ A L Q AQ P S HTQ S MQ P Q DL DL DL DL DL DL

K K K K K K

Q L ML Q L ML Q L ML Q L ML Q CML Q MM L

MW H P T WQ P MW H P MW H P MW H Q R WS P

V V V L I V

DNS DNS DNS D NQ DNR DR S

P P P P P P

T T T T I T

CG CG CG CG CG CG

AWE AWE AWE P WE P WE P WE

E E E E E E

N N N N N N

I I I I I I

V V V V V V

L L L L L L

RG RG RG RG RG RG

T V T T T K

DPQ YG DPQ YG DP T YG DP V YG DPQ NP DL KQ R

RQ RQ RQ RQ RQ KE

L L L L L L

F F F F F F

Q CG R Q CG R L CG R L CG R L CG R DCGQ

E E E E E T

NDV K HL NDV K V L NE V K L L D D V DQ L R E VQAL E DV F K L

V V V V V E

E R MM A L E R MM A L E R MM A L K R MM A L D K MM A L E E MM T L

K K K K K K

S S S S S S

K K K K K K

S S S S S S

G G G G G G

AC AC AC AC AC AC

V V V V V V

RG RG RG RG RG RS

P P P P P P

V V V V V V

E E E E E E

S S S S S S

TQ TQ TQ AQ PQ PQ

QQG QQG QQG QQG QPG QQG

F F F F F F

I I I I I V

K I NY K MS Y K I T Y KVAY KT VY CCS Y

MK MK MK MK MK L K

P P P P P P

I I I V I F

T P T P S P AL S L T P Q Q Q Q Q Q

E E E E E E

R R R R R R

L L L L L L

G G G G G G

T T T T T T

GG GG GG GG GG GG

F F F F F F

G G G G G G

NV NV NV NV YV NV

I I I I L T

R WH NQ R WH NQ R WH NQ R WH N K R W I HQ L WQ N K

AT VT E T E T DT E T

E K E K EQ EQ PQ E K

R R R R R R

L L L L L L

I I I I I V

HK HK HK HK HK HK

I I I I I I

I I I I I I

DL DL DL DL DL DL

G G G G G G

Y Y Y Y Y Y

T

P

K

P NG P NG P NG P NG NVG NK E

- - - - - SQ

R R R R P R

PQP PQP PQP P HP AHP VL P

E E E E E E

T T T T T T

D I D I D I D I DS D I

DL DL DL DL DL DL

DS MNV S DS MNV S DS MNA S E S M NM S DS L G T S DG G R T P

V V V V V V

E

S V S V S V S V S V N I Q Q Q Q Q Q

S C S C S C DY T V RF

G G G G G S

E E E E E I

Q Q Q Q Q Q

I I I V V I

A A A A A A

I I I I I I

S S S S S S

L L L L L L

C C C C C C

T T T T T T

S S S S S S

S

P

- - - - - EG

S F S F AF HF T Y PY

F F F F T S

QL R QMR QL R QL R HL R SQR

S177

AK AK AK AK AK AK

E E E E E E

L L L L L L

DQ G DQ G DQ G DQ G DQ G DQ S

E

E

S

K

E

I LQE P VLQE P I LQE P I L Q DP I L Q DP VQNE P

K K K K K K

R R R K R R

NL NL NL NL PL PL

- - - - - - - - - - DAE

R S P MG R S P MG R S P MG RS PLG R NP L G R Q P WR

R L S HPGQL R L S HPG HL R L SQPGQL R L NS PGQL R MP G P S Q L PAPT P -QL

MS MS MS L L L S MS

R R R R R -

KQGG KQGG KQGG KQGG K HRG -SGE

QP QP QP QV QP HR

S S S P P P

T

T L T L T L T L T L AL

S AC S AC T AS SGT AL S S HP

*

KQ KQ KQ KQ KQ KQ

C C C C C C

RQ RQ RQ RQ RQ RQ

E E E E E E

L L L L L L

S S S S S S

P P P P P V

VG VG VG VG VG VG

T T T T T T

L L L L L L

Q Q Q Q Q Q

Y Y Y Y Y Y

L L L L L L

- - - - - - - - - - DAP

K

- - - - - AP

C

* * S181

N R D R WC L N R D R WC L N R E R WC L N R D R WA L N R E R WC L N K G R WS L

E E E E E E

I I I I I I

Q Q Q Q Q Q

I I I I I I

AP AP AP AP AP AP

E E E E E E

K K K K K K

Y Y Y Y Y Y

T T T T T T

V V V V V V

T V T V T V T V T V AV

D Y WS D Y WS D Y WS D Y WS D Y WS D Y WS

F

C C C C C C

F F F F F F

RAL RAL KAL KAL QAL DE L

L L L L L L

NL NL NL NL S L NL

K K K K K K

L L L L L L

HV H I H I HV S V HV

L L L L L L

L L L L L L

E E E E E E

QQ QQ QQ QQ QK QQ

DD DD DD DD DS S H

K K K K R R

V WG Q V WG Q V WG Q V WG Q V WG Q T WG Q

V WH S V WH S V WH S I WH T T WQ T AWH T

I I I I I I

QT L K QT L K QT L K R ML K R AMK RAL K

E E E E E E

E E E E E E

EQ EQ EQ EQ EQ VK

ARE L ARE L ARE L ARE L AK DL AME L

Y Y Y Y Y F

R R R R R R

R R R R R K

L L L L L L

R R R R R R

E E E E E E

K K K K R K

- - - - AV - -

P P P S AQ PS

- - - - - DG

E S DK DS DK E PAK E S VR E YVK P HT E

K K K K K E

S S S S S S

E E E E E L

E L E L E L E L DL HV

V V V L L I

AE AE AE L E S E E E

DDL DDL DDL E DL DDL E T L

DS L DS L NS L YAL AS L S S P

- - - - - AK

F F F F F F

K K R R K K

D

WT

P P P P P P

R R R R R R

I I I I I I

DC NR DC NR DC NR DC NR DC AR D WQ R DQ DQ DQ DQ DQ EQ

AHAL AHAL AHNL SQKL S L KL S R I F

C C C C C E

MR MR MR MK MK MK

V V V L L V

R R R R K R

L L L L L L

NHP NV NHP NV T HP NV NHP NV NHP NV T HM N V F F F F F F

V V V V V V

DV DV DV DV E V DV

P P P P P P

E G MQ E G MQ E G MQ E G MQ DG L Q E E LQ

NL NL NL KL KL KL

AP AP AP AP AP AT

I I I I I I

T T T T T T

G G G G G G

F F F F F F

L L L L L L

N WQ N WQ N WQ N WQ N WQ T WQ

V I I V V I

HT Y HT Y HT Y HT Y HT Y F NY

P P P P P S

VT E VME VT E VT E VS E VQP

R R R R R R

NNS NNS NNS Y NS Y NT H NG

C C C T E T

L L L L L L

S S S S S S

K MK K MK K MK K MK KKK KQK

NAMA S NAMA S NS MA S NS MA S NL MT S NE MV S

LQ LQ LQ LQ IQ VQ

A IQ A IQ A IQ A IQ A IQ AVQ

S S S T S F

F F F F F Y

E E E E E E

K K K K K R

K K K K R K

R R R R I R

V I V I V I V I L I DF

Y Y Y Y Y Y

F T T L F T T L F T AL F MA L L MS L MQ I L

R

DWS DWS DWS DWS DWS E WE

WL WL WL WL WL WL

QME DE E R C S QME DE E R CG QT E E E E HS C QL HV E E R S S S QQ E E A S G T NG G Q D L A - -

G G G G G G

T T T T T T

L L L L L L

NM V NM V NM V NM V NM V NM T

T T T T S S

GT GT GT GT GK AE

A A MM S L A A MM N L A A MM N L A A MM N L T F MV NL V A I MS L

QQGQ QQGQ QQGQ QQGQ L HG Q QQGQ

T T T T T S

EG EG EG SG AG MG

DS Q E MV D S Q DM V DS Q E MV DS Q E I V DS NE MV DCQ E V I

R R R R R K

S RL E S RL E T L L E SQL E R QME NR L Q

S AL Q DT S AL Q DT NA I Q DT NVMHDT T S MMG F S L VQ DT

VKEQ V KQQ VREQ M K DQ MK E Q IQE T

L L L L L L

L L L L L V

AAR AAR AAR AAR S AR AAR

AF AF AF AF AF VF

L L L L L L

R R R R R R

R R R R R K

L L L L L L

L L L L L L

E E E E E E

DR DR DQ EQ DS E S

C C C C C C

S S S S S S

R R R R R R

P P P P P P

+/-

P P P P P P

P P P P P P

DE S L Q S L K DE S L Q S L K DE S L Q S L K E E T LQT VK NE NL QNL K E DDV AS L Q

+/+

ikk2m10

F F F F F F

NDL NDL NDL NDL NDL NDL

V V V V V L

P P P P P P

L L L L L L

CQGG CQGG CQGG CQGG CEGG CQGG

R R R R R R

R R K K K K

Y Y Y Y Y Y

L L L L L L

NQ NQ NQ NQ NQ NL

K K K K K K

S E VD S E VD S E VD S E L D S NE H HV DD

I I I I I I

V V V V V V

V V V V V V

S S S S Y Y

E E E E E E

L L P P P P

E T DQ E T DQ E E DQ E Q DQ E E EQ P A NQ

E E E E E E

L L L L L L

L L L L L L

Q Q Q Q Q L

E AG E AG E AG E AG ASG E AG

F F F F F F

QE RE QQ QS EQ AQ

DT DT DT DT DT DT

G G G G G G

I I I I I I

L L L L L L

K K K K K K

AK AK AK AK AK AT

L L L L L L

DF DF DF DF DF DF

T T T T T T

VVC VVC VVC VVC VAC MV C

I I I I L I

T AQQ T AQQ MS QQ L S QQ E F EQ M HQ R

TQL SQL TQL AQ L DQ L T HL

S S S S S S

K K K K K K

V RQ V RQ V RQ V RQ VRE L QQ

DL DL DL DL DL DL

T R T R AR AR S R E R

L L L P S -

KQK KQK KQK KQK K HK RQR EQ EQ EQ EQ P I - -

K K K K R G

T S T S T S T S NS AS

I I I I I L

Q I Q I Q I Q I Q I HV

DL DL DL DL DL D I

AL AL AL AL AL VL

E L E L E L E L E L AL

L L L F S L

P P P P P P

V V V V V V

K K K R K R

F F F L F V

DL DL DL DL DL DL

E E E E E S

E E E E E E E K KE EG

K K K K K K

E E E E E E

NC NC NC NC NC NC

NG NG NG SG MG GG L L L L L L

AV T V T V E V AV E V

VL VL AL AL AL AL

YK YR YS YK YS YQ

CG L R CG L R CG L R CG L R CG L K CGMR

E E E E E E

K K K K K R

F F F F F Y

S S S S S S

E E E E E E

GAV GA I GA I E A I GP I AAV

L L L L L L

S S S S S K

S L S L S L S L VL HL

F P F P YP CP T P NP

P P P P P P

T T T I T T

S S S S S Q

127 127 127 127 138 127

NNL NNL NNL NNL NHL HNL

265 265 265 265 276 265

L L L L L L

L L L L L I

L P DK P L P DK P I P DK P F TQK NP AQ P E PQNS

ATQ ATQ ATQ - - L TQ V MQ

C I S DS C I S DS C I S DG HT ADS F V V DC C A I DY

374 374 374 370 385 403

QT E F QT E F QT E F Q I E F QME F QRAT

I I I I I I

DK DK DK E K E K E K

V V S L MNE V V R L MNE V V S L MNE VMNL MNE VMNL MR E L V S AMA R

G G G G G G

DE DE DE DE DE S E

T S T S T S T S T S AS

RT V KT V KT V E T V K MV KQL

V V V V V L

L L L L L L

L L L F S S

512 512 512 508 523 541

RLQ RLQ RLQ RLQ K RQ HL Q

E E E E E E

648 648 648 644 661 675

L L L L L L

757 757 756 753 771 779

ACD ACD AS T HM T T T - - -

+/+

ikk2m379

+/-

****** *** *****

ikk2m379

+2

Y Y Y Y Y F

K K K K K K

WT

-/-

AME AME AME AME AME AME

V Q WH S V Q WH S V Q WH S V Q WH T V Q WHG V Q WH S

C ikk2m10

L L L L L L

**************

-/Δ7

Suppl Figure 1.

A

B

I

WT

ikk2-/III

D

C

II

ikk2-/IV

E

ikk2-/-

ikk2-/-

G

F WT

ntl

H ikk2-/-

ntl

ikk2-/-

ntl

Suppl Figure 2.

A

WT

A’

D

ikk2-/-

D’

B

WT

B’

E

ikk2-/-

E’

C

WT

C’

F

ikk2-/-

F’

Suppl Figure 3.

A

C

WT (male) B

WT (female) D

ikk2-/- (male)

ikk2 (female) -/-

G

J

90% epi

G’

90% epi

K M

WT (female) F

ikk2-/- (female)

I

MZikk2-/-

MZikk2-/-

WT

E

H WT I

MZikk2-/III

MZikk2-/-

L

MZikk2-/II

MZikk2-/-

N

IV

MZikk2-/-

Suppl Figure 4.

B

128-cell

C

sphere

D

30% epi

I’

MZikk2-/-

J

WT

MZikk2-/-

J’

shield

F

60% epi

C’

G

D’

75% epi

H

90% epi

K’

L

L’

M

M’

N

N’

O

O’

P

P’

Q

Q’

R

R’

S

S’

T

T’

F’

G’

H’

F’’

G’’

H’’

papc

E’

eve1

MZikk2-/-

ntl

WT

E

B’

chordin

A’

K

bmp2b

WT MZikk2-/-

I

WT

ﬂh

A

64-cell

Suppl Figure 5.

4

B

2

aurka-interacting genes

zebrafish ikk2 mRNA

2hr WT

3

MZikk2-/-

4hr WT

2h

MZikk2-/-

4h nFkB

rhoab Maternal

MBT

e2f7 bbs4

1

rxfp2a

ep

ib ol y

g 50 %

ob lo n

12 8ce ll

16 -c el l

0 1ce ll

Relative Fold Change (normalized against β -actin)

A

klhl21 cxcr5 vps4a becn1 tex14 pik3c3 sh3glb1 aurka uvrag

kif14 kif3b spast

Suppl Figure 6.

chmp4b e2f8 pik3r4

+ +

Table S1 Developmental events affected by loss-of-function of Ikk2

Defect in\LOF Cytokinesis YSL Gastrulation Notochord\axis formation Angiogenesis Survival as embryos Survival as adults

MZikk2-/√ √ √ √

Zikk2-/-

√ √

√

Anti-ikk2 MO

√ √ √

√

Supplementary Figures Legends.

Figure S1. Structure, expression and mutations of Ikk2. (A) Multiple sequence alignment between putative zebrafish Ikk2 protein and proteins from other species including human, mouse, rat, chicken and frog. Note that K44 of the catalytic active site and the two serine residues (S177/S181) of the activation loop (marked in red) are conserved between zebrafish and other species. Alignment is coloured using ClustalW alignment scheme. (B, C) Semiquantitative RT-PCR analysis of ikk2 expression during embryogenesis and in adult organs. Two different primer pairs amplifying ikk2 (n.t. 46-1000 and n.t. 952-1976) cDNA were used independently. actin was used as a loading control. (D) Example of sequence chromatograph of DNA regions containing mutations in the two ZFN-mediated zebrafish ikk2 mutant alleles. The ikk2m10 allele carries a two-nucleotide insertion, whereas the ikk2m379 allele carries a 7nucleotide deletion. All fish were genotyped by sequencing.

Figure S2. The hindbrain angiogenic vasculature of wild-type and zygotic ikk2-/embryos (obtained from heterozygotic parents) at 48 hpf. (A-F), ImageJ processed binary image of the confocal projection view of the hindbrain CtA vessels. I’-G’, corresponding output image from ImageJ Angiogenic Analyser Macro with corresponding vessel junctions, branches, master segments, isolated segments marked in different colours. Their length and numbers were analysed to calculate the angiogenic index using ImageJ Angiogenic Analyser Macro as shown in Figure 3, F-H.

Figure S3. Analysis of zygotic mutants at 24 hpf. (A-E) Bright-field lateral view of wildtype and

ikk2-/- (zygotic) embryos obtained from heterozygotic parents with notochord

defects at 24 hpf. A, wild-type embryos. B-E, ikk2-/- embryos classified into four categories

based on severity of the notochord defects. Class I and II show notochord defects and Ushaped somites. Class III and IV show mild notochord defects and increased turbidity of the brain suggestive of apoptosis. (F-H) Lateral view of embryos stained by in situ hybridization using ntl probe. ikk2-/- embryos have either crooked notochord or its truncation. Scale bar: 250 µm.

Figure S4. Morphological analysis of adult heterozygotic and embryonic homozygotic Ikk2 mutants obtained from heterozygotic parents. (A-D) Adult ikk2-/- fish with skeletal defects. Adult ikk2-/- fish are short with curved skeleton (B, D) as compared to wild types (A, C). (E, F) Adult ikk2-/- fish are prone to skin lesion accompanied by loss of scales and hypopigmentation. Scale bar in A-D: 1cm; scale bar in E,F: 0.1 cm. (G-N) In MZikk2-/escapers cells detach in embryonic shield (arrow) at late gastrula. (G, G’), later stage embryos contain increased cell debris under the chorion (I, arrowed) and show defects in posterior body (K-N). G, lateral view. G’, dorsal view. J-N, lateral view, 24 hpf. K-N, ikk2-/- embryos with defects in posterior body are classified into four categories (I to IV) based on severity of the notochord defects. This phenotype mimics that of zygotic ikk2-/- mutants (Figure S2, A-E). Scale bar in G-N: 500 µm.

Figure S5. Morphological and WISH analysis of MZikk2-/- embryos. (A-H) Bright field image of wild-type and MZikk2-/- embryos obtained from homozygotic parents at blastula and gastrula stages. MZikk2-/- embryos show disorganized blastoderms (A’-C’) unlike compact blastoderm of wild-type embryos (A-C). Epiboly is incomplete (D’-H’) and cells detach from yolk. F’’-H’’, due to delayed epiboly constriction starts before epiboly completion resulting in peanut-shaped embryos. All embryos shown in lateral view. (I-T) Wild-type and MZikk2-/embryos stained by WISH at shield stage. Embryos stained with dorsal markers (flh, chordin

in I-L), ventral markers (bmp2b in M-N) , and mesendoderm markers (ntl, papc, and eve1 in O-P). MZikk2-/- embryos show signs of correct dorsal-ventral patterning and mesendoderm differentiation. I, K, M, O, Q, S: lateral view with dorsal side to the right; J,L,N,P,R,T: animal pole view with dorsal side to the right. Scale bar in all panels: 200 µm.

Figure S6. (A) Real-time PCR (qPCR) quantification of ikk2 expression during early embryonic stages. qPCR results were normalized against actin (actb), and expressed as relative fold changes as compared to 1- cell stage using the ΔΔCt methods. Error bars represent mean ± SEM. (B) Heat-map representing RNAseq results of aurka and its interacting partners (green – positively-regulated gene, red – negatively-regulated gene ).