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1 SUPPLEMENTAL FIGURE LEGENDS Supplemental Fig. 1. Sequence analysis of the zebrafish ATX. (A) Amino acid sequences of human, mouse and zebrafish ATXs. The catalytic center is indicated by the red triangle. (B) Phylogenic analysis of NPP family in human, mouse and zebrafish. Zebrafish ATX (zgc:63550) is localized in the cluster of atx/npp2. Supplemental Fig. 2. ATX expression was attenuated by morpholino antisense oligonucleotides (MOs). (A) Schematic representation of zebrafish ATX gene with exons represented as black boxes. The positions of the morpholinos employed in this study are indicated by gray bars above exons 2 and 4, and the position of primers used for the RT-PCR analysis are indicated by arrows. (B) ATX MO1 caused the deletion of exon 2 and resulted in a 160-bp PCR fragment compared to the intact 263-bp fragment in ATX MO1 5-mis MO-injected embryos. (C) ATX MO2 caused the partial deletion of exon 4 and resulted in a 500-bp PCR fragment compared to the intact 514-bp fragment in untreated embryos. Supplemental Fig. 3. Sequence analysis of the zebrafish LPA receptors. (A) Amino acid sequences of human, mouse and zebrafish LPA 2-6 receptors. (B) Phylogenic analysis of LPA receptors in human, mouse and zebrafish. Supplemental Fig. 4. Each LPA receptor expression was attenuated by MOs. (A-D) Blocking of splicing by MOs was confirmed by RT-PCR. (A) LPA 1 MO caused the deletion of exon 2 and resulted in a 453-bp PCR fragment compared to the intact 1208-bp fragment in untreated embryos as shown previously (28). (B) LPA 2 a MO caused the deletion of exon 2 and resulted in a 456-bp PCR fragment compared to the intact 1382-bp fragment in untreated embryos. (C) LPA 2 b MO caused the deletion of exon 2 and resulted in a 476-bp PCR fragment compared to the intact 897-bp fragment in untreated embryos. (D) LPA 3 MO caused the partial deletion of exon 2 and resulted in a 819-bp PCR fragment compared to the intact 937-bp fragment in untreated embryos. (E-I) Translation blocking induced by MOs. Each MO against LPA 4 , LPA 5 a, LPA 5 b, LPA 6 a or LPA 6 b inhibited the translation of the egfp gene cloned downstream of the 5’-UTR of LPA 4 , LPA 5 a, LPA 5 b, LPA 6 a or LPA 6 b containing the target sequence of each MO respectively. Scale bar: 200 μm. Supplemental Fig. 5. Effects of LPA 1 /LPA 4 double MOs on vascular formation. Confocal images of fli1:EGFP embryos injected with LPA 4 MO2 alone (A) and LPA 1 MO with LPA 4 MO2 (B) at 48 hpf. Lateral views, anterior to the left. Embryos injected with LPA 4 MO2 did not show vascular defects, whereas embryos injected with both LPA 1 MO and LPA 4 MO2 showed severe vascular defects such as stalling (asterisk) and abnormal connection around horizontal myoseptum of SA (arrowhead) that were identical to those in ATX morphant embryos. Scale bar: 50 μm.

SUPPLEMENTAL FIGURE LEGENDS Supplemental Fig… · SUPPLEMENTAL FIGURE LEGENDS Supplemental Fig. 1. ... Schematic representation of zebrafish ATX gene with exons ... LPA 1 MO caused

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SUPPLEMENTAL FIGURE LEGENDS Supplemental Fig. 1. Sequence analysis of the zebrafish ATX. (A) Amino acid sequences of human, mouse and zebrafish ATXs. The catalytic center is indicated by the red triangle. (B) Phylogenic analysis of NPP family in human, mouse and zebrafish. Zebrafish ATX (zgc:63550) is localized in the cluster of atx/npp2. Supplemental Fig. 2. ATX expression was attenuated by morpholino antisense oligonucleotides (MOs). (A) Schematic representation of zebrafish ATX gene with exons represented as black boxes. The positions of the morpholinos employed in this study are indicated by gray bars above exons 2 and 4, and the position of primers used for the RT-PCR analysis are indicated by arrows. (B) ATX MO1 caused the deletion of exon 2 and resulted in a 160-bp PCR fragment compared to the intact 263-bp fragment in ATX MO1 5-mis MO-injected embryos. (C) ATX MO2 caused the partial deletion of exon 4 and resulted in a 500-bp PCR fragment compared to the intact 514-bp fragment in untreated embryos. Supplemental Fig. 3. Sequence analysis of the zebrafish LPA receptors. (A) Amino acid sequences of human, mouse and zebrafish LPA2-6 receptors. (B) Phylogenic analysis of LPA receptors in human, mouse and zebrafish. Supplemental Fig. 4. Each LPA receptor expression was attenuated by MOs. (A-D) Blocking of splicing by MOs was confirmed by RT-PCR. (A) LPA1 MO caused the deletion of exon 2 and resulted in a 453-bp PCR fragment compared to the intact 1208-bp fragment in untreated embryos as shown previously (28). (B) LPA2a MO caused the deletion of exon 2 and resulted in a 456-bp PCR fragment compared to the intact 1382-bp fragment in untreated embryos. (C) LPA2b MO caused the deletion of exon 2 and resulted in a 476-bp PCR fragment compared to the intact 897-bp fragment in untreated embryos. (D) LPA3 MO caused the partial deletion of exon 2 and resulted in a 819-bp PCR fragment compared to the intact 937-bp fragment in untreated embryos. (E-I) Translation blocking induced by MOs. Each MO against LPA4, LPA5a, LPA5b, LPA6a or LPA6b inhibited the translation of the egfp gene cloned downstream of the 5’-UTR of LPA4, LPA5a, LPA5b, LPA6a or LPA6b containing the target sequence of each MO respectively. Scale bar: 200 µm. Supplemental Fig. 5. Effects of LPA1/LPA4 double MOs on vascular formation. Confocal images of fli1:EGFP embryos injected with LPA4 MO2 alone (A) and LPA1 MO with LPA4 MO2 (B) at 48 hpf. Lateral views, anterior to the left. Embryos injected with LPA4 MO2 did not show vascular defects, whereas embryos injected with both LPA1 MO and LPA4 MO2 showed severe vascular defects such as stalling (asterisk) and abnormal connection around horizontal myoseptum of SA (arrowhead) that were identical to those in ATX morphant embryos. Scale bar: 50 µm.

Supplemental Fig. 1

C

mN

pp4

mNpp6

mN

pp7

hNPP

4

zNpp2/zATX

(zgc:63550)

zNpp5

zNpp

7b

hNPP1

mNpp1

A

Human Mouse

Zebrafish (zgc:63550)

60 59 55

120 119 115

180 179 175

240 240 235

300 300 295

360 360 355

420 420 415

480 480 474

540 540 534

600 600 588

660 660 648

720 720 708

780 780 767

840 839 827

863 862 850

B

Supplemental Fig. 2

514bp 263bp fw1-rv1

GAPDH

160bp fw2-rv2

GAPDH

A MO1 MO2

fw1 primer (exon1)

fw2 primer (exon2) rv2 primer (exon6)

rv1 primer (exon3)

1 2 4 5 6 25 3

1 kb

500bp

B C

A human LPA2 mouse LPA2 zebrafish LPA2a zebrafish LPA2b

human LPA5 mouse LPA5 zebrafish LPA5a zebrafish LPA5b

human LPA3 mouse LPA3 zebrafish LPA3

human LPA4 mouse LPA4 zebrafish LPA4

human LPA6 mouse LPA6 zebrafish LPA6a zebrafish LPA6b

mLPA4  

hLPA4  

zLPA3   mLPA1  

hLPA1  

zLPA5b  

mLPA

6  

zLPA

2b  

zLPA

6a  B

Supplemental Fig. 3

Exon 1 Exon 2 Exon 3

E 1 E 2 E 3 Normal :

Exon 1 Exon 2 Exon 3

E 1 E 2 E 3 Normal :

E 1 E 7

Normal :

E1 E2 E3

E1 E 2 E 3 Normal :

897bp

1208bp

937bp

E 1 E 3 MO treated :

GAPDH

453bp

E 2 E 3

476bp

E 4 E 5 E 6

E1 E 2 E 3 MO treated :

819bp

E 1 E 3 MO treated :

MO treated :

fw primer rv primer fw primer rv primer

fw primer rv primer fw primer rv primer

GAPDH

E 1 E 7 E 2 E 3 E 4 E 5 E 6

E 1 E 7 E 3 E 4 E 5 E 6

GAPDH GAPDH

1382bp

456bp

LPA2b MO LPA3 MO

LPA1 MO LPA2a MO

A B

C D

Supplemental Fig. 4

Phenol red LPA4 MO1

LPA4-EGFP mRNA (24 hpf)

Phenol red LPA6a MO Phenol red LPA6b MO

LPA4 MO2

LPA6a-EGFP mRNA (24 hpf) LPA6b-EGFP mRNA (24 hpf)

E

H I

Supplemental Fig. 4

Phenol red LPA5a MO Phenol red LPA5b MO

LPA5a-EGFP mRNA (24 hpf) LPA5b-EGFP mRNA (24 hpf) F G

LPA1 MO + LPA4 MO2

LPA4 MO2

* B

A

Supplemental Fig. 5

Supplemental Table 1

The list of oligonucleotides used for cloning ATX and LPA receptors

forward primer reverse primer

zATX 1st AGAGAGCCACCAGACGCACGTCAAC ACCTTGGACATGCTAATGCAGGGAG

zATX 2nd (pBluescript) GGGGTACCACCATGCTGCAACTTAAATGGGTATTC GAATTCTCAGATCTCACTTTCGTATGTGTGC

zATX (pCAGGS-MCS-

myc) GGAATTCCACCATGCTGCAACTTAAATGGGTATTC AAAACTCGAGGATCTCACTTTCGTATGTGTGCAAG

zATX (T205A) GGGTACCACCATGCTGCAACTTAAATGGGTATTC, GTACCCTACAAAAGCCTACCCAAATTTG

CAAATTTGGGTAGGCTTTTGTAGGGTAC, CCAATCACCTTATCAACATCCC

LPA1 1st ACAGAGCCGAGCAACAATC CCTGCTGGTTTATTACGTGAG

LPA1 2nd AAGGATGATAGACAATGCTAC CGGGGTACCTCAGACCACTGAATGGTCATTATG

LPA2a 1st AAACGGGCAGATGGACTTC CAGGGAGAAATGGTTTTTCCATC

LPA2a 2nd AAGGTATTTAACAGCTGTGAG CGGGGTACCTCAAGACAATGGGCTTTGTG

LPA2b 1st CCAATCACTGACCTCTGAGAC CTTTCAAGTCAAGGTCTCCAAATC

LPA2b 2nd AAGGTTTGCTACAACAACGAGAC CGGGGTACCTTCAGCTGTCGCTGGACTC

LPA3 1st GTCCTAGTGGGTTTTGTCCAAAG TCTCACCTGCAGCAGGATTG

LPA3 2nd AAGGCCAGGCACAACATCTGCTAC CGGGGTACCTCAGGACTTGAGCATCTCCTG

LPA4 1st AACAGGAGTACATCTACTGCAAG TGCTCTGTGAAAGGGTTTCACTG

LPA4 2nd AAGGCCAGTCTTGTTCTTAATGAG CGGGGTACCTCAGAACTGAGTCTCACCAAC

LPA5a 1st CAACACAGCACTTCTCTTAAACAC GTTTGGTTCTTCTTGCGTAACC

LPA5a 2nd AAGACTTCAAACAACACTACAGTC CGGGGTACCGGTTCTTTATTGTCCAGTCCAAC

LPA5b 1st CGGAACACCTTTAACCCAG CTTAGGAGCTCCGTAGAAAC

LPA5b 2nd AAGAATCAAAACTTCACCAACAC CGGGGTACCCAGCTGGTCAAGACCTATC

LPA6a 1st CGACTTCTGCGGAGAGGATTG CCCTATAAGAGCCCACCGTTTAC

LPA6a 2nd AAGTACAATACAAGTCTTGAGATGG CGGGGTACCCCTCATACGTGGGACTCATTG

LPA6b 1st GCAACCTCTGAACGGTAAGAC GTTATCAGTCTGCTATATCCTGTCC

LPA6b 2nd AAGATGACTGAGAGGAGCAACTG CGGGGTACCCCTATTTGCTGTGTGATCTTTGG

The recognition sites for restriction endonucleases used for cloning are underlined.

Supplemental Table 2

forward primer forward primer

LPA1 MO GCGAGTGATTTCTGGACCTTTCAGC GCCTTTTTCACAGTCTCTTCTTGCG

LPA2a MO CCAGATGAAGAGCGTTTCC CAGGGAGAAATGGTTTTTCCATC

LPA2b MO TGAGACTCGATTCCCTGAG GACGACGGTCTTCATCAG

LPA3 MO 1st ATGGAGAGTGAACCGAGAAC CGGGGTACCTCAGGACTTGAGCATCT

LPA3 MO 2nd ATGGAGAGTGAACCGAGAAC ATGGGGTTCATGACGGAGTTG

ATX MO 1 1st GTGCCGAGGTTTGAGTAG ACAGTGGTCATCAAAGTCTG

ATX MO 1 2nd TCGTTCAGTGTGGAAGAGAG ACAGGTTGTCACAGCGAC

ATX MO 2 1st AGTAGTCGTTCAGTGTGGAAGAGAG TCAGGATAAAGCCCCGTTG

ATX MO 2 2nd TCCGTTATGTGGCTTTTCTCA ACCGTGCCTCCTCTTTTCA