Forced expression of α2,3-sialyltransferase IV rescues impaired heart development in α2,6-sialyltransferase I-deficient medaka

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Legends to figures

Fig. 1. Localization and significance of α2,6- and α2,3-sialosides in medaka embryos at 01 dpf. A. Structures of Siaα2,6Gal (upper) and Siaα2,3Gal (lower). R, other monosaccharides;

B. Whole mount immunofluorescent staining of 16~32-cell-stage embryos using FITC-SSA

and FITC-MAA. DIC, differential interference contrast. A dotted square is enlarged at right

panel; C. Cartooning of lectins injection into PVS of 1~4-cell embryos (upper). Fluorescence

stayed at the PVS at 2 dpf (lower right), after FITC-dextran was injected there at 0 dpf (lower

left); D. SNA, MAA, and mIgG (control) were PVS-injected in 1~4-cell embryos and the effects

were observed at gastrula embryos at 18 h-post-injection. Embryos of dead, abnormal, and

normal morphology were counted. n, number of embryos tested.

Fig. 2. Distribution of Siaα2,6Gal epitope and expression profiles of ST6Gal I and ST6Gal

II in WT. A. SNA lectin blotting of 9 dpf embryos before (-) and after (+) PNGase F treatment

(LB: SNA). IB: β-actin, loading control; Human transferrin (hTf) was used as a control for the

lectin blotting; B. Developmental expression of ST6Gal I and ST6Gal II by qPCR. The

expressions were normalized with the β-actin expression. The bars represent standard

deviations from the triplicated experiments.

Fig. 3. Phenotypes of the ST6Gal I-KO and ST6Gal II-KO medaka embryos. A. Diagrams

of the target site in the ST6Gal I and ST6Gal II genes and their base sequences; B. Genotyping

of the (+/+), (+/-), and (-/-) medaka for ST6Gal I (upper) and ST6Gal II (lower) genes. N.C.,

water instead of the templates; C. Upper, DIC images of WT (left), ST6Gal I-KO (middle), and

ST6Gal I-KO (right) embryos at 7 dpf. Scale bars, 85 µm. Lower, Fluorescent images

(mCherry) of Tg941 WT (left), ST6Gal I-KO (middle), and ST6Gal II-KO (right) embryos at 7

dpf. A, atrium; V, ventricle. Scale bars, 85 µm; D. Monitoring of heartbeat of Tg941 WT (left),

ST6Gal I-KO (middle), and ST6Gal II-KO (right) embryos at 7 dpf for 2 s. The heartbeats of

atrium (black) and ventricle (gray) were analyzed by the FIJI software. Relative area stands for

their sizes, and most dilated ones were set to 1.0.



Fig. 4. Rescue experiments of ST6Gal I-KO by forced expression of ST6Gal I- and ST6Gal

II-mRNAs. A, B, C. DIC images of the embryos at 7 dpf that were injected with DsRed mRNA

(control, A), ST6Gal I mRNA (B), and ST6Gal II mRNA (C) at 1-cell embryos. Scale bars, 85

µm; D. Summary of the heart phenotypes of ST6Gal I-KO embryos at 7 dpf injected with DsRed,

ST6Gal I, and ST6Gal II mRNAs. n, number of experiments; Dead, Abnormal, Cardiac

abnormality, and Normal stand for lethality, severe anomalous morphologies, cardiac

abnormalities, and normal embryos; E. ST6Gal I (left) and ST6Gal II (right) expression in

DsRed, ST6Gal I, and ST6Gal II mRNAs-injected ST6Gal I-KO embryos as evaluated by qPCR.

The expression level was normalized by that of β-actin. The bars represent standard deviations

from the triplicated experiments. **, p < 0.005; ***, p < 0.0005; F. (upper)SNA lectin blotting

of DsRed, ST6Gal I, and ST6Gal II mRNAs-injected ST6Gal I-KO embryos at 7 dpf before (-)

and after (+) sialidase treatment (LB: SNA). IB: β-actin, loading control; hTf was used as a

control for the lectin blotting. (Lower) quantification of SNA epitopes/β-actin values calculated

from the above data. The bars represent standard deviations from the triplicated experiments.

Fig. 5. Rescue experiments of ST6Gal I-KO by forced expression of ST3Gal IV- and

ST3Gal V-mRNAs. A, B, C. DIC images of the embryos at 7 dpf that were injected with DsRed

mRNA (control, A), ST3Gal IV mRNA (B), and ST3Gal V mRNA (C) at 1-cell embryos. Scale

bars, 85 µm; D. Summary of the heart phenotypes of ST6Gal I-KO embryos at 7 dpf injected

with DsRed, ST3Gal IV, and ST3Gal V mRNAs. n, number of experiments; Dead, Abnormal,

Cardiac abnormality, and Normal stand for lethality, severe anomalous morphologies, cardiac

abnormalities, and normal embryos; E. ST3Gal IV (left) and ST3Gal V (right) expression in

DsRed, ST3Gal IV, and ST3Gal V mRNAs-injected ST6Gal I-KO embryos as evaluated by

qPCR. The expression level was normalized by that of β-actin. The bars represent standard

deviations from the triplicated experiments. *, p < 0.05; ***, p < 0.0005. F. MAA lectin blotting

(upper) of DsRed and ST3Gal IV mRNAs-injected ST6Gal I-KO embryos at 7 dpf before (-)

and after (+) sialidase treatment (LB: MAA). IB: β-actin, loading control; Ft was used as a

control for the lectin blotting. (Lower) quantification of MAA epitopes/β-actin values. The bars

represent standard deviations from the triplicated experiments; G. MAA lectin blotting (upper)



of DsRed and ST3Gal V mRNAs-injected ST6Gal I-KO embryos at 7 dpf. (Lower)

quantification of MAA epitopes/β-actin values. For details, see the legend for F.



Fig. 1

HOH2C

HO

AcHN

HO

OH

HO

COO

OH

HOH2C

HO

AcHN

COO

OH

OH

6 CH2

OH

OH 6

CH2OH

OH















Glass needle

FE

Embryo

Ratio of phenotype

PVS

(%)

100

n=9

n=20

n=17

n=37

n=16

80

Dead

60

Abnormal

40

20

Normal

250

0 dpf

2 dpf

n=19

500

mIgG

250

500

SNA

250

500 (μg/mL)

MAA

Fig. 2

WT

PNGase F

(kDa)

210

140

100

70

55




− + − +

0.016

0.014

0.012

0.010

0.008

0.006

0.004

0.002

LB: SNA

Copy numbers

40

35

IB: β-actin

ST6Gal I

ST6Gal II

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

dpf

Fig. 3

E1 E2 E3

E4 E5 E6 E7E8

E9

ST6Gal I

E2 E3

E4E5 E6



ST6Gal I



  





WT CTCT

GCAT

ST6Gal I (Ins8) CTCT CATGCCTC GCAT

E1






  

E7

ST6Gal II







WT TGAG CAGT GGGA

ST6Gal II (Del4) TGAG

GGGA

ST6Gal I-KO

ST6Gal II-KO

mCherry

DIC

WT

Relative area

WT

ST6Gal I-KO

ST6Gal II-KO

1.1

1.1

1.1

1.0

1.0

1.0

0.9

0.9

0.9

0.8

0.8

0.8

0.7

0 0.5 1.0 1.5 2.0

0.7

0.7

0 0.5 1.0 1.5 2.0

Time (s)

0 0.5 1.0 1.5 2.0

ST6Gal I-KO

+ DsRed mRNA

Phenotype ratio

ST6Gal I-KO

+ ST6Gal I mRNA

(%)

n=6

n=14

ST6Gal I-KO

+ ST6Gal II mRNA

n=6

100

Dead

80

60

Abnormal

40

Cardiac abnormality

20

Normal

DsRed

ST6Gal I ST6Gal II

ST6Gal I

ST6Gal II

***

**

0.0025

0.0020

0.0015

0.0010

0.0005

0.0015

0.0010

0.0005

DsRed

ST6Gal I

DsRed

sialidase − + − + − +

(kDa)

DsRed

ST6Gal I

ST6Gal II

−+ −+ −+

ST6Gal II

−+ −+ −+

hTf



210

140

100

70

55

40

35

SNA/β-actin value

Copy numbers

Fig. 4

−+

70

55

LB: SNA

IB: β-actin

SNA epitope

SNA epitope

1.5




1.0




0.5




DsRed

ST6Gal I

DsRed

ST6Gal II

Phenotype ratio

ST6Gal I-KO

+ DsRed mRNA

ST6Gal I-KO

+ ST3Gal IV mRNA

(%)

n=6

n=7

80

60

Abnormal

Cardiac abnormality

40

20

Normal

DsRed ST3Gal IV ST3Gal V

ST3Gal V

***

0.08

0.0008

0.06

0.0006

0.04

0.0004

0.02

0.0002

DsRed

ST3Gal IV

ST6Gal IV

sialidase − + − + − +

(kDa)

−+ −+ −+

Ft

(kDa) − +

210

140

100

70

55

LB: MAA

40

35

IB: β-actin

MAA epitope

1.0

0.5

ST3Gal IV

ST3Gal V

DsRed

70

55

DsRed

DsRed

ST6Gal V

sialidase − + − + − +

(kDa)

−+ −+ −+

Ft

(kDa) − +

210

140

100

70

55

70

55

LB: MAA

40

35

MAA/β-actin value

Copy numbers

ST3Gal IV

DsRed

n=5

Dead

ST6Gal I-KO

+ ST3Gal V mRNA

100

MAA/β-actin value

Fig. 5

IB: β-actin

MAA epitope

1.5

1.0

0.5

DsRed

ST3Gal V

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Conflict of interest

The authors declare no conflicts of interest regarding this manuscript

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