1 Perturbation of Retinoid Homeostasis Increases Malformation Risk in Embryos Exposed to Pregestational Diabetes Short title: Retinoid homeostasis in diabetic embryopathy Leo M.Y. Lee, 1,2,3 Maran B.W. Leung 1 , Rachel C.Y. Kwok 1 , Yun-Chung Leung 3 , Chi-Chiu Wang 1,2 , Peter J. McCaffery 4 , Andrew J. Copp 5 and Alisa S.W. Shum 1 1 School of Biomedical Sciences, 2 Department of Obstetrics and Gynaecology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong 3 Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong 4 Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom 5 Newlife Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, United Kingdom Corresponding author: Alisa S.W. Shum School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong Tel: (852) 39436840 Fax: (852) 39420990 e-mail: [email protected](7 Figures, 1 Table, 7 Supplementary Figures, 3 Supplementary Tables) Page 1 of 47 Diabetes Diabetes Publish Ahead of Print, published online January 13, 2017
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1
Perturbation of Retinoid Homeostasis Increases Malformation Risk in
Embryos Exposed to Pregestational Diabetes
Short title: Retinoid homeostasis in diabetic embryopathy
Leo M.Y. Lee,1,2,3
Maran B.W. Leung1, Rachel C.Y. Kwok
1, Yun-Chung Leung
3,
Chi-Chiu Wang1,2
, Peter J. McCaffery4, Andrew J. Copp
5 and Alisa S.W. Shum
1
1School of Biomedical Sciences,
2Department of Obstetrics and Gynaecology, Faculty
of Medicine, The Chinese University of Hong Kong, Hong Kong
3Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University, Hong Kong
4Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United
Kingdom
5Newlife Birth Defects Research Centre, Institute of Child Health, University College
London, London WC1N 1EH, United Kingdom
Corresponding author:
Alisa S.W. Shum
School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong
Figure 1 - Embryos of diabetic mice exhibit specific down-regulation of Cyp26a1. A-D,F
and H: Whole-mount ISH patterns of Cyp26 genes in embryos of manifestly diabetic
(MD) mice compared with those of non-diabetic (ND) mice. Cyp26a1 mRNA transcripts
are seen in extraembryonic endoderm (red arrowhead) and headfold mesenchyme (yellow
arrowhead) of E7 conceptuses (A), cranial mesenchyme (green arrowhead) and caudal
neural plate (orange arrowhead) of E8 embryos (B), craniofacial, cervical and branchial
arch mesenchyme (circled) (C), and tailbud (blue arrowhead) of E9 embryos (D).
Cyp26b1 (F) and Cyp26c1 (H) are expressed in the cranial but not in the tailbud region of
E9 embryos. At least 20 embryos from 5-6 litters were examined for each group. Scale
bar = 0.05 mm (A), 0.1 mm (B), 0.7 mm (C), 0.2 mm (D), 0.7 mm for whole embryo and
0.35 mm for caudal region (F and H). E,G,I: Quantification of mRNA levels of Cyp26a1
(E), Cyp26b1 (G) and Cyp26c1 (I), normalized to β-actin, and expressed relative to ND,
which was set as 1 (n = 5 from 5 litters). *P < 0.05, Student’s t test. Error bars represent
mean ± SEM.
Figure 2 - Embryos of diabetic mice show reduced efficiency of RA catabolism. A: In
vitro RA degrading efficiency, presented as % of RA in the medium being degraded by
the tailbud lysate, in the presence or absence of cofactor (NADPH) and reducing agent
(DTT) for optimal activity of CYP26 enzymes, and varying concentrations of R115866
(CYP26 inhibitor) (n = 18 in ND and MD groups with NADPH-DTT, and n = 3-9 in
other groups, from 20 ND and 18 MD litters). *P < 0.001, Student’s t test; †R
2 = 0.742
Page 26 of 47Diabetes
27
and P = 0.001, linear regression. B: In vivo RA clearance measured as the amount of RA
released from individual tailbuds, using a RA reporter cell line, at hourly intervals after
injection of 50 mg/kg RA at E9 (n = 16-42 from 3-8 litters). *P < 0.001 vs ND; Student’s
t test and nonlinear regression. Error bars represent mean ± SEM.
Figure 3 - Tailbuds of E9 embryos of diabetic mice have increased levels of endogenous
RA detected using a RA reporter cell line. A: Figure to illustrate the caudal-most portion
of the tailbud (boundary marked by dotted line) as excised for detection of bioactive RA.
B: Percentage of tailbuds excised from embryos of non-diabetic (ND) and manifestly
diabetic (MD) mice that have induced different numbers of positively stained cells in the
RA reporter cell line (n = 19 for ND and 22 for MD from 3 and 4 litters respectively). C:
Representative figures of excised tailbuds that were placed directly on the RA reporter
cells and have induced different numbers of stained cells in the RA reporter cell line.
Figure 4 - Tailbuds of E9 embryos of diabetic mice exhibit a greater magnitude of
suppression of key genes for caudal development induced by RA. A: Quantification of
mRNA levels of various caudal regulatory genes and RA nuclear receptors, normalized to
β-actin, and expressed relative to ND, which was set as 1, in tailbuds of embryos of
non-diabetic (ND) and manifestly diabetic (MD) mice at E9 (n = 5 from 5 litters). **P <
0.01, Student’s t test. B and C: Quantification of mRNA levels of Cyp26a1 (B), and
various caudal regulatory genes and RA nuclear receptors (C), normalized to β-actin, and
expressed relative to ND (CON), which was set as 1, in tailbuds of embryos of ND and
MD mice 4 h after maternal injection of 50 mg/kg RA (50RA) or vehicle as control (CON)
Page 27 of 47 Diabetes
28
at E9 (n = 5 from 5 litters). *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA
followed by Bonferroni test. Error bars represent mean ± SEM.
Figure 5 - Cyp26a1 loss of function genotype interacts with diabetic maternal
environment to influence RA degrading efficiency and susceptibility to RA teratogenesis.
A: Whole-mount ISH patterns of Cyp26a1 expression in Cyp26a1 heterozygous null (+/-)
embryos and their wild-type (+/+) littermates from non-diabetic (ND) and manifestly
diabetic (MD) mice at E9. At least 30 embryos from 6-8 litters were examined for each
group. Scale bar = 0.25 mm. B: Quantification of Cyp26a1 mRNA, normalized to β-actin,
and expressed relative to ND (+/+), which was set as 1, in tailbuds of embryos from
different genotype-maternal environment combinations at E9 (n = 5 from 5 litters). C: In
vitro RA degrading efficiency, presented as % of RA in the medium being degraded by
the tailbud lysate in the absence or presence of 100 nM R115866 (CYP26 inhibitor) (n =
8-13 from 10-14 litters). D: Quantification of RA released from individual tailbuds, using
a RA reporter cell line, at 3 h after in vivo challenge with 25 mg/kg RA (25RA) at E9 (n =
22-28 from 6-7 litters). E: Maternal injection of 25 mg/kg RA (25RA) or vehicle as
control (CON) at E9. Caudal truncation, measured in terms of the ratio of tail
length/crown-rump length (TL/CRL), was examined in E13 embryos (n = 41-62 from 10
litters). F: Maternal injection of 40 mg/kg RA (40RA) or CON at E9. Near-term E18
fetuses were examined for renal malformations (n = 11 litters). *P < 0.05; **P < 0.01;
***P < 0.001, one-way ANOVA followed by Bonferroni test. Error bars represent mean ±
SEM.
Page 28 of 47Diabetes
29
Figure 6 - Pre-conditioning with low dose RA up-regulates Cyp26a1, increases RA
degrading efficiency, and protects against RA-induced caudal truncation. A:
Quantification of Cyp26a1 mRNA, normalized to β-actin, and expressed relative to ND
(CON), which was set as 1, in tailbuds of E9 embryos from non-diabetic (ND) and
manifestly diabetic (MD) mice 2 h after maternal oral feeding with low dose RA of 0.625
mg/kg (0.625RA) or 1.25 mg/kg (1.25RA), or with vehicle as control (CON) (n = 5 from
5 litters). B: In vitro RA degrading efficiency, presented as % of RA in the medium being
degraded by the tailbud lysate from embryos with or without preconditioned with
0.625RA (n = 5 from 3-5 litters). C: Embryos, with or without preconditioned with low
dose RA, were maternally challenged with a teratogenic dose of 25 mg/kg RA (25RA) at
E9, and examined for caudal truncation, measured in terms of the ratio of tail
length/crown-rump length (TL/CRL) at E13 (n = 34-60 from 3-5 litters). *P < 0.01; **P <
0.001, Student’s t test. †R
2 = 0.900 and P < 0.001;
††R
2 = 0.869 and P < 0.001;
†††R
2 =
0.610 and P < 0.001; ††††
R2 = 0.729 and P < 0.001, linear regression. Dotted line
represents no significant difference between the two groups. Error bars represent mean ±
SEM.
Figure 7 - Increased susceptibility of embryos of diabetic mice to RA-induced neural
tube defects is abolished by pre-conditioning with low dose RA. A and B: Incidence rates
of exencephaly (A) and spina bifida (B) in E13 Cyp26a1 heterozygous null (+/-) and
wild-type (+/+) embryos from non-diabetic (ND) and manifestly diabetic (MD) mice (n =
8-9 litters). Effect of pre-conditioning by oral feeding of low dose 0.625 mg/kg RA
(0.625RA) is compared with vehicle-fed control (CON) or no treatment (NT) group, 2 h
Page 29 of 47 Diabetes
30
prior to in vivo challenge with a teratogenic dose of 25 mg/kg RA (25RA) at E8. *P <
0.05; **P < 0.01; ***P < 0.001, one-way ANOVA followed by Bonferroni test. Dotted line
represents no significant difference between the two groups. Error bars represent mean ±
SEM.
Page 30 of 47Diabetes
0.0
0.4
0.8
1.2
0.0
0.4
0.8
1.2
ND MD
0.0
0.4
0.8
1.2
ND MD
ND MD
E7
ND MD
E8
E9E9
Rel
ativ
e ex
pres
sion
of
Cyp
26a1
/ -a
ctin
A B
C D
E ND MD
E7Conceptus
E8Embryo
E9Embryo
E9Tailbud
Cyp
26b
1 C
yp26
a1
F
ND MD
Cyp
26c1
H I
G
E9
E9
* * * *
E9
Figure 1
Rel
ativ
e ex
pres
sion
of
Cyp
26b1
/ -a
ctin
Rel
ativ
e ex
pres
sion
of
Cyp
26c1
/-a
ctin
ND MD
ND MD
Page 31 of 47 Diabetes
0
20
40
60
80
100
0
2
4
6
8
0 1 2 3 4 5 6 7 8 9
Tailbud lysate
NADPH-DTT
CYP26 inhibitor (nM)
ND
-
-
ND
+
-
ND
+
1
ND
+
10
ND
+
100
MD
+
-
MD
+
100
% R
A in
med
ium
bei
ng d
egra
de
d
†
A
Am
ount
of
RA
rel
ease
d fr
om
indi
vidu
al t
ailb
ud (
nM)
No. of hours post-injection of RA
BMDND
**
ND MD
Figure 2
*
*
*
**
*
*
*
Page 32 of 47Diabetes
0
20
40
60
80
100
ND MD
(-) (+) (++) (+++)
A B
C
> 40 stained cells (+++)
21-40 stained cells (++)
10-20 stained cells (+)
< 10 stained cells (-)% o
f ta
ilbud
s
Figure 3
ND MD
Page 33 of 47 Diabetes
0
2
4
6
8
10
12
14
Cyp26a1
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fgf 8 Wnt3a Cdx2 RARg RXRa
Figure 3
Rar RxrFgf8 Wnt3a Cdx2 Cyp26a1
BND MD
ND (CON)
MD (CON) ND (50RA) MD (50RA)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fgf 8 Wnt3a Cdx2 RARg RXRaRxrCdx2Fgf8 Wnt3a Rar
***
***
*C
**
******
**
*
**
*** ***
Rel
ativ
e ex
pres
sion
of
targ
et g
ene/-
actin
Rel
ativ
e ex
pres
sion
of
Cyp
26a1
/-a
ctin
Rel
ativ
e ex
pres
sion
of
targ
et g
ene/-
actin
***** **
***A
Figure 4
Page 34 of 47Diabetes
0.0
0.2
0.4
0.6
0.8
1.0
ND SD
0
10
20
30
40
50
60
70
ND SD Inhibitor0.0
0.2
0.4
0.6
0.8
1.0
1.2
ND MD
**
0
20
40
60
80
100
ND MD
ND (+/+) MD (+/+) MD (+/-)ND (+/-)
A
B
ND MD
**
****
ND (+/+) ND (+/-) MD (+/+) MD (+/-)
Rel
ativ
e ex
pres
sion
of
Cyp
26a1
/ -a
ctin
***
TL/
CR
L
E
0.0
0.1
0.2
0.3
0.4
0.5
0.6
ND MD ND MDND NDMD MD(CON) (CON) (25RA) (25RA)
***
******
***
****
D
ND MD
******
******
(25RA) (25RA)
***C
% R
A in
med
ium
bei
ngde
grad
ed
ND MD
**
**
***
***%
em
bryo
s pe
r lit
ter
with
re
nal m
alfo
rmat
ions
ND(40RA)
MD(40RA)
******
***
**
F
Figure 5
***
Am
ount
of
RA
rel
ease
d fr
om in
divi
dual
tai
lbud
(nM
)
ND +CYP26 inhibitor
Page 35 of 47 Diabetes
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
CON
0.62
5RA
1.25
RACO
N
0.62
5RA
1.25
RA
0.0
0.1
0.2
0.3
0.4
0.5
CON
1.25
RA
CON +
25R
A
0.62
5RA + 2
5RA
1.25
RA + 2
5RACO
N
1.25
RA
CON +
25R
A
0.62
5RA + 2
5RA
1.25
RA + 2
5RA
0
20
40
60
80
100
CON
0.62
5RACO
N
0.62
5RA
% R
A in
med
ium
be
ing
degr
ade
d
TL/
CR
L
***
††† ††††
TL/
CR
L
**
†††
*A B
C
**
ND MD
Figure 6
Rel
ativ
e ex
pres
sion
of
Cyp
26a1
/ -a
ctin
** *** *
Page 36 of 47Diabetes
0
20
40
60
80
100
CTL CTL CTL-OF SD SD SD-OF
0
20
40
60
80
100
CTL CTL CTL-OF SD SD SD-OF
A
% e
mbr
yos
per
litte
r w
ith
spin
a bi
fida
% e
mbr
yos
per
litte
r w
ith
exen
cep
hal
y
**
**
***
B**
**
*
**
**
*
**
** **
ND (+/+)
ND (+/-)
MD (+/+) MD (+/-)
Figure 7
***
***
*
*
*
Page 37 of 47 Diabetes
0.0
0.5
1.0
1.5
2.0
2.5
-12-11-10-9-8-7-6
OD
600
Log RA concentration (M)
Supplementary Figure 1. A representative standard curve showing the response ofthe RA reporter cell line to serially diluted RA solutions from 10-6M to 10-11M. Alinear dose-response is present from 10-7M to 10-10M. RA concentration of the samplewas plotted against the standard curve within the linear range. R2 represents thecoefficient of determination for linear regression analysis.
R2 = 0.9994
Page 38 of 47Diabetes
0
2
4
6
8
10
12
Immediatelyfrozen
37 °C 20hr
Supplementary Figure 2. RA was stable at 37°C for 24 h. RA was added to theculture medium at a concentration of 10 nM. The medium was either immediatelyfrozen and stored at -80°C (n = 4) or incubated at 37°C for 24 h in a 5% CO2 incubator(n = 4). The RA concentrations of the two groups of samples were then determined bythe RA reporter cell line. There was no significant degradation of RA in the samplethat had been incubated at 37°C for 24 h when compared with the immediately frozensample.
Am
ount
of
RA
(nM
)
Immediatelyfrozen
37oC for 24 h
Page 39 of 47 Diabetes
0
1
2
3
4
ND MD
Am
ount
of
prot
ein
per
tailb
ud (
µg)
Supplementary Figure 3. No significant difference in the protein content of theexcised tailbuds from embryos of non-diabetic (ND) and manifestly diabetic (MD)mice. Total protein in individual tailbuds measured using Bradford assay (n = 18-20from 3 litters). Error bars represent mean ± SEM.
ND MD
Page 40 of 47Diabetes
Supplementary Figure 4. The midbrain, which does not express any of the threeCyp26 genes, exhibits no difference in RA levels between embryos of diabetic andnon-diabetic mice after RA treatment. A: In situ hybridization on E9 embryosdemonstrates that all three Cyp26 genes (Cyp26a1, Cyp26b1 and Cyp26c1) do notexpress in the midbrain (MB) with or without (No RA) being maternally treated with50 mg/kg RA (50RA). Scale bar = 0.4 mm. B: Amount of RA in the tailbud andmidbrain of E9 embryos 3 h post-injection of 50RA, measured using HPLC (n = 7-9from 7-9 litters). The tailbud of embryos of manifestly diabetic (MD) mice withreduced Cyp26a1 expression had a significantly greater amount of RA than inembryos of non-diabetic (ND) mice. In contrast, the midbrain, without Cyp26expression, showed no difference in the amount of RA between embryos of MD andND mice. *P < 0.001, Student’s t test. Error bars represent mean ± SEM.
0
4
8
12
16
Tailbud Midbrain
MB MB
Cyp26a1 Cyp26b1 Cyp26c1
MB
No
RA
50R
A
MBMB MB
MBMB
Am
ount
of
RA
(nM
)
A
B *
ND MD
Tailbud(50RA)
Midbrain(50RA)
Page 41 of 47 Diabetes
Supplementary Figure 5. Heterozygosity for Cyp26a1 loss of function exacerbatesincreased susceptibility to RA-induced caudal truncation in embryos exposed todiabetes. Morphology of the caudal region of E13 Cyp26a1 heterozygous null (+/-)embryos and their wild-type (+/+) littermates from non-diabetic (ND) and manifestlydiabetic (MD) mice that were challenged in vivo with 25 mg/kg RA (25RA) or vehicleas control (CON) at E9. Embryos of different genotype-maternal environmentcombinations showed prominent differences in susceptibility to RA-induced caudaltruncation. Scale bar = 0.1 cm.
ND (+/-) MD (+/-)
ND (+/+) MD (+/+)
ND (+/+) MD (+/+)
25R
AC
ON
Page 42 of 47Diabetes
Supplementary Figure 6. Oral feeding of low dose RA does not cause any change inexpression of key genes for caudal development. Quantification of mRNA levels ofvarious caudal regulatory genes, normalized to β-actin, and expressed relative to ND(CON), which was set as 1, in tailbuds of E9 embryos of non-diabetic (ND) andmanifestly diabetic (MD) mice, 2 h after oral feeding with low dose 0.625 mg/kg RA(0.625RA) or vehicle as control (CON) (n = 5 from 5 litters). No significant changesin mRNA levels were associated with low dose RA treatment. *P < 0.05, one-wayANOVA followed by Bonferroni test. Error bars represent mean ± SEM.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fgf8 Wnt3a Cdx2Fgf8 Wnt3a Cdx2
* * ND (CON)
MD (CON) ND (0.625RA) MD (0.625RA)
Rel
ativ
e ex
pres
sion
of
targ
et g
ene/-
actin
Page 43 of 47 Diabetes
0
5
10
15
20
25
30
35
No RA 25RA
% e
mbr
yos
per
litte
rw
ith e
xenc
eph
aly
Supplementary Figure 7. Incidence of exencephaly in E13 embryos (ICR♂x ICR♀genetic background) from non-diabetic (ND) and manifestly diabetic (MD) mice withor without (No RA) in vivo challenge with 25 mg/kg RA (25RA) at E8 (n = 9-12litters). In untreated conditions, only 3% of embryos of MD mice developedexencephaly. However, when challenged with 25RA, a dose that only inducedexencephaly in 3% of embryos of ND mice, embryos of MD mice were significantlymore susceptible to RA teratogenesis and exhibited a nine-fold increase in theincidence rate of exencephaly. *P < 0.001, one-way ANOVA followed by Bonferronitest. Error bars represent mean ± SEM.
***
ND MD
No RA 25RA
Page 44 of 47Diabetes
Supplementary Table 1. Blood glucose levels of non-diabetic (ND) and manifestly diabetic (MD) mice before pregnancy and on the day of embryo collection
ND
Before pregnancy
ND Day of embryo collection (E9)
MD Before
pregnancy
MD Day of embryo Collection (E9)
Blood glucose levels (mmol/L) mean ± SEM
Not determined
6.08 ± 0.12 (n = 12 )
21.56 ± 0.32 (n = 101)
27.53 ± 0.51* (n = 67)
Remarks: The blood glucose levels of MD mice on the day of embryo collection at E9 were significantly higher than MD mice before pregnancy (*P < 0.001, Student’s t test), with none of them having blood glucose levels lower than 16.7 mmol/L. Similarly, there were hardly any MD mice that exhibited blood glucose levels lower than 16.7 mmol/L on the day of embryo collection at other stages (E8, E13 and E18). These findings supported that embryos of MD mice were exposed to a hyperglycemic milieu throughout development.
Page 45 of 47 Diabetes
Supplementary Table 2. PCR conditions and primer sequences
The PCR conditions included initiation at 95oC for 10 minutes, followed by 40 cycles comprising of denaturation at 95°C for 15 seconds, annealing at 55°C for 30 seconds and extension at 72°C for another 30 seconds. Primers sequences, designed by the Primer Express Software (Applied Biosystems), for detecting various mouse genes were:
Gene Primer Sequences
β-actin forward: 5’-TGT TAC CAA CTG GGA CGA CA-3’
reverse: 5’-GGG GTG TTG AAG GTC TCA AA-3’
Cdx2 forward: 5’-AAA CTC CAC TGT CAC CCA GT-3’
reverse: 5’-CCT GAG GTC CAT AAT TCC AC-3’
Cyp26a1 forward: 5’-CAG TGC TAC CTG CTC GTG AT-3’
reverse: 5’-AGA GAA GAG ATT GCG GGT CA-3’
Cyp26b1 forward: 5’-TTC AGT GAG GCA AGA AGA CA-3’
reverse: 5’-CTG GGA GGA GGT GCT AAG TA-3’
Cyp26c1 forward: 5’-GGG ACC AGT TGT ATG AGC AC-3’
reverse: 5’-AGC CAA CTC CTT CAG CTC TT-3’
Fgf8 forward: 5’-AGA GAT CGT GCT GGA GAA CA-3’
reverse: 5’-AAG GGC GGG TAG TTG AGG AA-3’
Rarγ forward: 5’-AGG CAG CAG ACT GAC CAT TT-3’
reverse: 5’-TTC TGG TAG GTG TGC AGC AG-3’
Rxrα forward: 5’-TCA CCA TCC TCG CCA TCT TT-3’
reverse: 5’-CTC CAA ACA GAG GTG CCA TG-3’
Wnt3a forward: 5’-CTG GCA GCT GTG AAG TGA AG-3’
reverse: 5’-GCC TCG TAG TAG ACC AGG TC-3’
Page 46 of 47Diabetes
Supplementary Table 3. RA in individual tailbuds of embryos of non-diabetic (ND) and manifestly diabetic (MD) mice 3 h post-injection of 50 mg/kg RA at E9, measured using the RA reporter cell line or HPLC
Amount of RA per tailbud (nM)
Method of measurement ND MD Difference between
ND and MD
RA reporter cell line 4.21 0.13 (n = 36)*
6.71 0.20 (n = 42)*
59.38%
HPLC 7.88 0.62 (n = 7)**
12.80 0.79 (n = 9)**
62.44%
* One tailbud in each sample ** Tailbuds from one litter of embryos were pooled as one sample Remarks: The data for the RA reporter cell line was extracted from Fig. 2B. The HPLC experiment was conducted in a separate study to validate the result in Fig. 2B. In analyzing the result, it is more relevant to compare the difference between ND and MD groups using the two RA detection methods, rather than the absolute amount of RA in the tailbud.