Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information
Fig.
S1
Otic-enriched transcriptional regulators.
A.
Transcripts enriched in the otic
placode were identified by comparison of the otic transcriptome to the whole embryo at ss3
and known and putative transcription factors were extracted. Numbers of otic enriched
transcription factors (TF) at each stage are indicated in the lower right corner of each
diagram. A few known otic regulators are labelled.
B, C.
Validation of enrichment of 10 otic
transcription factors by RT-qPCR. At both 5-6ss (B) and 11-12ss (C), fold change was
calculated relative to the expression in whole embryo at 3ss. Fold change from RNAseq is
plotted alongside for comparison of consistency between two assays.
D.
Expression of 52
otic genes evaluated by NanoString. The genes are ranked based on their count. 46/52 otic
enriched genes pass the count >300 threshold at 11-12ss.
Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information
Fig.
S2
Summary of the expression patterns of 95 otic genes.
This table was assembled
using data from the literature and our own new data. References are provided in the table.
a
P
P
R
p
P
P
R
N
P
N
N
E
A
M
O
E
P
D
N
C
C
N
P
A
M
O
E
P
D
N
C
C
N
T
A
M
O
P
E
P
E
P
I
D
N
C
C
N
T
A
R
I
D
3
A
S
3
F
i
g
E
x
p
r
e
s
s
i
o
n
A
R
I
D
3
C
w
S
3
F
i
g
N
o
e
x
p
r
e
s
s
i
o
n
A
T
N
1
w
S
3
F
i
g
N
o
t
c
h
e
c
k
e
d
B
A
C
H
2
w
w
m
R
N
A
-
s
e
q
d
a
t
a
(
n
o
t
s
h
o
w
n
)
w
=
w
e
a
k
e
x
p
r
e
s
s
i
o
n
w
P
R
D
M
1
F
i
g
2
A
n
t
e
r
i
o
r
/
L
e
n
s
A
B
M
P
4
S
t
r
e
i
t
a
n
d
S
t
e
r
n
,
1
9
9
9
;
B
o
t
h
e
e
t
a
l
.
,
2
0
1
1
M
e
d
i
a
l
/
T
r
i
g
e
m
i
n
a
l
M
c
M
Y
B
w
w
B
e
t
a
n
c
u
r
e
t
a
l
.
,
2
0
1
1
A
n
t
e
r
i
o
r
N
e
u
r
a
l
P
l
a
t
e
a
N
P
C
R
E
B
B
P
w
w
S
3
F
i
g
P
o
s
t
e
r
i
o
r
/
O
E
P
D
O
E
P
D
D
A
C
H
2
O
L
R
i
m
S
3
F
i
g
O
t
i
c
P
l
a
c
o
d
e
O
P
D
L
X
3
K
h
u
d
y
a
k
o
v
a
n
d
B
o
n
n
e
r
-
F
r
a
s
e
r
,
2
0
0
9
E
p
i
b
r
a
n
c
h
i
a
l
P
l
a
c
o
d
e
E
P
D
L
X
5
O
L
S
t
r
e
i
t
,
2
0
0
2
E
p
i
d
e
r
m
i
s
E
P
I
D
E
T
S
1
G
e
i
s
h
a
e
x
p
r
e
s
s
i
o
n
p
a
t
t
e
r
n
;
S
3
F
i
g
A
n
t
e
r
i
o
r
N
e
u
r
a
l
R
i
d
g
e
A
N
R
E
T
S
2
m
R
N
A
-
s
e
q
d
a
t
a
(
n
o
t
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h
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w
n
)
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e
u
r
a
l
P
l
a
t
e
/
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r
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t
N
C
C
E
T
V
4
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L
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u
n
n
e
t
a
l
.
,
2
0
0
7
N
e
u
r
a
l
F
o
l
d
/
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u
b
e
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T
E
T
V
5
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L
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u
n
n
e
t
a
l
.
,
2
0
0
7
O
l
f
a
c
t
o
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y
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l
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d
e
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L
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Y
A
1
w
w
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h
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r
a
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t
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l
.
,
2
0
0
8
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Y
A
2
M
c
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a
r
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t
a
l
.
,
2
0
0
3
;
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s
h
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h
a
r
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t
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l
.
,
2
0
0
8
E
Y
A
4
w
w
w
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i
m
S
3
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g
F
O
X
D
3
K
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y
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k
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v
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n
d
B
o
n
n
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-
F
r
a
s
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r
,
2
0
0
9
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O
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G
1
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a
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n
p
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F
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I
2
K
h
a
t
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a
n
d
G
r
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v
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s
,
2
0
1
3
F
O
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I
3
w
K
h
a
t
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n
d
G
r
o
v
e
s
,
2
0
1
3
F
O
X
P
1
S
3
F
i
g
F
O
X
P
4
l
S
3
F
i
g
F
S
T
L
4
w
u
n
p
u
b
l
i
s
h
e
d
G
A
T
A
3
S
h
e
n
g
a
n
d
S
t
e
r
n
,
1
9
9
9
;
B
o
t
h
e
a
n
d
D
i
e
t
r
i
c
h
,
2
0
0
3
G
B
X
2
N
i
s
s
a
n
d
L
e
u
t
z
,
1
9
9
8
;
S
h
a
m
i
m
a
n
d
M
a
s
o
n
,
1
9
9
8
;
P
a
x
t
o
n
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t
a
l
.
,
2
0
1
0
H
M
G
1
S
3
F
i
g
H
O
M
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R
2
O
L
O
L
O
L
u
n
p
u
b
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H
O
X
A
2
P
a
x
t
o
n
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t
a
l
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,
2
0
1
0
H
O
X
A
3
P
a
x
t
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n
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t
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l
.
,
2
0
1
1
I
R
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1
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4
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2
0
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0
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1
9
M
c
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r
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t
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l
.
,
2
0
0
3
;
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e
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K
L
F
7
S
3
F
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g
K
L
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8
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3
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g
K
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w
w
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r
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F
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r
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o
,
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3
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,
1
9
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t
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r
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2
0
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d
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t
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l
.
,
2
0
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4
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a
r
d
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t
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l
.
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2
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1
1
M
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B
B
e
t
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n
c
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r
P
e
t
a
k
.
,
2
0
1
1
M
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h
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d
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n
d
B
o
n
n
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r
a
s
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r
,
2
0
0
9
N
F
K
B
1
u
n
p
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b
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5
.
1
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1
9
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2
0
0
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;
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t
r
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t
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o
t
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7
;
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2
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;
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h
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;
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1
6
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2
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3
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2
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N
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3
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3
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g
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N
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w
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t
a
(
n
o
t
s
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w
n
)
Z
N
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(
n
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R
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1
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2
Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information
Fig.
S3
Expression patterns of new otic and epibranchial transcription factors
.
Arid3a
(
A, B
),
FoxP4
(
C, D
),
Hmg1
(
E, F
),
Irx5
(
G, H
) and
FoxP1
(
I, J
) are expressed from OEP
stages and remain widely expressed in the otic and epibranchial territories, except for
FoxP4
which becomes confined to the epibranchial region (
D
).
Klf7
(
K, L
) is already expressed at
6ss and becomes restricted to the epibranchial domain at 12ss (
L
).
Tfap2e
(
M, N
) is
expressed in neural crest cells and later in the epibranchial placodes. Late onset genes
(after 11-12ss) include
Arid3c
(
O, P
),
Atn1
(
Q, R
),
Znf384
(
S, T
)
Klf8
(
U, V
),
Prep2
(
W, X
),
Tead3
(
Y, Z
),
Bach2
(
AA, AB
).
Sox11
(
AC, AD
),
Tbx10
(
AE, AF
),
Eya4
(
AG, AH
),
Lef1
(
AI,
AJ
),
Ets1
(
AK, AL
) and
Dach2
(
AM, AN
) are localized at the ridge the otic cup and/or in
neural crest cells (
Lef1
,
Ets2
). Most factors are also expressed in other domains like the
neural tube and underlying mesoderm.
Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information
Fig.
S4
Otic genes are partitioned into different clusters. A.
6 clusters are revealed by
hierarchical clustering (C1-6). The top enriched biological process and signalling pathway
are indicated for each cluster.
B-D
expression profiles of signalling pathway components in
cluster C2 (
B
), C5 (
C
) and C6 (
D
). * in C indicates genes whose expression is plotted
against the second axis on the right hand side, while * in D indicates that Log2(RPKM) is
used to plot DTX4 expression.
Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information
Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information
The table summarizes the interactions described in the otic gene regulatory network (Fig
.7)
including the appropriate references. Gene names are colour-coded as in the networks and
use chick nomenclature; names used in other species are shown in brackets.
*
Exper
i
mentally verified direct interactions. References are listed in the final section of
supplementary information.
Fig.
S5
Known interactions from the literature.
Fig.
S6
Summary of perturbation experiments. A.
Summary of Etv4, Pax2 and Lmx1a
morpholino knockdown experiments evaluated with qPCR, NanoString (NS) and
in situ
hybridization (ISH).
B, C
. Evaluation of Pax2 and Etv4 knockdown by qPCR. * significantly
affected genes (p-value<0.05).
Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information
Table.
S1
Otic
-enriched genes.
Genes enriched in the otic placode at 5-6ss, 8-9ss and 11-
12ss using fold change > 1.5 and a padj<
0.1. Genes labelled “NA” are removed because
their RPKM is <4 or count number <300.
Table.
S2
Otic
-enriched genes.
Genes enriched in the otic placode at 5-6ss, 8-9ss and 11-
12ss using fold change >1.5 as cut-
off. Genes labelled “NA” are removed because their
RPKM is <4 or count number <300.
Table.
S3
Time course of differentially expressed genes.
Genes labelled “NA” are
removed because RPKM is <4 or count number <300.
Click here to Download Table S1
Click here to Download Table S2
Click here to Download Table S3
Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information
Table.
S4
Otic
transcription factors used for cluster analysis.
The data sheet contains
transcription factors enriched in otic territory (see Fig 1) and differentially expressed from
time-course analysis (see Fig 3) except those downregulated at 5-6ss relative to HH6. The
fold change was calculated with DEseq relative to HH6 and the normalized RPKM was
acquired from Cuffdiff2.
Table
. S5 Genes affected by Etv4 and Pax2 knockdown.
NanoString nCounter data
showing changes in gene expression after Etv4 and Pax2 knockdown. Genes with a mean
expression count of less than 300 in either condition are excluded from any analysis.
Click here to Download Table S4
Click here to Download Table S5
Development 144: doi:10.1242/dev.148494: Supplementary information
Development • Supplementary information