Developmental Cell
Supplemental Information
Axud1
Integrates
Wnt
Signaling
and
Transcriptional Inputs
to
Drive Neural Crest Formation
Marcos Simões
-
Costa, Michael Stone, and Marianne
E. Bronner
Figure S1, related to Figure 2.
S
pecificity of Axud1 morpholinos
.
(A
-
C)
Electroporation of both
splice
-
(Axud1SBMo) and translation
-
blocking (Axud1TBMo) morpholinos into the right side of stage
HH5 embryos resulted in strong down
-
regulati
on of
FoxD3
, whe
n compared to the control side
.
(D
-
F)
C
o
-
electroporation of an Axud1 expression vector with either Axud1SBMo
(E)
or Axud1TBM
o
(F)
resulted in recovery of phenotype associated
with loss of FoxD3 expression
. (G) Quantitation of loss
-
of
-
functi
on an
d rescue experiments.
The total number of embryos analyzed for each experiment is
represented in parenthes
e
s.
(H
-
L)
Axud1 loss
-
of
-
function does not
cause
cell death in dorsal neural
folds.
(H
-
I
) Whole mount views of embryo electroporated with Axud1
morpholino (g
reen) and stained
with an anti
-
a
ctive Caspase
-
3 antibody (red). (
J
-
K
) Histological analysis indicates that electroporation of
Axud1 morpholino does not alter the number of cells positive to active Caspase
-
3 (shown with
arrowheads) in the dorsa
l neural folds.
(L
) Average number of cells in the neural tube positive
for
active
Caspase
-
3. Histological sections of six embryos electroporated with control and Axud1 morpholinos were
used for this analysis
(n = 6 embryos)
.
Error bars represent standard
deviation.
Active Casp3: a
nti
-
active
Caspase
-
3 antibody
,
CoMo: Control morpholino,
Axud1SBMo: A
xud1 splice
-
blocking morpholino,
Axud1TBMo: Axud1 translation
-
blocking morpholino
.
Figure S2
, related to Figure 4.
Loss of Axud1 results in upregulation of neuronal and placodal
markers in the dorsal neural folds
. (A) Whole mount views of embryo electroporated with Axud1
morpholino (green) and control morpholino (blue). (B)
Cross section of the cephalic region of an embryo
electroporated with control and Axud1 morpholinos.
Immuno
histochemistry with the neuronal marker
Sox2 antibody reveals
Axud1 knockdown results in upregulation
of this protein in the
dorsal neural folds
(n = 6/6)
.
(C)
Consistent with the results of Nanostring analysis (Fig
.
4), Axud1
loss
-
of
-
function also
causes
a medial expansion of the
Six1 positive
domain
(n = 5/6). The arrowhead shows the medial limit
of Six1 expression, at hindbrain
-
level sections. Mo: Morpholino.
Figure S3, related to Figure 5
.
The
Wnt/Axud1
pathway is
required for neural crest
specification.
Whole mount views of embryos (A
-
D) after
β
-
catenin morpholino (green) and control morpholino (blue)
were electroporated into the right and left neural fold
s, respectively. (E
-
H) Expression of neural crest
specifiers in the same embryo represented in A
-
D. Knock
down of
β
-
catenin
caused loss of multiple
neural crest specifier genes, including
FoxD3
(A, E),
Sox9
(B, F),
Snai2
(C, G) and
Sox10
(D
-
H). (I)
Quantit
ation of
β
-
catenin
loss
-
of
-
function experiments.
The total number of embryos analyzed for each
experiment is represented in parenthes
e
s.
(J
-
R)
Axud1 is sufficient to rescue
expre
ssion of
SoxE
genes,
Sox9
and
Sox10
,
following Wnt1 knoc
kdown
.
Morpholino
-
mediated Wnt1 loss
-
of
-
function results in
loss of neural crest specification, as assayed by
FoxD3
(Fig. 5). Neural crest specifiers
Sox9
(
J
,
L
),
Sox10
(
N
,
P
) are also strongly
downregulated following Wnt1 knockdown. C
o
-
electroporation of an Axud
1
expression vector with the Wnt1 morpholino
resulted in recovery of
Sox9
(
K
,
M
) and
Sox10
(
O
,
Q
)
expression
.
(
R
) Quantitation of loss
-
of
-
function and rescue experiments. The total number of embryos
analyzed for each experiment is represented in parenthes
e
s.
β
-
cat Mo:
β
-
catenin morpholino.
CoMo:
Control m
orpholino
, Wnt1Mo: Wnt1 morpholino.
Figure S4
, relate
d to Figure 6
.
Proximity ligation assay indicates putative interactions between
Axud1 and neural plate border specifiers in dorsal neural tube
.
(A, B) Proximity ligation assays
(PLA) puncta visualized in transverse sections through the neural tube
of HH9 embryos indicate a large
number of putative interactions between Axud1/Pax7 and Axud1/Msx1 in the region where the ne
ural
crest becomes specified.
(B) High magnification images of dorsal neural folds highlight the presence of
PLA puncta in the nucleus of the cells residing in the dorsal neural tube (white arrowheads).