S
UPPLEMENT
AL INFORMA
TION
List of Supplemental Materials
Figures S1 – S4
Movies S1, S2
Figure S1.
Figure
S1.
TUNEL
assays,
hid
expression
in
WT
vs
E2f1
mutant
backgr
ound,
rescue
of
htl
mutant
cell
death
by
constitutively-active
Ras,
and
quantification
of
CVM
cell
number
at
stage
11.
Related
to
Figure
1.
(A)
Stage
12
and
13
WT
embryos
immunostained
with
GFP
antibody
(red)
to
visualize
CVM
cell
membranes,
FasIII
(cyan)
to
visualize
TVM,
and
TUNEL
fluorescein
signal
(green)
to
visualize
cell
death.
In
WT
embryos,
some
cell
death
is
observed
in
CVM
cells
that
have
lost
adhesion
to
the
TVM
(white
arrowhead).
(B)
Widespread
cell
death
around
the
posterior
turn
in
stage
12
htl
mutant
embryos,
which
persists
into
late
stage
12.
(C-E’)
Stage
13
embryos
immunostained
with
Tey
antibody
(white
or
red)
to
visualize
CVM
cell
nuclei
and
FasIII
(cyan)
to
visualize
TVM.
(E-E’)
CVM
cell
death
phenotype
is
attenuated
via
tissue-specific
rescue
with
constitutively-active
Ras.
(F-H)
Imaging
of
stage
11
embryos
immunostained
with
Tey
antibody
to
visualize
CVM
cell
nuclei.
(I-J)
2
FISH
in
WT
and
E2f1
mutants
with
hid
riboprobe
(green)
and
immunostained
with
Tey
antibody
(purple).
(I)
In
WT
embryos,
hid
expression
is
observed
in
the
CVM
at
stage
12
and
is
maintained
through
stage
13.
(J)
In
E2f1
mutant
embryos,
there
is
a
significant
delay
in
the
onset
of
hid
expression,
such
that
little
to
no
mRNA
is
observed
in
the
CVM
at
stage
12,
and
a
reduced
level
is
observed
at
stage
13.
(K)
Quantification
of
CVM
cell
number
in
stage
11
WT,
htl
hid
,
and
htl
stg
mutants.
The
number
of
Tey-positive
nuclei
in
both
left
and
right
sides
was
counted
and
tabulated
for
each genotype (G, n=5 per genotype). p<0.05. Scale bars=20
μ
M.
3
Figure S2.
Figure
S2.
stgVM-lacZ
expression
dynamics
and
quantification
of
lacZ
riboprobe
fluorescence
intensity
.
Related
to
Figure
2.
(A-A’)
FISH
with
lacZ
riboprobe
(green)
in
stage
12
embryo
4
expressing
the
stgVM-lacZ
reporter
construct
in
a
WT
background,
co-stained
with
Bin
antibody
(blue)
to
visualize
the
visceral
mesoderm
(CVM
and
TVM,
white
arrowhead).
(B-C’)
FISH
with
lacZ
riboprobe
(green)
in
stage
11
and
stage
13
embryos
expressing
the
stgVM-lacZ
reporter
construct
in
a
WT
background,
co-stained
with
Tey
antibody
(purple)
to
visualize
the
CVM.
(B,B’)
In
stage
11
embryos,
there
is
a
lack
of
reporter
expression
around
the
CVM,
suggesting
that
this
enhancer
is
not
active
before
reaching
the
posterior
turn
at
stage
12.
(C,C’)
In
stage
13
embryos,
there
is
persistent
reporter
expression
in
the
posteriormost
CVM
cells.
(D)
FISH
with
lacZ
riboprobe
in
stage
12
embryo
expressing
a
dsRNA
construct
targeting
tkv
specifically
in
the
CVM,
with
majority
of
CVM
lacking
lacZ
mRNA
puncta.
(E)
FISH
with
lacZ
riboprobe
in
stage
12
embryo
expressing
constitutively-active
Tkv
specifically
in
the
CVM,
showing
increased
lacZ
mRNA
puncta
(yellow
arrowheads).
(F)
Mean
fluorescence
intensity
quantification
of
lacZ
riboprobe
signal
in
Stage
12
WT,
tkv
7
,
and
G447>tkv
CA
embryos.
(n=23-25
CVM
cells,
three
individuals
per
genotype).
p<0.05.
Scale
bars=20
μ
M.
Images
in
panels
A-E each depict a single plane.
5