Cell Reports, Volume
17
Supplemental Information
cMyc Regulates the Size of the Premigratory
Neural Crest Stem Cell Pool
Laura Kerosuo and Marianne E. Bronner
Stage 4
Stage13
Stage11
Dlx5
cMYC
Stage 4
Stage 6
Stage 7
cMYC
C’
D’
Stage 7’
Stage 6’
A
B
C
D
E
F
D’
C’
Supp fig. 1
Supplemental Figure 1.
cMyc
Is Not Expressed At The Neural Plate Border, Related to Figure 1.
(A
-‐
B)
cMyc
RNA is not expressed at the neural plate border (marked with
Dlx5
) but rather more
lateral to it as depicted with the blue arrows
(C
-‐
D, D’)
and is absent from the neural folds at HH
stages 6 and 7.
(C’)
The anterior expression is of endodermal origin.
(E
-‐
F)
cMyc expression extends
far laterally in the migrating cranial neural crest and is also emerging in the premigratory / early
migrating ce
lls at vagal and trunk levels at stages 11 and 13. Expression is also seen in blood islands
in the extra
-‐
embryonic tissue. Scale bar for whole embryos 150
μ
m; sections 20
μ
m.
CoMO
cMYCMO
Sox2
Pax7
cMycMO
cMycMO
CoMo
CoMo
*
*
cMYC
1.2 mM cMYCMO Stage 7 / 8-
Stage 8+ /
FOXD3
1.5 mM cMYCMO Stage 8+/10
Stage 9
27%
18%
73%
0
20
40
60
80
100
NT closure failure/
degradation
NT closed
Stage 10
SOX10
Stage 9
SOX10
E’
cMYCMO
CoMO
A
B
C
D
E
E’
F
CoMO
cMYCMO
cMYCMO
CoMO
tubulin
19%
81%
mM
Supp fig. 2
H
CoMO
cMYCMO
Stage 8+ /
nMYC
H
H’
D’
CoMO
cMYCMO
G
HH 9/10
CoMO
cMYCMO
1.5
1.5
1.2
N=20
N=21
N=24
82%
Supplemental Figure 2. cMyc Morpholino Is Specific And Functions In A Dose Dependent Fashion,
Related to Figure 2 (A)
Western blot showing reduced levels of cMyc expression in embryonic head
lysates after 1.2mM cMycMO as compared to control morpholino
lysate.
(B, C)
cMyc morpholino does not
affect PAX7 immunopositive neural crest cells during induction at the neural plate border or
Sox2
positive
neural stem cells prior to cMyc expression at HH stage 7 (n=5/5).
(D
-
E, E’,D’)
Higher dose (1.5mM) of
cMyc m
orpholino causes a more severe phenotype with intensive degradation of the dorsal neural tube at
stages 8
-
9 (D’) visualized with the FITC labeled morpholino. Note that the chromogenic
in situ
staining
masks the FITC labeled CoMo in the control side of the
dorsal neural tube.
(F
-
G)
At stage 9
-
10,
degradation causes neural tube (NT) closure failure (black arrow) seen in 73% of the cMyc deficient
embryos injected with 1.5mM cMycMO as compared to 1.2mM cMycMO levels that predominantly have
closed neural tubes c
omparable to control embryos.
(H)
cMyc
knockdown is not compensated by increased
nMyc
expression levels in the premigratory neural crest cells (n=7/7). Scale bar 20
μ
m.
PHENOTYPE
NO PHENOTYPE
100
80
60
40
20
%
100
80
60
40
20
%
FL
FL
V394D
V394D
Neural tube closure failure/
degradation
Prolonged emigration
D
Pax7
Pax7
Dapi
Supp fig. 3
A
B
CO
FLMyc
cMycMo
CoMo
P27
Stage 9+
Stage 8
PH3
F
PH3
Dapi
G
E
Dapi
cMycRFP
merge
cMyc binding domain I
cMyc binding domain II
C
NESGSTDSQENSGETRLLRSGTYSDRTESKAYGSVTHKC
EESAGTDSQELGMEGQNLRSGTYGDRTESKAYGSIIHKC
-E-LATDSD---SQHLGSGATPYPDRNESRPYGSTTHKC
NESAGTDSQELGSEARGLRSGTYGDRTESKAYGSVIHKC
Miz1 chicken
Miz1 mouse
Miz1 frog
Miz1 human
KAGMKILEPEDG----SELNIVTVASDDMVTLATEALAATAVTQLTVVPVAAAVTADETEALKAEITKAVKQVQEADPNTQIL
KAGIKILEPEDG----GEVSVVTV--DDMVTLATEALAATAVTQLTVVPVAAAVTADETEVLKAEISKAVKQVQEEDPNTHIL
KAGMKVLRPEDGELDEDEVNIVTVASDEMVTLTTEAIAATAVTELTVVPVTTSVAADETEALKAEISKAVKQVQEADPNTQIL
KAGIKILEPEDG----SEVSVVTV--DDMVTLATEALAATAVTQLTVVPVAAAVTADETEVLKAEISKAVKQVQEEDPNTHIL
Miz1 chicken
Miz1 mouse
Miz1 frog
Miz1 human
x
x
x
conserved
conserved / similar AA switch
not conserved
Supplemental Figure 3. Related to figures 4,5 and 6. (A)
Antibody against Phospo Histone 3 shows
proliferative cells in the neural tube.
(B)
In situ hybridization shows lower levels of p27 mRNA in the
neural crest cell domain (circled) than in the ventral
neural tube.
(C)
The two well
-
characterized cMyc
binding domains of the Miz1 (ZBT17) protein are highly conserved among species from human to frog,
which trongly suggest that the chcken Miz1 can bind to the human cMyc protein construct. The cMyc
binding d
omain I corresponds to the area (AA 269
-
308 in Peukert et al., 1997) immediately before the first
zinc finger; and the second domain (AA 641
-
715 in Peukert et al., 1997) is located in between the last (12
th
)
zinc finger and the zinc finger
–
like domain. Th
e conserved areas are highlighted in yellow. The blue letters
indicate conservation with a switch into another amino acid of the same functional subgroup. The
sequences correspond to: Miz1_Gga GI: 171919772, Miz1_Mm GI: 443609492, Miz1_Xtr GI: 54261587
and
Miz1_Hsa GI: 62906906.
(D)
FL cMyc overexpression causes prolonged emigration of newly
produced neural crest cells from the dorsal neural tube in about 24% of all embryos roughly equivalent to
half of the embryos that show a phenotype of increased neural
crest cells in Fig. 6C) as compared to the
contralateral side electroporated with a control plasmid.
This is not seen in embryos overexpressing
V394DMyc.
(E)
70% of embryos that overexpress (2μg/μl) FL cMyc have neural tube closure defects
compared to 11%
of the V394DMyc overexpressing embryos, further showing that the Miz1 binding
mutant does not cause a phenotype in the neural crest cells (FLMyc n=25; V394DMyc n=27 for both D and
E).
(F)
Electroporation of a lower dosage (0.5 μg/μl) of cMyc FL is sufficie
nt to increase the neural crest
cell pool size as demonstrated by more PAX7 immunopositive cells migrating further laterally (n=4/5).
(G)
Transfection efficiency to chick crestospheres was 10
-
20% per sphere. Scale bar 20 μm.