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Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
twist1
|
|
laevis
|
NF stage 18
|
neural crest
|
twist1
|
|
laevis
|
NF stage 24
|
mandibular crest
,
hyoid crest
,
cranial neural crest
,
migratory neural crest cell
|
zic1
|
|
laevis
|
NF stage 18
|
neural crest
,
neural plate border
|
pax3
|
|
laevis
|
NF stage 18
|
neural crest
,
neural plate
,
neural plate border
|
sox2
|
|
laevis
|
NF stage 18
|
neural plate
|
snai2
|
|
laevis
|
NF stage 14
|
neural crest
,
neural plate border
|
snai2
|
|
laevis
|
NF stage 18
|
neural crest
,
neural plate border
|
hes4
|
|
laevis
|
NF stage 18
|
neural crest
,
neural plate
|
msx1
|
|
laevis
|
NF stage 18
|
neural crest
,
neural plate border
|
myod1
|
|
laevis
|
NF stage 18
|
paraxial mesoderm
|
krt12.4
|
|
laevis
|
NF stage 18
|
epidermis
,
non-neural ectoderm
|
|
|
Fig. 3. PFKFB4 low-level depletion delays NC
early specification, causes retention of NB
character, and impairs NC late specification and
migration. (A) At st.14, snail2 expression was
severely reduced, or abolished, on the injected side.
(B) At st.18, sibling embryos had recovered snail2
expression. (C,D) Whereas sox10 expression was
mainly unaffected, twist1 was severely impaired.
(E-H) In contrast, expression of the immature NC
marker hes4 was expanded, as were some NB
markers, either strongly ( pax3) or moderately (zic1).
Other NB markers were unperturbed (msx1).
(I-K) Neural plate (sox2), non-neural ectoderm (ep.
ker.) and paraxial mesoderm (myod) seemed to be
unaffected. A-K: dorsal views. (L) Percentage of
embryos with each phenotype, i.e. diminished,
increased or normal expression. Snail2 score at
st.14 is indicated as the first bar, then several gene
scores at st.18 are indicated in the following ten
bars. (M-R) St.24 tailbud embryos exhibited a
severe NC migration defect (M-Q). Sox2 expression
appeared grossly unaffected, despite marginal
reduction of optic vesicle size (R). The injected side
(inj) is compared with the control side (co) in side
views (M,N,P,Q, anterior to the right) or frontal views
(O,R; red arrow on injected side). (S) Co-injection of
pfkfb4 mRNA with PFKFBMO rescued both
sox10/twist1 alterations of expression and NC
migration defects in a significant proportion of the
embryos, compared with PFKFB4MO injections
alone. sox10 and twist1 expression were restored or
increased a majority of the embryos. Scale bar:
500 μm. Phenotype scores are shown in Table S8. |
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Fig. 7. Glycolysis and PI3K-AKT signaling impact NC migration similarly
to PFKFB4 low-level depletion, and activating AKT signaling rescues
PFKFB4 downregulation. (A-H) When glycolysis (2DG) or PI3K-AKT
pathway (LY294002) were blocked during EMT and migration (st.18-24) both
treatments severely affected NC migration at st.24 (as revealed by twist1 and
sox10 expression). (I-L) At st.45, tadpoles treated during NC migration then
grown in control medium, exhibited general head morphology defects,
including eye defects and smaller branchial cartilages. (I,K) Sibling controls; (J)
2DG; (L) LY294002. A-H show side views; I-L dorsal views. (M-P) At tadpole
st.45, morphant sides were severely affected (M,P), whereas activation of Akt
signaling (N,P) did not affect overall craniofacial morphogenesis. Tadpoles coinjected
with PFKFB4MO and caAkt (O,P) were largely rescued, with 66% of
embryos with injected side symmetrical to contralateral side. (M) Red bar
indicates eye distance from the midline on the morphant side; blue bar
indicates control distance. Both bars are aligned below for comparison. (N,O)
On both sides, the same blue bar measures eye distance from the midline.
Arrowheads indicate the injected side. Scale bar: 500 μm. Phenotype scores
are shown in Tables S11, S12. |
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Fig. S6. Glycolysis and PI3K-Akt signaling control neural crest migration. Glycolysis blockade (AJ)
and PI3K-Akt blockade (K-T) during EMT and neural crest migration (stage 18 to stage 24) affects
neural crest migration as shown by the expression of Sox9, Sox10 and Twist. Embryos rinsed at stage
24 and grown until stage 45 display craniofacial and eye development defects, albeit the presence of
all individual cartilage elements. (A-C,F-H,K-M,P-R, U-W) Side views, anterior is to the left. (D,I,N,S,X)
dorsal views st 45. (E,J,O,T,Y) Ventral views of alcian blue stained visceral cartilages, st 45. Scale bar=
500μm. |