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Fig. 4. A dominant negative Fzd7 induces cardia bifida phenotype.
(A-Ai and F, Fi) Lateral and ventral views of wild type embryos at stage 29 showing
normal tnnic (A, Ai) and nkx2-5 (F-Fi) expression in the heart. (B, Bi and G, Gi) Lateral
and ventral views of embryos injected in the dorsal blastomeres at 4 cell stage with
dominant negative fzd7 (fzd7 CRD). The cardia bifida phenotype is shown by tnnic (B,
Bi) and nkx2-5 (G-Gi) expression. These embryos were fixed at the same stage as the
control embryos in (A and F). (C) Graph showing fzd7 CRD cardia bifida phenotype
percentages indicated by tnnic expression. (D, Di). Lateral and ventral views of
embryos injected in the dorsal blastomeres (DB) at 4 cell stage with full length of fzd7
showing normal heart tube. Note that embryos in (D) and (G) are showing severe
convergent extension defects but cardia bifida phenotype is only induced by fzd7 CRD.
(E, Ei). Lateral and ventral views of injected embryo in the DB at 4 cell stage with fzd3
dominant negative form (fzd3 CRD) showing normal heart looping (at stage 38)
indicating that fzd7 CRD cardia bifida phenotype is specific to Fzd7. Magnification 20x. (H and I). Lateral (H) and ventral (I) views of embryos injected in the DB at 4 cell stage
with full length of fzd7 showing normal heart tube indicated by nkx2-5 expression. |
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Fig. 5. Activation of non-canonical wnt signalling rescues fzd7 CRD induced cardia
bifida.
(A, Ai) Wild type control embryos showing normal tnnic expression in the heart. (B, Bi)
fzd7 full length overexpression (500pg) injected into the dorsal blastomeres (DB) at the
4 cell stage show normal heart expression of tnnic despite suffering a severe extension
movement defect. (C). Embryos injected with 500pg fzd7 CRD show cardia bifida
phenotype, note that embryos have normal to mild convergent extension defects. (D).
Rescue of the fzd7 CRD (250pg) cardia bifida phenotype with 250pg full length fzd7,
embryos show normal morphology as well as tnnic expression. (F). Graph of fzd7 CRD
cardia bifida phenotype rescue with fzd7 Full length. (E, Ei). Rescue of fzd7 CRD
(500pg) cardia bifida phenotype with 1.25ng dishevelled1-Delta-N (Dvl1N) indicating
that fzd7 is required for the non-canonical signalling in the heart. (G). Graph of fzd7
CRD cardia bifida phenotype rescue with dvl1N, (Gi) is the key for the cardia bifida
phenotype scoring in (G). Magnification 20x. |
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Fig. 2. fzd7 expression coincides with expression of the early heart markers nkx2-5,
tnnic and gata6. Lateral view of Xenopus laevis embryos at stage 31 showing fzd7
expression detected in red (A-Aii) and co-localised by double in situ hybridisation with
other heart markers in dark blue including nkx2-5 (B-Bii), tnnic (C-Cii) and gata6 (DDii).
(Ai, Bi, Ci, Di). Magnified lateral view of the same embryos in (A, B, C and D)
respectively. (Aii, Bii, Cii, Dii). Cross section through the heart region of the embryos
in (A, B, C, D) respectively. fzd7 is expressed in the myocardium and pericardium (Aii)
and in other structures including neural crest, eye, pronephric duct and tail bud. fzd7
expression shows a high degree of overlapping with the heart markers in the
myocardium but not in the pericardium (Bii, Cii, Dii). h: heart, c: cement gland, e: eye,
nc: neural crest, pnd: pronephric duct, tb: tail bud, mc: myodcardium, lpm: lateral plate of mesoderm. Magnification: 20x in (A, B, C, D), 30x in (Ai, Bi, Ci, Di), 200x in (Aii, Bii,
Cii, Dii). |
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Fig. 3. fzd7 is required for Xenopus heart development
(A, Ai and D, Di) Lateral and ventral views of embryos injected in the dorsal
blastomeres (DB) at 4 cell stage with control morpholino (CMO) showing normal nkx2-
5 (A-Ai) and tnnic (D, Di) expression. (Aii, Dii). Cross sections in the heart region of the
embryos in (A) and (D) respectively showing normal nkx2-5 and tnnic expression in
the myocardium. (B, Bi and E, Ei). Lateral and ventral views of embryos injected in the
DB at 4 cell stage with fzd7 MO showing loss of nkx2-5 (B-Bi) and tnnic (E , Ei)
expression. (Bii, Eii). Cross sections in the heart region of the embryos in (B) and (E)
respectively showing loss of the heart. (C) Graph showing that fzd7 MO phenotype
leads to reduction/loss of nkx2-5 expression in a dose-dependent manner. (F, Fi). Fzd7
MO phenotype can be rescued by fzd7 SDM full length, (Fi) is the key for the phenotype
scoring. Red staining in B, Bi and Bii is due to lac-Z lineage tracing using Red-Gal. |
