Abu-Elmagd M et al. (2017), Frizzled-7 is required for Xenopus heart develo...

XB-ART-54239

Biol Open 2017 Dec 15;612:1861-1868. doi: 10.1242/bio.026963.

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Frizzled-7 is required for Xenopus heart development.

Abu-Elmagd M , Mulvaney J , Wheeler GN .

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Wnt signalling regulates cardiogenesis during specification of heart tissue and the morphogenetic movements necessary to form the linear heart. Wnt11-mediated non-canonical signalling promotes early cardiac development whilst Wnt11-R, which is expressed later, also signals through the non-canonical pathway to promote heart development. It is unclear which Frizzled proteins mediate these interactions. Frizzled-7 (fzd7) is expressed during gastrulation in the mesodermal cells fated to become heart, and then in the primary heart field. This expression is complementary to the expression of wnt11 and wnt11-R We further show co-localisation of fzd7 with other early- and late-heart-specific markers using double in situ hybridisation. We have used loss of function analysis to determine the role of fzd7 during heart development. Morpholino antisense oligonucleotide-mediated knockdown of Fzd7 results in effects on heart development, similar to that caused by Wnt11 loss of function. Surprisingly, overexpression of dominant-negative Fzd7 cysteine rich domain (Fzd7 CRD) results in a cardia bifida phenotype, similar to the loss of wnt11-R phenotype. Overexpression of Fzd7 and activation of non-canonical wnt signalling can rescue the effect of Fzd7 CRD. We propose that Fzd7 has an important role during Xenopus heart development.

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Species referenced: Xenopus laevis
Genes referenced: fzd7 gata6 nkx2-5 tnni3 wnt11 wnt11b
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	Fig. 1. Endogenous expression of fzd7 in Xenopus heart and relative to wnt11 and wnt11-R expression. (A, B): stage 10.5 (mid-gastrula) fzd7 and wnt11 expression detected at the dorsal side of the embryo and appear complementary in the presumptive heart region. (C-Cii and D-Di): fzd7 and wnt11-R expression at stage 25. fzd7 is seen in the heart field and wnt11-R in the anterior endoderm. fzd7 and wnt11- R expression are complementary in the heart region (Cii, Dii). (E, Ei and F, Fi): stage 29 embryos with fzd7 and wnt11-R expression in the heart field. hf: heart field, ae: anterior endoderm. Magnification 20x.
	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).
	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.
	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.
	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 (Dvl1N) indicating that fzd7 is required for the non-canonical signalling in the heart. (G). Graph of fzd7 CRD cardia bifida phenotype rescue with dvl1N, (Gi) is the key for the cardia bifida phenotype scoring in (G). Magnification 20x.
	Fig. S1. Cardiac development is independent on the convergent extension movement defects caused by overexpression of fzd7. (A, B). fzd7 full length (250pg) injected into the dorsal blastomeres at 8 cell stage and incubated till stage-32 showing detectable tnnic (A) and nkx2-5 (B) expression in both normal embryos and those with convergent extension movement defects (arrow heads in A and B).

References [+] :

Abu-Elmagd, Frizzled7 mediates canonical Wnt signaling in neural crest induction. 2006, Pubmed, Xenbase