Rigo-Watermeier T et al. (2012), Functional conservation of Nematostella Wnts in...

XB-ART-46296

Biol Open 2012 Jan 15;11:43-51. doi: 10.1242/bio.2011021.

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Functional conservation of Nematostella Wnts in canonical and noncanonical Wnt-signaling.

Rigo-Watermeier T , Kraft B , Ritthaler M , Wallkamm V , Holstein T , Wedlich D .

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Cnidarians surprise by the completeness of Wnt gene subfamilies (11) expressed in an overlapping pattern along the anterior-posterior axis. While the functional conservation of canonical Wnt-signaling components in cnidarian gastrulation and organizer formation is evident, a role of Nematostella Wnts in noncanonical Wnt-signaling has not been shown so far. In Xenopus, noncanonical Wnt-5a/Ror2 and Wnt-11 (PCP) signaling are distinguishable by different morphant phenotypes. They differ in PAPC regulation, cell polarization, cell protrusion formation, and the so far not reported reorientation of the microtubules. Based on these readouts, we investigated the evolutionary conservation of Wnt-11 and Wnt-5a function in rescue experiments with Nematostella orthologs and Xenopus morphants. Our results revealed that NvWnt-5 and -11 exhibited distinct noncanonical Wnt activities by disturbing convergent extension movements. However, NvWnt-5 rescued XWnt-11 and NvWnt-11 specifically XWnt-5a depleted embryos. This unexpected 'inverse' activity suggests that specific structures in Wnt ligands are important for receptor complex recognition in Wnt-signaling. Although we can only speculate on the identity of the underlying recognition motifs, it is likely that these crucial structural features have already been established in the common ancestor of cnidarians and vertebrates and were conserved throughout metazoan evolution.

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Species referenced: Xenopus
Genes referenced: atf2 cdc42 gap43 mapk8 mapre1 pcdh8 pcdh8.2 pik3cb wnt1 wnt11b wnt2 wnt3a wnt5a wnt7b

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	Fig. 1. NvWnt-1 has an axis induction capacity in Xenopus.(A) Wildtype embryos stage 20. Double axis formation with (B) MWnt-1 (20 pg) and (C) NvWnt-1 (200 pg) but not with (D) NvWnt-2 (500 pg), (E) NvWnt-3 (500 pg), (F) NvWnt-5 (400 pg), (G) NvWnt-7b (500 pg) and (H) NvWnt-11 (200 pg). (I) Quantification of embryos with double axes. (J) Radioactive TNT assay shows the successful synthesis of all used Wnt constructs. # number, *p<0.005 to wildtype embryos. Error bars indicate standard error; scale bars 900 μm.
	Fig. 2. NvWnt-5 and NvWnt-11 act in noncanonical Wnt-signaling by disturbing convergent extension movements.(A; H) Wildtype DMZ explants with normal elongation and constriction. (B) XWnt-5a (100 pg) overexpression impairs constriction of DMZ explants, while (D) XWnt-11 (20 pg) overexpression completely inhibits their elongation. (C) NvWnt-5 expression has both an effect on constriction and elongation. (E) NvWnt-11 expressing DMZ explants elongate but the constriction fails. (I) NvWnt-1 (200 pg), (J) NvWnt-2 (400 pg) and (K) NvWnt-3 (400 pg) expressing DMZ explants show normal elongation and constriction. (F, G, L) Quantification of elongated DMZ explants (black) and of elongated DMZ explants showing normal constriction (gray). # number, p<0.005 to wildtype DMZ explants, *p<0.05 to wildtype DMZ explants. Error bars indicate standard error; scale bars 750 μm.
	Fig. 3. NvWnt-5 and NvWnt-11 show inverse concentration-dependent noncanonical activities in Xenopus.(A) Wildtype DMZ explants with normal elongation and constriction. XWnt-5a MO explants elongate without constriction (B) and cannot be rescued by NvWnt-5 (C), but by NvWnt-11 expression (D). (E) XWnt-11 MO explants with blocked elongation that is rescued by NvWnt-5 (G) but not by NvWnt-11 (F). (H, I) Quantification of elongated DMZ explants (black) and elongated DMZ explants showing normal constriction (gray). # number, *p<0.005 to wildtype DMZ explants, p<0.005 to MO DMZ explants. Error bars indicate standard error; scale bars 750 μm.
	Fig. 4. NvWnt-11 rescues XPAPC expression in XWnt-5a morphants.XPAPC ISH of single-side injected () Xenopus embryos (stage 12.5). (A) Depletion of XWnt-5a leads to reduction of XPAPC expression. (C) Coinjection of NvWnt-11 (200 pg), (B) but not of NvWnt-5 (400 pg) rescues XPAPC expression. (D) Quantification of embryos with normal XPAPC expression. # number, *p<0.005 to MO. Error bars indicate standard error; scale bars 460 μm.
	Fig. 5. NvWnt-5 rescues microtubule orientation in XWnt-11 morphants.(A–E) In vivo imaging of microtubule growth and cell polarization in DMZ explants expressing EB1-eGFP (500 pg) and GAP43-mCherry (200 pg). Scale bar 20 μm. (A′–E′) Microtubule growth directions of the indicated cells statistically rendered in rose diagrams. (A–E, A′–E′) 30 minutes after explant isolation (stage 10.25) microtubules grow from the cell centre in all directions. After 4 hours (stage 12.5), microtubule growth gets bipolar (A, A′) in control and (B, B′) in XWnt-5a depleted explants, while (C, C′) in absence of XWnt-11 microtubules keep on growing in all directions. (E, E′) NvWnt-5 (400 pg) but not (D, D′) NvWnt-11 (200 pg) expression in XWnt-11 morphants rescues microtubule orientation. (F) Quantification of cells with bipolar microtubule orientation. # number, *p<0.005 to wildtype, p<0.005 to MO. Error bars indicate standard error.
	Fig. 6. Alignment of vertebrate and Nematostella noncanonical and canonical sequences.The evolutionary conserved backbone of Wnt residues is indicated by black bars. Wnt-5 specific residues are drawn in red, Wnt-11 specific in blue. ‘Crosshomology’ is indicated by red bars for the Wnt-5 subfamily and blue bars for the Wnt-11 subfamily. Note that some of the Wnt-5 or Wnt-11 specific residues are also shared by the canonical NvWnt-1 (outgroup). (For further details see text).

References [+] :

Broun, Formation of the head organizer in hydra involves the canonical Wnt pathway. 2005, Pubmed

	Fig. 1. NvWnt-1 has an axis induction capacity in Xenopus.(A) Wildtype embryos stage 20. Double axis formation with (B) MWnt-1 (20 pg) and (C) NvWnt-1 (200 pg) but not with (D) NvWnt-2 (500 pg), (E) NvWnt-3 (500 pg), (F) NvWnt-5 (400 pg), (G) NvWnt-7b (500 pg) and (H) NvWnt-11 (200 pg). (I) Quantification of embryos with double axes. (J) Radioactive TNT assay shows the successful synthesis of all used Wnt constructs. # number, *p<0.005 to wildtype embryos. Error bars indicate standard error; scale bars 900 μm.
	Fig. 2. NvWnt-5 and NvWnt-11 act in noncanonical Wnt-signaling by disturbing convergent extension movements.(A; H) Wildtype DMZ explants with normal elongation and constriction. (B) XWnt-5a (100 pg) overexpression impairs constriction of DMZ explants, while (D) XWnt-11 (20 pg) overexpression completely inhibits their elongation. (C) NvWnt-5 expression has both an effect on constriction and elongation. (E) NvWnt-11 expressing DMZ explants elongate but the constriction fails. (I) NvWnt-1 (200 pg), (J) NvWnt-2 (400 pg) and (K) NvWnt-3 (400 pg) expressing DMZ explants show normal elongation and constriction. (F, G, L) Quantification of elongated DMZ explants (black) and of elongated DMZ explants showing normal constriction (gray). # number, p<0.005 to wildtype DMZ explants, *p<0.05 to wildtype DMZ explants. Error bars indicate standard error; scale bars 750 μm.
	Fig. 3. NvWnt-5 and NvWnt-11 show inverse concentration-dependent noncanonical activities in Xenopus.(A) Wildtype DMZ explants with normal elongation and constriction. XWnt-5a MO explants elongate without constriction (B) and cannot be rescued by NvWnt-5 (C), but by NvWnt-11 expression (D). (E) XWnt-11 MO explants with blocked elongation that is rescued by NvWnt-5 (G) but not by NvWnt-11 (F). (H, I) Quantification of elongated DMZ explants (black) and elongated DMZ explants showing normal constriction (gray). # number, *p<0.005 to wildtype DMZ explants, p<0.005 to MO DMZ explants. Error bars indicate standard error; scale bars 750 μm.
	Fig. 4. NvWnt-11 rescues XPAPC expression in XWnt-5a morphants.XPAPC ISH of single-side injected () Xenopus embryos (stage 12.5). (A) Depletion of XWnt-5a leads to reduction of XPAPC expression. (C) Coinjection of NvWnt-11 (200 pg), (B) but not of NvWnt-5 (400 pg) rescues XPAPC expression. (D) Quantification of embryos with normal XPAPC expression. # number, *p<0.005 to MO. Error bars indicate standard error; scale bars 460 μm.
	Fig. 5. NvWnt-5 rescues microtubule orientation in XWnt-11 morphants.(A–E) In vivo imaging of microtubule growth and cell polarization in DMZ explants expressing EB1-eGFP (500 pg) and GAP43-mCherry (200 pg). Scale bar 20 μm. (A′–E′) Microtubule growth directions of the indicated cells statistically rendered in rose diagrams. (A–E, A′–E′) 30 minutes after explant isolation (stage 10.25) microtubules grow from the cell centre in all directions. After 4 hours (stage 12.5), microtubule growth gets bipolar (A, A′) in control and (B, B′) in XWnt-5a depleted explants, while (C, C′) in absence of XWnt-11 microtubules keep on growing in all directions. (E, E′) NvWnt-5 (400 pg) but not (D, D′) NvWnt-11 (200 pg) expression in XWnt-11 morphants rescues microtubule orientation. (F) Quantification of cells with bipolar microtubule orientation. # number, *p<0.005 to wildtype, p<0.005 to MO. Error bars indicate standard error.
	Fig. 6. Alignment of vertebrate and Nematostella noncanonical and canonical sequences.The evolutionary conserved backbone of Wnt residues is indicated by black bars. Wnt-5 specific residues are drawn in red, Wnt-11 specific in blue. ‘Crosshomology’ is indicated by red bars for the Wnt-5 subfamily and blue bars for the Wnt-11 subfamily. Note that some of the Wnt-5 or Wnt-11 specific residues are also shared by the canonical NvWnt-1 (outgroup). (For further details see text).