Torres MA et al. (1996), Activities of the Wnt-1 class of secreted signa...

XB-ART-18159

J Cell Biol 1996 Jun 01;1335:1123-37.

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Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development.

Torres MA , Yang-Snyder JA , Purcell SM , DeMarais AA , McGrew LL , Moon RT .

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When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.

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Species referenced: Xenopus laevis
Genes referenced: actl6a cdh2 gsc gsk3b mtor prl.1 spon1 tbx2 wnt1 wnt4 wnt5a wnt8a

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	Figure 1. Xwnt-5A antagonizes Wnt-1 class activity. (A) Control embryos possess single axes at stage 25. (B) Embryos injected with prolactin followed by Xwnt-8 RNA have duplicated axes. (C) Embryos injected with Xwnt-5A followed by Xwnt-8 RNA have single axes. (D) Uninjected stage 10 control embryos possess a single site of gsc expression. (E) Embryos injected with prolactin followed by Xwnt-8 RNA display multiple sites of gsc expression. (F) Embryos injected with Xwnt-5A followed by Xwnt-8 RNA have a single site of gsc expression. (Arrows) Axes (A, B, and C) and sites of gsc expression (D, E, and F).
	Figure 2. Detection of gsc transcripts by RT-PCR in stage 10 embryos ventralized by UV irradiation. (A) Xwnt-5A inhibits the rescue of gsc expression by Xwnt-8. (B) AN-cadherin blocks the rescue of gsc expression by Xwnt-8. ( C) Neither Xwnt-ll nor AN-cadherin block the rescue of gsc expression by BVgl. (A, B, and C; lane 1) Control embryos. (A, B, and C; lane 2) Uninjected embryos UV irradiated to eliminate endogenous gsc expression. (Lane 3) UV-irradiated embryos injected with prolactin followed by Xwnt-8 RNA (A and B) or prolactin followed by BVgl RNA (C). (Lane 4) UV-irradiated embryos injected with Xwnt-5A (A), AN-cadherin (B), or Xwnt-ll (C) RNA followed by Xwnt-8 (A and B) or BVgl (C) RNA. (Lane 5) UV-irradiated embryos injected with wild type N-cadherin (B) or AN-cadherin RNA (C) followed by Xwnt-8 (B) or BVgl RNA (C). All EFla lanes serve as controls for RT-PCR (see Materials and Methods).
	Figure 3. Xwnt-4 antagonizes the activity of Xwnt-8 after MBT. The ability of CSKA-Xwnt-8 to interfere with notochord formation (Christian and Moon, 1993) was scored in stage 25 embryos by anti-Tor 70 whole mount immunocytochemistry to detect the notochord and by F-spondin in situ hybridization to detect the floorplate. (A) Uninjected embryos possess a normal notochord and floorplate. (B) Transcription of Xwnt-8 from the cytoskeletal actin promoter vector after MBT leads to loss of notochord and floorplate staining. (C) Xwnt-4 interferes with the Xwnt-8 activity, restoring the formation of the notochord and the floorplate. (Arrowheads) F-spondin floorplate staining. (Straight arrows) Tor 70 notochord staining. (Curved arrows) anterior limit of the notochord.
	Figure 4. Overexpression of Xwnt-5A inhibits open face dorsal lip explant elongation and decreases Ca2+-dependent cell reaggregation. (A) Uninjected control dorsal lip explants elongate normally. (B) Dorsal lip explants overexpressing Xwnt-5A RNA do not elongate. (C) Xwnt-8 does not inhibit dorsal lip explant elongation. (D) Xwnt-5A blocks dorsal lip explant elongation when coexpressed with Xwnt-8 at a 1:1 ratio of injected RNAs. (E) Dissociated control dorsal lip explants reaggregate in a CaZ÷-dependent manner. In the absence of Ca 2÷, reaggregation is inhibited (inset). (F) Xwnt-5A inhibits Ca2÷-dependent cell reaggregation, which can be rescued by co-expressing Xwnt-5A with N-cadherin (inset). (G) Xwnt-8 has no appreciable effect on caa÷-dependent cell reaggregation. (H) Xwnt- 5A blocks Ca2÷-dependent cell reaggregation when coexpressed with Xwnt-8 at a 1:1 ratio of injected RNAs. dN-cadherin blocks Ca 2÷dependent cell reaggregation in a manner similar to Xwnt-5A (inset).
	Figure 5. The inhibition of elongation of the gastrula organizer explants by Xwnt-5A does not require Xwnt-5A to be expressed in all cells of the explant. Embryos were injected into the marginal zone of one dorsal cell at the four-cell stage with either fl-galactosidase RNA mixed with rhodamine dextran (A), or with Xwnt-5A RNA mixed with rhodamine dextran (B). Open face explants of the entire dorsal marginal zone were prepared at stage 10 and visualized by fluorescence microscopy after control embryos had developed to stage 13-15. Control explants (A) elongate extensively (6 of 8 explants), while explants expressing Xwnt-5A (B) display no convergence and extension movements (10 of 15 explants), or reduced elongation (3 of 15 explants). In both panels, pseudocolor imaging reveals that the injected dextran and RNAs were restricted to the yellow-red cells.
	Figure 6. AN-cadherin but not N-cadherin inhibits the induction of ectopic gsc expression by Xwnt-8 as assayed by in situ hybridization for gsc in stage 10 gastrula embryos. (A) Uninjected control embryos possess a single site of gsc expression. (B) Embryos injected with prolactin followed by Xwnt-8 RNA possess two sites of gsc expression. (C) Embryos injected with AN-cadherin followed by Xwnt-8 RNA possess a single site of gsc expression. (D) Embryos injected with N-cadherin followed by Xwnt-8 RNA possess two sites of gsc expression. (Arrows) Representative sites of gsc expression.
	Figure 7. Members of the Wnt-5A class and AN-cadherin do not block the induction of ectopic gsc expression by BVgl, as assayed by in situ hybridization for gsc in stage 10 gastrula embryos. (A) Uninjected control embryos possess a single site of gsc expression. (B) Embryos injected withprolactin followed by BVgl RNA possess two sites ofgsc expression. (C) Embryos injected with Xwnt-ll or AN-cadherin (D) RNA followed by BVgl RNA also possess multiple sites of gsc expression. (Arrows) Sites of gsc expression. Tortes et al. Wnt Antagonism 1133 Downloaded from jcb.rupress.org on May 8, 2015 Published June 1, 1996

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