Schneider PN et al. (2012), Differential role of Axin RGS domain function i...

XB-ART-45835

PLoS One 2012 Jan 01;79:e44096. doi: 10.1371/journal.pone.0044096.

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Differential role of Axin RGS domain function in Wnt signaling during anteroposterior patterning and maternal axis formation.

Schneider PN , Slusarski DC , Houston DW .

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Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover.

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Species referenced: Xenopus laevis
Genes referenced: axin1 chrd ctnnb1 dlx2 myc nodal3.1 notch1 pax6 rgs4 sia1 szl wnt8a

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	Figure 2. Axin1Q162A does not interact with Gna at the plasma membrane.(A–D) Immunostaining against Myc showing localization of Axin1-Myc and Axin1(Q162A)-myc in zebrafish embryos, without (A, B) or with coexpression of Gnao (C, D). The arrowhead in C indicates membrane localization of Axin1 upon Gnao expression. Embryos were counterstained with TOPRO3 to show nuclei (purple).
	Figure 3. Overexpression of Axin1Q162A blocks axis formation and Wnt signaling in Xenopus embryos.(A–C) Representative phenotypes of stage 40 control uninjected embryos (A), axin1-injected embryos (500 pg), (B) and axin1(Q162A)-injected embryos (500 pg) (C). (D) Representative realtime RT-PCR of sia1, nr3.1 and chd expression in control whole embryos (un WE), control animal caps (un cap) and caps injected with wnt8 (1 pg) and Axin constructs (500 pg). (E) Realtime RT-PCR of nr3.1 expression in animal caps injected with wnt8 and different doses of Axin constructs (100 pg, 300 pg, 500 pg; indicated below sets of bars).
	Figure 4. Both axin1 and axin1(Q162A) rescue hyperdorsalization in maternal axin1-depleted embryos.(A–D) Representative phenotypes of control and axin1-depleted Xenopus embryos obtained following host transfer. Chart showing distribution of phenotypes is inset in each panel, percentages are indicated; black = normal, grey = dorsalized. (A) control uninjected stage 37 embryo, (n = 40) (B) axin1-depleted embryo (4 ng oligo; n = 44), (C) axin1-depleted embryo injected with 60 pg axin1 mRNA (n = 32), (D) axin1-depleted embryo injected with 60 pg axin1(Q162A) mRNA (n = 21). (E) Representative realtime RT-PCR of sia1, nr3.1 and chd expression in control whole embryos (un), axin1-depleted embryos (axin-) and axin- embryos injected with axin constructs.
	Figure 5. axin1 but not axin1(Q162A) rescues anterior defects in Axin1-depleted embryos.(A–D′) Representative phenotypes of control and Axin1-depleted zebrafish embryos. (A) control uninjected embryo, (B) Axin1-depleted embryo (4 ng axin1-MO), (C) MO injected embryo coinjected with 25 pg axin1 mRNA, (D) MO injected embryo coinjected with 25 pg axin1(Q162A) mRNA. (E) Summary table of morphological defects.
	Figure 6. axin1 but not axin1(Q162A) rescues telencephalic defects in Axin-depleted embryos.(A–D) Representative examples of pax6 expression in control and Axin1-depleted Xenopus embryos. Arrows indicate the presumptive telencephalon region of the eye field, visible as a narrowed medial notch in the area of pax6 expression. (A) Control uninjected stage 20 embryo, (B) Axin1-depleted embryo (20 ng axin1-MO), (C) MO injected embryo coinjected with 60 pg axin1 mRNA, (D) MO injected embryo coinjected with 60 pg axin1(Q162A) mRNA. (E) Summary table of pax6 expression data.
	Figure 7. axin1 but not axin1(Q162A) rescues telencephalic defects in Axin1-depleted and in mbl−/− embryos.(A–H) Representative examples of dlx2 expression in control and Axin1-depleted zebrafish embryos (A–D) and in wildtype and mbl−/− embryos (E–H). (A) control uninjected embryo at 24 hpf, (B) axin1-MO-injected, (C) axin1-MO+xx axin1, (D) axin1-MO+axin1(Q162A), (E) wildtype embryo at 24 hpf, (F) mbl−/−, (G) mbl−/−;+axin1 (H) mbl−/−;+axin1(Q162A). (I) Summary of dlx2 expression data. Arrows indicate areas of reduced or absent dlx2 in the telencephalon.
	Figure 8. CAAX-tagged Axin1 is sufficient to inhibit axis formation and to promote Axin1 protein turnover.(A–D) Localization of CAAX-tagged and untagged Axin1 proteins in Xenopus animal caps. (E–G) Uninjected control embryos (E) and embryos expressing Axin1-CAAX (F) and Axin1(Q162A)-CAAX (G) at the tailbud stage. (H) Immunoblots of stage 9 embryo lysates injected with FLAG-axin1 (100 pg, 300 pg) and FLAG-axin1-caax (100 pg, 300 pg). The top panel shows anti-FLAG blotting, the bottom panel shows a non-specific band (n.s.) detected by the anti-FLAG antibody to confirm equivalent loading. (I) Representative realtime RT-PCR of sia1, nr3.1 and sizzled (szl) expression in control embryos (un) and in embryos injected with FLAG-axin1 (100 pg, 300 pg) or FLAG-axin1-caax (100 pg, 300 pg).
	Figure 1. Structure-function analysis of the Axin1 RGS domain.(A) Alignment of RGS domains from human (hAXIN1), mouse (mAxin1), Xenopus (xAxin1) and zebrafish Axin1 (zAxin1) with human RGS4. Blue bars = APC binding interface; orange bars = Gna binding interface. * = residues required for GAP activity in RGS4. Arrow = residue required for RGS4 GAP activity, mutated in this study. (B) Immunoblots showing equivalent protein expression in 24 hpf zebrafish embryos injected with axin1-myc and axin1(Q162A)-myc. (C) Immunoprecipitation of FLAG-tagged Axin1 constructs with HA-tagged Gnao, showing reduced binding of FLAG-Axin1(Q162A) to overexpressed Gnao. (D) FLAG-Axin1 and FLAG-Axin1(Q162A) immunoprecipitate endogenous APC equivalently. (E) FLAG-Axin1 and FLAG-Axin1(Q162A) immunoprecipitate HA-tagged APC-SAMP3 equivalently.

References [+] :

Brannon, A beta-catenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus. 1997, Pubmed, Xenbase