Paulson AF et al. (1999), Misexpression of the catenin p120(ctn)1A pertur...

XB-ART-13417

Dev Biol 1999 Mar 15;2072:350-63. doi: 10.1006/dbio.1998.9158.

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Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification.

Paulson AF , Fang X , Ji H , Reynolds AB , McCrea PD .

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Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120(ctn) (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120(ctn) contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. However, little is known about the function of p120(ctn) within the cadherin complex. We examined the role of p120(ctn)1A in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120(ctn)1A, in contrast to beta-catenin, did not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor did p120(ctn) affect mesoderm induction. Rather, dorsal misexpression of p120(ctn) specifically perturbed gastrulation. Lineage tracing of cells expressing exogenous p120(ctn) indicated that cell movements were disrupted, while in vitro studies suggested that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120(ctn) are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120(ctn) has a role in cadherin function and that heightened expression of p120(ctn) interferes with appropriate cell-cell interactions necessary for morphogenesis.

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Species referenced: Xenopus
Genes referenced: cad cdh3 ctnnb1 ctnnd1 gsc h4c4 isyna1 jup mrc1 not sia1 tbxt

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	FIG. 1. Xenopus p120ctn is expressed in early development and associates with C-cadherin. (A) The expression of p120ctn in Xenopus embryos was detected by RT-PCR, using as template total RNA isolated from embryos at the indicated stages. The Xp120ctn-specific primers were generated from Xp120ctn sequence cloned from a stage 17 Xenopus cDNA library. Primers for histone H4 were used as a control for reaction conditions. (2RT indicates control reaction without reverse transcriptase.) (B) Xenopus embryo lysates were immunoprecipitated with a C-cadherin polyclonal antibody or a mixture of p120ctn monoclonal antibodies and blotted for p120ctn (positive control, mouse p120ctn1A-injected whole-cell lysate) or immunoprecipitated with a mixture of the p120ctn monoclonal antibodies and blotted for C-cadherin [positive control whole-cell extract of L cells expressing C-cadherin (Brieher and Gumbiner, 1994)]. (C-cad, C-cadherin, IP, immunoprecipitation.)
	FIG. 2. Expression of in vitro-transcribed p120ctn mRNA injected into Xenopus embryos and the association of p120ctn with Xenopus C-cadherin. (A) Western blot analysis of uninjected embryos and embryos injected with 0.2 ng of p120ctn mRNA at the four-cell stage, extracted at 1, 2, 6, 24, and 48 h following injection, and blotted with a polyclonal antibody against p120ctn. This p120ctn polyclonal antibody did not detect the endogenous p120ctn. (B) Embryos were injected with 0.2 ng of p120ctn or DR8-10 mRNA into the animal pole of both blastomeres at the two-cell stage and extracted at stage 12–13. Lanes 1, 3, 5, and 7 are p120ctn-injected embryos and lanes 2, 4, and 6 are DR8-10-injected embryos. Lanes 1 and 2 are whole embryo extracts blotted for p120. Lanes 3 and 4 are p120ctn IPs blotted for p120ctn. Lanes 5 and 6 are C-cadherin IPs blotted for p120ctn. Lane 6 is rabbit serum IgG control. (WC, whole-cell extract, C-cad, C-cadherin, IP, immunoprecipitation.) (C) Quantitation of p120ctn and DR8-10 bands in B. (D) Diagram of full-length p120ctn1 and the deletion construct DR8-10.
	FIG. 3. p120ctn does not induce expression of Siamois or goosecoid. (A) Detection of Siamois expression in dorsal and ventral halves of stage 10 embryos with RT-PCR. Embryos were injected vegetally with 0.2 ng of p120ctn or 2 ng of b-catenin mRNA into one ventral blastomere at the four-cell stage. The embryos were dissected into ventral and dorsal halves at stage 10 and analyzed for Siamois expression by RT-PCR. Control embryos are uninjected. (2RT is control reaction without reverse transcriptase.) (B) Embryos were injected with 0.2 ng of p120ctn (A) or 2 ng of b-catenin (B) mRNA into the marginal zone of one ventral blastomere at the four-cell stage. Whole-mount in situ hybridization was undertaken using digoxigenin-labeled cRNA probes to detect gsc expression in stage 10 embryos. The embryos are viewed from the vegetal side, with the dorsal lip at top. The solid arrows refer to endogenous gsc expression found in the organizer region, while the white arrow refers to exogenous ventral expression of gsc.
	FIG. 4. Effect of p120ctn misexpression on early Xenopus development. Embryos were injected with 0.2 ng of p120ctn or DR8-10 mRNA into one dorsal blastomere at the four-cell stage. (A–D) Embryos injected with p120ctn into the dorsal marginal zone and (E–H) embryos injected with DR8-10 into the dorsal marginal zone. A and E are gastrula-stage embryos, B and F are neurula-stage embryos, C and G are tailbud-stage embryos, and D and H are tadpole-stage embryos. Lineage tracing of p120-expressing cells suggests that p120ctn perturbs convergent extension movements in the whole embryo. Embryos (I) injected with 2 ng of b-galactosidase mRNA or (J) co-injected with 2 ng b-galactosidase mRNA and 0.2 ng p120 mRNA at the four-cell stage were allowed to develop until late gastrula (12–12.5), fixed in MEMFA, and processed with X-gal to stain for b-galactosidase.
	FIG. 5. Misexpression of p120ctn does not alter expression of mesodermal markers Xbra, Xnot1, or gsc. Embryos were injected with 0.2 ng of p120ctn (B, D, F, H, J, L) or DR8-10 (A, C, E, G, I, K) mRNA into one dorsal blastomere at the four-cell stage. Embryos were permitted to develop to the appropriate stage, fixed in MEMFA, and subjected to whole-mount in situ hybridization with antisense mRNA probes to Xbra (A–F), Xnot1 (G–J), and gsc (K, L). Stage 10 (K, L) and stage 10.5 embryos (A, B and G, H) are viewed vegetally with dorsal side up. Stage 12.5 embryos (C, D and I, J) are viewed dorsally with the anterior at the top and posterior at the bottom. Stage 22 embryos are viewed laterally (E) and dorsally (F) with the anterior side on the left and posterior on the right. The closed arrows refer to the tailbud, and the open arrows refer to the head.
	FIG. 6. p120ctn decreases adhesion between reaggregated animal cap blastomeres. Embryos were injected with 0.2 ng of p120ctn or DR8-10 mRNA into the animal hemisphere of each blastomere at the two-cell stage. Animal pole explants were isolated from stage 8 embryos and dissociated in 13 CMF-MMR. Calcium was added to the medium to 2 mM, and pictures were taken of random samples at 60 min of aggregation. The number of cells per aggregate was quantified for each condition.