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Difference in XTcf-3 dependency accounts for change in response to ß-catenin-mediated Wnt signalling in Xenopus blastula

Fiona S. Hamilton, Grant N. Wheeler^* and Stefan Hoppler

Division of Cell and Developmental Biology, School of Life Sciences, The Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, UK
^* Present address: School of Biological Sciences, University of East Anglia, Norwich NR4 7T3, UK

View larger version (28K): [in a new window]   Fig. 1. Alternative Wnt signal transduction pathways. (A) The canonical Wnt signal transduction pathway, which functions in many tissues. (B) The signal transduction cascade that Wnt7a uses to control axonal remodelling in the developing mouse brain. (C) Frizzled patterns cell polarity during Drosophila eye development through a JNK cascade. (D) The Wnt5a class of ligands exerts its effects via a Ca2+-dependent signal transduction pathway.

View larger version (56K): [in a new window]   Fig. 2. Lithium-mediated inhibition of GSK3ß in late blastula shows involvement in ventrolateral-promoting Wnt signalling. Xenopus embryos analysed for marker gene expression with whole-mount RNA in situ hybridisation carried out at stage 11. Control embryos (A-D) were treated with NaCl and experimental embryos (E-H) were treated with LiCl at stage 9.5. In all embryos dorsal is towards the top. Control embryos (A-D) show normal molecular marker expression. The molecular markers used were chordin (Chd) and Xnot, which are dorsal specific, and XmyoD and Xpo, which mark ventral and lateral. Chordin acts as a control as it has previously been shown that it is unaffected by late blastula Wnt signalling. The expression of chordin therefore remains unchanged in the dorsal side of the embryo by lithium treatment (compare E with A). The expression of the dorsal marker Xnot is greatly reduced after inhibition of GSK3ß by lithium (F) when compared with the control embryo (B). Expression of the ventral and lateral markers XmyoD (G) and Xpo (H) are expanded into the dorsal midline of the embryo after lithium treatment whereas in control embryos (C,D) no expression is seen in the dorsal midline.

View larger version (81K): [in a new window]   Fig. 3. Effects of lithium treatment carried out at a series of successive stages in Xenopus embryonic development. Stages indicate the stage at which lithium treatment was carried out (stage 6.5-10). Whole-mount RNA in situ hybridisation was carried out on all embryos at gastrulation stage 11. In all embryos dorsal is towards the top. The molecular markers used were chordin (Chd) and Xnot, which are dorsal specific, and XmyoD and Xpo, which mark ventral and lateral. Treatment with lithium at early blastula stages (A,G,M,S) results in dorsalisation of the embryo with expansion of Chd and Xnot into the ventral and lateral sides of the embryo and reduction or loss, respectively of the ventral and lateral markers Xpo and XmyoD. Treatment with lithium at early gastrula stage (F,L,R,X) results in ventralisation of the embryo with loss or reduction of the dorsal marker Xnot and expansion of the ventral and lateral markers XmyoD and Xpo into the dorsal midline. Chordin acts as a control during lithium treatment of late blastula stages as it has previously been shown that chordin is unaffected by late blastula Wnt signalling (see also Fig. 2E). Therefore the expression of chordin remains unchanged in the dorsal side of the embryo. Analysis of molecular markers shows the change of response to Wnt signalling from early dorsal-promoting to later ventrolateral-promoting is different for different Wnt responsive genes. The dorsal markers Chd and Xnot show ectopic expression in early to mid-blastula stages of development (A-C,G-I) with normal expression when lithium treated at stage 9.5 for Chd (E) and reduced expression at stage 10 for Xnot (L). Lithium treatment at stage 7.5 causes a reduction in the expression of XmyoD dorsolaterally (N). Treatment at stages 9-10 results in ventrolateral promoting response with ectopic expression in the dorsal side of the embryo (P-R). Treatment with lithium, even at the very early stages of development, results in the expansion of expression of the ventrolateral marker Xpo into the dorsal midline (T-X).

View larger version (53K): [in a new window]   Fig. 4. Overexpression of ß-catenin shows involvement in late blastula Wnt signalling. GFP, ß-catenin and marker gene expression in gastrulation stage Xenopus embryos. Using a double construct (hsp::ß-catenin::hsp::GFP) to generate transgenic embryos, both ß-catenin and GFP can be overexpressed to high levels (see Materials and Methods). This increase in levels can be seen in the western blot carried out with stage 10 embryos (A). GFP positive embryos (1-4) also have high levels of exogenous ß-catenin protein. In non-transgenic embryos (5-8) only low levels of GFP and exogenous ß-catenin protein is detectable, which is probably due to cytoplasmic expression from unincorporated plasmid DNA. (B-K) Detection of ubiquitous GFP expression under UV light (G) therefore allows transgenic embryos overexpressing ß-catenin (G-K) to be selected from control embryos (B-F). (G) GFP expressing embryo under UV-light (compare with non-GFP-expressing embryo in B). (H) Morphological phenotype of an embryo overexpressing ß-catenin (compare with control embryo in C and to Xwnt-8 overexpressing embryo in Fig. 7G). The embryos in D-F show normal molecular marker expression after whole-mount RNA in situ hybridisation is carried out at stage 11 and embryos I-K show molecular marker expression in ß-catenin overexpressing embryos. In all embryos dorsal is at the top. Expression of Xnot, a dorsal molecular marker (D) is repressed in ß-catenin overexpressing embryos (I), whereas the ventral and lateral mesodermal markers XmyoD and Xpo, are ectopically expressed in the dorsal midline of these ß-catenin overexpressing embryos (compare J and K with E and F). (L-Q) As a control, transgenic embryos using a single construct (hsp::GFP) to overexpress only GFP (O,Q) develop a normal morphology (P), as compared to non-transgenic siblings (L-N).

View larger version (62K): [in a new window]   Fig. 5. N-Tcf-3 inhibits dorsalising but not ventrolateral-promoting Wnt signalling when expressed in Xenopus embryos. (A) N-Tcf-3 construct used in this experiment. The morphology and molecular marker expression of uninjected control embryos are shown (B-F). N-Tcf-3 RNA was injected into the dorsal marginal zone (DMZ, G-K) or the ventral marginal zone (VMZ, L-P) at the two-cell stage and embryos were analysed by morphology at stage 31 (G,L) and molecular marker expression at stage 11 (H-K,M-P). Dorsally injected embryos have a strong ventralised phenotype with reduced dorsal structures (G). Analysis of molecular markers show that there is a repression of dorsal markers (chordin, H) and Xnot (I) (compared with C,D) and ectopic expression of ventral and lateral markers XmyoD (J) and Xpo (K) in the dorsal midline (compared with E,F). Ventrally injected embryos are unaffected by injection of N-Tcf-3 RNA and show the same morphology and molecular marker expression as the control embryos (compare L-P with B-F). (Q) Western blot detecting HA-tagged N-XTcf-3 protein at stages 6 and 10 of development after injection of the RNA at the four-cell stage into the dorsal marginal zone (DMZ) or the ventral marginal zone (VMZ). As a control, uninjected embryos show no N-XTcf-3 protein present; however, injected embryos show comparable levels of protein at stages 6 and 10 with both ventral and dorsal injection of N-XTcf-3 RNA.

View larger version (62K): [in a new window]   Fig. 6. N-XTcf-3 inhibits dorsalising and ventrolateral-promoting Wnt signalling when expressed in Xenopus embryos. (A) N-XTcf-3 construct used in this experiment. The morphology and molecular marker expression of uninjected control embryos are shown (B-F). N-XTcf-3 RNA was injected into the dorsal marginal zone (DMZ; G-K) or the ventral marginal zone (VMZ; L-P) at the two-cell stage and embryos were analysed by morphology at stage 31 (G and L) and molecular marker expression at stage 11 (H-K,M-P). Dorsally injected embryos have a ventralised phenotype with reduced dorsal mesodermal structures (G). Analysis of molecular markers show that there is a repression of the dorsal marker (chordin, H; compare with C) and a reduction of Xnot (I; compare with D). Ectopic expression of the ventral and lateral markers XmyoD (J) and Xpo (K) in the dorsal midline is also seen (compared with E,F). Ventrally injected embryos are affected by injection of N-XTcf-3 with normal dorsal mesodermal structures but reduced ventral structures. This can be seen both morphologically (L compared with B) and with molecular markers (M-P compared with C-F). The expression of the dorsal marker Chd is comparable with the control expression (M,C) and Xnot, a notochord specific dorsal marker shows a slight expansion (N) into the ventral cells, which express N-XTcf-3 (compare with normal expression pattern, D). Reduced ventral development is also shown by the reduction of ventrolateral gene expression of XmyoD and Xpo in the ventral cells which express N-XTcf-3 (O,P compared with E,F). (Q) Western blot detecting HA-tagged N-XTcf-3 protein at stages 6 and 10 of development after injection of the RNA at the four-cell stage into the DMZ or the VMZ. As a control, uninjected embryos show no N-XTcf-3 protein present, however, injected embryos show comparable levels of protein at stages 6 and 10 with both ventral and dorsal injection of N-XTcf-3 RNA.

View larger version (82K): [in a new window]   Fig. 7. Effects of N-XTcf-3 and N-XTcf-3 RNA on ectopic Wnt signalling. ß-catenin RNA-mediated axis duplication is rescued by N-XTcf-3 and N-XTcf-3, while only N-XTcf-3 can rescue the CSKA Xwnt-8 DNA-mediated effects on dorsal development. (A) Uninjected control embryo. (B) Misexpression of ß-catenin RNA ventrally mimics ectopic Wnt signalling in early blastula stage embryos resulting in a duplicated axis. However, when ß-catenin is misexpressed ventrally in combination with N-XTcf-3 and N-XTcf-3 rescue occurs (C,D). The rescue with N-XTcf-3 (D) does not result in a wild-type phenotype (A) as with N-XTcf-3 (C), as ventral N-XTcf-3 injection affects ventral mesodermal development (compare with Fig. 6L). (E) Injection of ß-catenin and N-XTcf-3 RNA into different but adjoining ventral blastomeres at the four-cell stage does not rescue ß-catenin-mediated axis duplication, confirming that N-XTcf-3 functions cell autonomously. (F) Uninjected control embryo. (G) Misexpression of CSKA Xwnt8 DNA dorsally causes ectopic ventrolateral-promoting Wnt signalling resulting in embryos with reduced dorsal midline structures and expanded lateral structures. (H) When CSKA Xwnt8 is expressed dorsally in combination with N-XTcf-3, no rescue occurs. (I) The CSKA Xwnt-8 DNA-mediated effects on development are rescued when CSKA Xwnt8 is expressed in combination with N-XTcf-3. Co-injection of CSKA Xwnt8 DNA and N-XTcf-3 RNA results in a phenotype (H) that is a combination of the phenotype caused by CSKA Xwnt8 DNA (G) and the one caused by N-XTcf-3 RNA (Fig. 5G). The rescue with N-XTcf-3 (I) does not result in a wild-type phenotype (F), as ventral N-XTcf-3 injection affects dorsal development (compare with Fig. 6G).