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Fig. 1. MOs against Tcf/Lef factors produce different and specific phenotypes. (A) XlTcf1, XlLef1 or XlTcf3 MOs specifically inhibit protein synthesis from its corresponding DNA construct in in-vitro transcription and translation assays, while not affecting significantly translation of other Tcf constructs or a control luciferase DNA construct. Injection of 60 ng control MO into LMZs of both blastomeres at the 2-cell stage, or the marginal zones of two dorsal blastomeres (DMZ) or two ventral blastomeres (VMZ) does not affect the phenotype significantly (B,C,D). Injection of 20 ng Tcf1 MO into the LMZ causes a severe developmental arrest phenotype in the majority of embryos, and in the rest (E) or when only 15 ng Tcf1 MO is injected it interferes with both dorsal and ventral development (S). Injection of 20 or 15 ng of Tcf1 MO into the DMZ causes a severe dorsal bend at approximately the position of hindbrain (F,S), and into the VMZ causes an anteriorized phenotype (G,S). Injection of 60 ng Lef1 MO into the LMZ interferes slightly with both dorsal and ventral development (H,S), into the DMZ causes a slight dorsal bend (I,S), and into the VMZ causes a mild defect in ventral tissue development and a significant defect of tail development (J,S). Injection of 60 ng Tcf3 MO into the LMZ interferes with both dorsal and ventral development (K,S), but to a lesser degree than 20 or 15 ng of Tcf1 MO does. Injection of 60 ng Tcf3 MO into the DMZ causes a complete headless phenotype (L,S), and into the VMZ causes significant ventral development defects in both anterior and posterior regions (M,S). (N-R) Vegetal view of chordin (Xchd) expression in stage 10.5 embryos, dorsal towards the top, injections into the right side. The expression pattern and level of Xchd are not significantly affected by injection of 60 ng control MO (N), 20 ng Tcf1 MO (O), 60 ng Lef1 MO (P), 60 ng Tcf3 MO (Q) or 60 ng Tcf4 MO (R). (S) Numerical summary illustrating penetrance of morphological phenotypes caused by Tcf/Lef MOs, indicating dorsoanterior defects (i.e. clearly identifiable defects in the dorsal axis and the head and neck region), ventrolateral defects and combinations of these defects (but note that the detailed nature and severity of defects vary between Tcf1 MO, Lef1 MO and Tcf3 MO experiments, as illustrated in panels B-M).
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Fig. 2. Tcf1 and Tcf3 are non-redundantly required for mesoderm induction. Vegetal view of brachyury (Xbra) expression in stage 10.5 embryos, dorsal towards the top, injections into the right side. Xbra expression is not affected by injection of 60 ng control MO (A), Lef1 MO (B) or Tcf4 MO (C), but is completely blocked by injection of 20 ng Tcf1 MO (D) and significantly downregulated by injection of 60 ng Tcf3 MO (G) in the injected tissue. Blocking of Xbra expression by XlTcf1 MO is rescued by co-injection of 0.3 ng XtlTcf1 mRNA (E), but is not rescued by injection of 0.5 ng HA-Tcf3 mRNA (F). Downregulation of Xbra expression by XlTcf3 MO is rescued by co-injection of 0.5 ng HA-XlTcf3 mRNA (I), but is not rescued by co-injection of 0.3 ng XtlTcf1 mRNA (H). (J-N) Numerical summary illustrating penetrance of effects of Tcf/Lef MOs and Tcf/Lef mRNA on Xbra expression in stage 10.5 embryos, indicating absent, reduced and normal or almost normal Xbra expression detected at the site of injection. Control MO, Lef1 MO and Tcf4 MO do not significantly affect Xbra expression (J). mRNA injection-mediated overexpression of relatively low amounts of Tcf1 or Tcf3 hardly affects Xbra expression, while higher amounts of either Tcf1 or Tcf3 inhibit Xbra expression more dramatically and in a dose-dependent way (K). Xbra expression is strongly inhibited by Tcf1 MO in the vast majority of injected embryos, but is rescued by co-injection of relatively low amounts of Tcf1 mRNA; however, increasing amounts of Tcf1 mRNA result in an apparently less successful rescue, and Tcf3 mRNA fails to rescue Xbra expression altogether (L). Xbra expression is reduced by Tcf3 MO in the vast majority of injected embryos, but is dramatically rescued by relatively low amounts of Tcf3 mRNA and hardly rescued by relatively low amounts of Tcf1 mRNA; however, increasing amounts of Tcf3 or Tcf1 mRNA result in an apparently less successful rescue (M). At relatively low doses, Tcf1 MO or Tcf3 MO also inhibit Xbra expression to a lesser degree; however, Xbra expression is not further inhibited by co-injection of these two MOs, but is rescued to some extent (N).
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Fig. 3. Tcf1 and Lef1 are required for ventrolateral mesoderm development. Vegetal view of Xpo (A-O) and XmyoD (A′-O′) expression in stage 10.5 embryos, dorsal towards the top, injections into the right side. Xpo and XmyoD expression are not affected by injection of 60 ng control MO (B,B′) or Tcf4 MO (C,C′), but are both significantly downregulated by injection of 60 ng Tcf3 MO (D,D′), 20 ng Tcf1 MO (F,F′) or 60 ng Lef1 MO (K,K′). Co-injection of 0.1 ng Xbra mRNA rescues the downregulation of both Xpo and XmyoD expression by Tcf3 MO (E,E′), but does not rescue their downregulation caused by Tcf1 MO (G,G′) or Lef1 MO (L,L′), indicating that while Tcf3 is required for ventrolateral mesoderm development only because it is required for normal mesoderm induction, which is a prerequisite for ventrolateral mesoderm development; Tcf1 and Lef1 are required for ventrolateral mesoderm development independent of any requirement in mesoderm induction. Downregulation of Xpo and XmyoD expression by XlTcf1 MO or XlLef1 MO are both significantly rescued by co-injection of either 0.3 ng XtlTcf1 mRNA (H,H′; M,M′) or HA-XlLef1 mRNA (I,I′; N,N′), but cannot be rescued by co-injection of 0.3 ng HA-XlTcf3 mRNA (J,J′; O,O′). (P,P′) Numerical summary illustrating penetrance of effects of Tcf/Lef MOs (as labelled) or the co-injection of these MOs with Tcf/Lef mRNAs or Xbra mRNA (as labelled), indicating reduced or absent Xpo expression and XmyoD expression, respectively.
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Fig. 4. Tcf3 is predominantly required as a transcription repressor in mesoderm induction, while Tcf1 and Lef1 are predominantly required as transcription activators in mesoderm development. (A) Schematic representation of mutated XlTcf3 constructs used as molecular tools in this study. The β-catenin binding domain, the DNA-binding HMG box, and the Grg- and CtBP-binding domains are as indicated. Tcf3δN represents a constitutive repressor form, Tcf3δgrgδC a β-catenin-dependent active form and TVGR a constitutive activator form of XlTcf3. (B-I) Vegetal view of brachyury (Xbra) expression at stage 10.5 embryos, dorsal towards the top, injections into the right side. Blocking of Xbra expression by Tcf1 MO (B) is hardly rescued by co-injection of 0.3 ng Tcf3δN mRNA (C), but is significantly rescued by co-injection of 0.3 ng Tcf3δgrgδC mRNA (D) or 1 pg TVGR mRNA (induced at stage 9) (E). Downregulation of Xbra expression by XlTcf3 MO (F) is rescued by co-injection 0.3 ng HA-XTcf3δN mRNA (G) but is hardly rescued by co-injection of 0.3 ng HA-XTcf3δgrgδC mRNA (H) or 1 pg TVGR mRNA (induced at stage 9) (I). (J-Q) Vegetal view of Xenopus posterior (Xpo) expression at stage 10.5 embryos, dorsal towards the top, injections into the right side. Downregulation of Xpo expression by Lef1 MO (J) is not rescued by co-injection of 0.3 ng XTcf3δN mRNA (K), but is rescued by co-injection of 0.3 ng XTcf3δgrgδC mRNA (L) or 1 pg TVGR mRNA (M, induced at stage 9). Similarly, downregulation of Xpo expression by Tcf1 MO (N) is not rescued by co-injection of 0.3 ng XTcf3δN mRNA (O), but is rescued by co-injection of 0.3 ng XTcf3δgrgδC mRNA (P) or 1 pg TVGR mRNA (Q, induced at stage 9). (R) Numerical summary illustrating the penetrance of rescue effects of Tcf3 mutants mRNAs with distinct activity for downregulation of Xbra expression caused by Tcf1/Tcf3 MOs, indicating absent and reduced Xbra expression at the site of injection. (S) Numerical summary illustrating the penetrance of rescue effects of mRNAs encoding different Tcf3 mutated constructs in Tcf1 MO- or Lef1 MO-injected embryos, indicating absent or reduced Xpo expression at the site of injection.
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Fig. 5. The central motifs are crucial for the repressive role of Tcf3 in mesoderm development. (A) Schematic representation of the wild-type and mutated constructs used in this study. The β-catenin binding domain, the HMG box, LVPQ and SXXSS motifs are as indicated. (B-M) Vegetal view of brachyury (Xbra) expression in stage 10.5 embryos, dorsal towards the top, injections into the right side. Blocking of Xbra expression by injection of 20 ng Tcf1 MO (B) is hardly rescued by co-injection of 0.3 ng XTcf3δC (C) or 0.3 ng Tcf4A mRNA (D), but significantly rescued by 0.3 ng Tcf4C mRNA (E), 0.15 ng XTcf3δL-SA (F) or 0.3 ng Lef1 mRNA (G). By contrast, downregulation of Xbra expression by injection of 60 ng Tcf3 MO (H) is rescued by co-injection of 0.3 ng XTcf3δC (I) or 0.3 ng Tcf4A mRNA (J), but is not rescued by 0.3 ng Tcf4C mRNA (K), 0.1 ng XTcf3δL-SA (L) or 0.3 ng Lef1 mRNA (M). (N-S) Vegetal view of Xpo expression in stage 10.5 embryos, dorsal towards the top, injections into the right side. Downregulation of Xpo expression by injection of XlLef1 MO (N) is not rescued by co-injection of 0.3 ng XTcf3δC (O) or 0.3 ng Tcf4A mRNA (P), but is rescued by 0.3 ng Tcf4C mRNA (Q), 0.15 ng XTcf3δL-SA (R) or 0.3 ng HA-Lef1 mRNA (S). (T) Numerical summary illustrating the penetrance of rescue effects of mRNAs of Lef1, Tcf4 isoforms or Tcf3 mutated constructs in Tcf1 MO- or Tcf3 MO-injected embryos, indicating reduced or absent Xbra expression. (U) Numerical summary illustrating the penetrance of rescue effects of mRNAs of Lef1, Tcf4 isoforms or Tcf3 mutated constructs in Lef1 MO-injected embryos, indicating reduced or absent Xpo expression.
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