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In Xenopus gastrula stage embryos, four isoforms of Tcf1 (B, C, D and E) are present with high amino acid sequence conservation compared to fish, mice and human. We studied possible functional differences between these Tcf1 isoforms during early Xenopus development. After overexpression of single Tcf1 isoforms, two distinct phenotypes were observed. Overexpression of the B or D isoforms of Tcf1, which both lack a C-clamp, enhances early canonical Wnt signaling and induces ectopic dorsal mesoderm at the expense of ventrolateral mesoderm prior to gastrulation, causing severe antero-dorzalization of embryos. Overexpression of the E-isoform, which contains a complete C-clamp, does not induce ectopic dorsal mesoderm, but rather leads to severe caudal truncation. Overexpression of the C-isoform, which contains a partial C-clamp, induces a similar phenotype. Mutation of a single amino acid in the C-clamp, known to produce a hypomorphic mutant in D. melanogaster, led to a gain of function in inducing ectopic organizertissue, as observed after overexpression of the B or D isoforms of Tcf1. Depletion of the C-clamp exon from the zygotic mRNA pool, by injection of a morpholino oligo that targets the splice acceptor site of the exon containing the C-clamp, caused a severe shortening of the AP-axis. Furthermore, embryos showed poor development of the CNS, paraxial mesoderm and primary blood vessels. In situ hybridization analysis showed that Lef1 expression was downregulated at the mid-hindbrain boundary, in the otic vesicles and the branchial arches. The results indicate that in post-gastrula stage Xenopus embryos, the E-tail of Tcf1 is required for expression of Lef1 and for blood vessel formation.
Fig. 1. Overexpression of single XTcf1 isoforms induces two distinct phenotypes dependent on the presence or absence of a (partial) C-clamp. (A) Schematic drawing of the genomic organisation of XTcf1 showing exons X to XII generating different XTcf-1 splice variants. The two conserved motifs CR1 and CR2 located on exons XI and XII comprise the C-clamp (Atcha et al., 2003). NLS: nuclear localization signal. HMG: high-mobility group DNA binding domain. (B) amino acid sequences of the different splice variants of XTcf1 downstream of exon X. (C-G) X. tropicalis embryos after lateral injection of the four different Tcf1 isoforms at the 2-cell stage. NIC, non-injected control; white arrow head, cement gland; yellow arrow head, heart.
Fig. 2. Effect of XTcf1 isoform overexpression on the expression of organizer genes and ventrolateral genes during gastrulation after injection of single isoforms of XTcf1. Whole mount in situ hybridisation
of gastrula stage embryos after injection of different XTcf1 isoforms. Overexpression of isoforms B and D induces ectopic dorsal mesoderm at the expense of ventrolateral tissue, as indicated by the expansion of
Chordin and Goosecoid and the absence of Wnt8 and Cdx1.
Fig. 3. Mutation of the C-clamp of XTcf-1E leads to a loss of isoform specificity. (A) XTcf1E-CRVRF holds a mutation in the C-clamp changing alanine into valine in the CR1 motif. XTcf1-deltaC has a premature stop codon directly 3’ of the NLS. (B) Injection of 400 pg mRNA XTcf1- CRVRF induced ectopic dorsal mesoderm as indicated by the expansion of Chordin expression at the gastrula stage as does XTcf1-deltaC. (C,D) XTcf1E-CRVRF induces double axis formation in 29% of the injected embryos.
Fig. 3. Mutation of the C-clamp of XTcf-1E leads to a loss of isoform specificity [continued] (C,D) XTcf1E-CRVRF induces double axis formation in 29% of the injected embryos
Fig. 4. Depletion of XTcf1 C and E isoforms from the zygotic mRNA
pool by inhibiting splicing using a morpholino oligo (MO). (A) position
of the splice morpholino targeting the exon that carries the CR1 motif.
RT-PCR analysis of the mRNA of non-injected control (NIC) embryos and
embryos injected with 20 ng splice MO using PCR primers amplifying exon X and XI. (B) Control stage 39 embryo. (B’) Phenotype of an embryo after injection of 20 ng splice MO. (C) Histological sections of stage 39 NICembryo showing clear vessels. (C’) CR1 MO injected embryo showing lack of major blood vessels (cf Roël et al., 2009). Arrow indicates expected level of the posterior cardinal vein (pcv). (D) In situ hybridization of XLef1 in NIC stage 32 embryo and XTcf1- CR1 splice MO (10 ng) injected embryo (D’).XLef1 expression is downregulated in the branchial arches and the midbrain.
