Xenopus frizzled-7 morphant displays defects in dorsoventral patterning and convergent extension movements during gastrulation.

Wnt signaling has been implicated in the process of gastrulation in Xenopus laevis. Overexpression of Wnt molecules, such as a putative dominant-negative form of Wnt8 (Hoppler et al., 1996), Wnt11 (Tada and Smith, 2000), as well as wild type Wnt5A and Wnt11 proteins (Du et al., 1995; Gradl et al., 1999; Moon et al., 1993) interferes with convergent extension movements during gastrulation. Overexpression of some other Wnt signaling pathway members such as full-length and truncated forms of Xenopus frizzled-7 (Djiane et al., 2000; Sumanas et al., 2000), a truncated form of Xenopus disheveled (Sokol, 1996) also causes similar defects. However, evidence for the requirement of a specific Wnt pathway protein in the gastrulation process is still missing. Here we investigated the function of Xenopus frizzled-7 (Xfz7) utilizing the morpholino phosphorodiamidate antisense oligonucleotides approach (Heasman et al., 2000; Summerton, 1999). A mixture of two morpholinos designed against two different orthologous copies of Xfz7 was injected into unfertilized oocytes. The resulting embryos (Xfz7 morphants) failed to properly close the blastopore, whereas the more severely affected embryos exogastrulated. The gastrulation defect was caused by inhibition of convergent extension movements as demonstrated by an animal cap cell motility assay (Smith et al., 1990). Adding synthetic Xfz7 RNA alleviated the observed gastrulation defect arguing for the specificity of the morpholino-based targeting. At tadpole stages, Xfz7 morphants displayed loss or reduction of anterior structures. Molecular analysis showed that approximately half of Xfz7 morphants had reduced expression of a dorsal marker Xnr3 at the gastrula stage. Xnr3 is known to be the direct target of the maternal Wnt axis induction pathway (Smith et al., 1995; McKendry et al., 1997) and has previously been shown to be under maternal Xfz7 control (Sumanas et al., 2000). The results presented here argue for two different Xfz7 functions. They confirm our previous findings that Xfz7 is required for the dorsoventral Wnt/b-catenin axis induction pathway (Sumanas et al., 2000) and demonstrate a Xfz7 requirement in a zygotic Wnt pathway that regulates morphogenetic movements during gastrulation. This is the first evidence for a vertebrate Frizzled protein to act in two distinct Wnt-signaling pathways, as known to be the case for Drosophila Frizzled-1 (Adler, 1992; Bhanot et al., 1999; Chen and Struhl, 1999). To study Xfz7 function during development, we designed two different morpholinos against the 5’UTR of two distinct orthologous copies of the Xfz7 gene present due to the allotetraploid nature of Xenopus (Kobel and Du Pasquier, 1986). One ortholog has been reported by Djiane et al. (2000)., Medina et al. (2000), and Sumanas et al. (2000), and the other ortholog of Xfz7 was reported by Wheeler and Hoppler (1999). These two Xfz7 orthologs are 97% identical in amino acid sequences, but contain more significant differences in the UTR sequences (data not shown). Both orthologs have similar expression patterns at all stages reported. A mixture of the two different morpholinos (MO-1 and MO-2) was injected into unfertilized oocytes, and the oocytes were subsequently fertilized using the host–transfer method (Heasman et al., 1994). Injection of individual morpholinos up to 20 ng resulted in no significant developmental defects, whether delivered into early embryos or into oocytes followed by host transfer fertilization (data not shown). We used doses of 5 ng (2.5ng each MO) and 10 ng (5ng each MO) of the morpholino mixture to simultaneously knockdown both orthologous copies of Xfz7. Xfz7 morphants developed seemingly normally with only a slight developmental delay until the start of gastrulation. During gastrulation, Xfz7 morphants formed greatly enlarged blastopores, which subsequently failed to involute properly, resulting in exogastrulating embryos at higher doses of morpholino used (Fig. 1a–c, i). Microinjecting synthetic Xfz7 RNA into oocytes significantly reduced the severity of the phenotype, resulting in a greater percentage of normal and weakly affected embryos (Fig. 1d, i). At tailbud and tadpole stages, Xfz7 morphants displayed defects in dorsoanterior structures. They commonly had a reduced or missing head and cement gland