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Zebrafish frizzled‐2 morphant displays defects in body axis elongation

Wnt signaling has been implicated in many patterning processes in a vertebrate embryo including morphogenetic cell rearrangements during gastrulation. Pipetail (wnt5) and silberblick (wnt11) zebrafish mutants undergo abnormal gastrulation with an undulated notochord ( pipetail) and cyclopic embryos ( silberblick) at later stages of development (Hammerschmidt et al., 1996; Heisenberg et al., 1996; Heisenberg et al., 2000; Rauch et al., 1997). Wnt proteins are known to transmit signals via the Frizzled seven-pass transmembrane receptor family (Bhanot et al., 1996). We have isolated cDNA encoding a new Frizzled family protein, zebrafish Frizzled-2 (Fz2). Zebrafish frizzled-2 is a likely ortholog of human frizzled-2 by the sequence homology alignments and mapping data analysis. Fz2 is expressed zygotically within the notochord, somitic and posterior paraxial mesoderm. For protein knockdown studies, we utilized morpholino phosphorodiamidate antisense oligonucleotides (morpholinos, MOs). Injection of two different morpholinos into early zebrafish embryos targeted to fz2 caused similar developmental defects. Fz2-morpholino injected embryos (morphants) are shorter than controls and display an undulating or kinked notochord, neural tube, and hypochord. Similar, although weaker, undulations of axial structures have been observed in pipetail mutant embryos, raising possibility that fz2 may function as a receptor in the wnt5 pathway regulating gastrulation movements. We isolated a novel zebrafish frizzled homolog from gastrula stage cDNA library by the polymerase chain reaction (PCR). The isolated gene encodes a putative Frizzled protein with highest homology to the frizzled-2 subfamily as evident from BLAST and CLUSTAL alignments (Fig. 1a, b). Radiation hybrid mapping using panel LN54 (Hukriede et al., 1999) places fz2 on linkage group 3, linked to the marker Z22516 (distance 0.00cR, LOD score 22.2). To confirm that we isolated true frizzled-2 ortholog from zebrafish, we performed synteny analysis between corresponding zebrafish and human chromosome regions. Zebrafish fz2 maps to the marker Z22516, which is located between the hoxb9a and dlx8 genes on linkage group 3 in the LN54 panel (Hukriede et al., 1999; marker list at http://zfin.org/ZFIN/). This region of zebrafish linkage group 3 is syntenic to the human chromosome 17 region. Human fz2 maps by FISH to 17q21.1 (Zhao et al., 1995), human hoxB9 maps to 17q21-q22 (Acampora et al., 1989; Apiou et al., 1996; HUGO database), and human Dlx4, an ortholog of zebrafish Dlx8, maps to 17q21.33 (Nakamura et al., 1996; Stock et al., 1996). Thus, human frizzled-2 is likely to be the true ortholog of the zebrafish frizzled-2 gene. Using Northern blotting, we detected a single, zygotically expressed transcript of fz2, with maximal expression level approximately at the five-somite stage (Fig. 1c). In situ hybridization revealed weak and ubiquitous expression starting from the shield stage (data not shown). During early somitogenesis, fz2 RNA is detected within the forming somites as well as in two longitudinal stripes within the paraxial posterior mesoderm (Fig. 1, d–g). There is also weak expression within notochordal cells anterior to fz2 expression in the forming somites. At the 20-somite stage, fz2 expression is detected in the posterior part of the notochord and in the posterior somitic mesoderm (Fig. 1h, i). At 26 hpf, fz2 is localized within the tailbud mesoderm (Fig. 1k). Injecting 100–500 pg fz2 RNA into fish embryos at the 1–2 cell stage caused severe hyperdorsalization in .90% of experimental embryos (data not shown), as noted previously for other Frizzled proteins (Nasevicius et al., 1998). A number of fz2-overexpressing embryos displayed secondary axes (data not shown). This illustrates that fz2 is capable of activating dorsoventral axis induction pathway when overexpressed in zebrafish embryos. To study fz2 function, we utilized an antisense morpholino oligonucleotide approach (Nasevicius and Ekker, 2000; Summerton, 1999). Two nonoverlapping morpholinos were designed against the fz2 sequence. Injecting either morpholino against fz2 produced similar phenotypes, indicating a likely high specificity of targeting to fz2 transcripts. The resulting embryos (fz2 morphants) display characteristic defects not commonly seen with other unrelated morpholinos. At 70% epiboly, fz2 morphants have an oblong shape compared with