Morpholino phenocopies of sqt, oep, and ntl mutations.

Abstract

We have used antisense morpholino oligonucleotides (MOs) to target three genes whose loss of function were previously known to cause embryonic phenotypes in zebrafish: squint (sqt), one-eyed pinhead (oep), and no tail (ntl). One-eyed pinhead is an EGF-CFC protein and Sqt is a Nodal-related protein (Feldman et al., 1998; Zhang et al., 1998). Embryos homozygous for oep or sqt mutations have cyclopia and axial mesendoderm defects (Heisenberg and Nusslein-Volhard, 1997; Schier et al., 1996). Homozygous mutants for ntl, the zebrafish homologue of brachyury, have tail truncations and notochord deficiencies (Halpern et al., 1993; Schulte-Merker et al., 1994). Embryos injected with moderate doses of a ntl MO displayed a classic ntl mutant phenotype (class I ntl morphants, Table 1 and Fig. 1b), as previously reported for a partially overlapping ntl MO (Nasevicius and Ekker, 2000). Additionally, morphants with higher doses of ntl MO have an apparent excess of cell death near the head (class II ntl morphants, Table 1). Antibody staining of mid-gastrula embryos injected with ntl MO or a control MO revealed a specific and complete loss of Ntl in ntl MO-injected embryos (Fig. 1i–j), as shown previously by Western blot (Nasevicius and Ekker, 2000). The sqt mutation has low penetrance (data not shown). Consistent with this, only a fraction of sqt MOinjected embryos display the sqt phenotype, suggesting frequent rescue by another factor such as Cyclops (Feldman et al., 1998). These morphants were morphologically identical to sqt mutants (Table 1 and Fig. 1f). We also co-injected ntl and sqt MOs. Consistent with individual MO injections, most embryos had the ntl phenotype and a fraction of these also displayed sqt defects (Table 1 and Fig. 1d). Injection of oep MOs yielded variable results. At low doses, cyclopia and mesendoderm defects, but not floor plate defects, were phenocopied (class I oep morphants, Table 1 and Fig. 1c). A second class of oep morphants (class II oep morphants) lacking eyes was also observed, and higher doses of oep MO increased class II frequency (Table 1 and Fig. 1e). The presence of floor plate—a later differentiating structure—in class I and II oep morphants suggests the oep MO loses effectiveness by the second day of development. Eyeless and cyclopean oep morphant phenotypes were previously reported by Nasevicius and Ekker, using an identical oep MO. The same authors reported a third class of morphants resembling maternal-zygotic oep embryos (Gritsman et al., 1999; Nasevicius and Ekker, 2000). We never obtained this phenotype, even when embryos were injected immediately after fertilization.