Morpholino phenocopies of the bmp2b/swirl and bmp7/snailhouse mutations

Bone morphogenetic proteins (BMPs), members of the TGF-b superfamily, are essential for dorsoventral patterning in the early embryo. In zebrafish, mutations in the bmp2b/swirl and bmp7/snailhouse genes produce dorsalized embryos in which paraxial mesoderm and the neural plate are expanded at the expense of ventral structures, demonstrating that bmp2b/swirl and bmp7/ snailhouse are required for specification of ventral cell fates (Dick et al., 2000; Kishimoto et al., 1997; Mullins et al., 1996; Nguyen et al., 1998; Schmid et al., 2000). Here, we report phenocopies of bmp2b/swirl and bmp7/snailhouse mutations produced by antisense morpholino oligonucleotide injection. A bmp2b/swirl antisense morpholino (bmp2b-MO) was injected into 1–4 cell-stage wild-type (TL strain) embryos. Injection with 210 pg bmp2b-MO caused phenotypes similar to those of bmp2b/swirl mutants at high frequency (Table 1). At the end of gastrulation and at early segmentation stages, the embryos displayed an elongated shape with a broadened notochord (Fig. 1C). Most of these embryos lysed during somitogenesis, and the rest of them had a truncated and twisted body axis at 30 h postfertilization (Fig. 1G). These correspond to class 5 (C5) and class 4 (C4) dorsalized phenotypes, respectively, according to the classification of Mullins et al. (1996). When injected with a lower amount (110 pg) of bmp2b-MO, embryos were weakly dorsalized (Fig. 1H, I; Table 1), mimicking the phenotype of bmp2b/ swirl heterozygotes (Mullins et al., 1996). However, embryos injected with higher amounts (840 pg to 3.4 ng) of bmp2b-MO exhibited an epiboly defect and usually lysed before 24 h. In these embryos, epiboly progressed slowly and usually failed to reach completion. Embryos injected with 840 pg bmp2b-MO developed a notochord and somites at 11 h without completing epiboly (Fig. 1D), whereas those injected with 3.4 ng bmp2b-MO failed to develop a notochord or somites at 11 h (Fig. 1E). bmp2b/swirl mutants have no epiboly defect even if maternal bmp2b/swirl gene function is inactivated (Kishimoto et al., 1997; Mullins et al., 1996; Nguyen et al., 1998), suggesting that the epiboly defect is not the result of depletion of Bmp2b but is a nonspecific effect of morpholino injection. To further examine the phenotypes of embryos injected with bmp2b-MO, we assayed marker gene expression by wholemount in situ hybridization. At midgastrulation (75% epiboly, 8 h), a ventral ectoderm marker gata2 was not detectable in embryos injected with 210 pg bmp2b-MO (Fig. 1K), whereas the neurectoderm marker otx2 was expanded ventrally (Fig. 1M). Early in segmentation (3-somite stage, 11 h), pax2.1 expression in midbrain–hindbrain boundary was expanded ventrally, whereas expression of this gene in pronephros and pronephric duct was not detectable (Fig. 1O). These phenotypes are indistinguishable from those of bmp2b/ swirl mutants (Kishimoto et al., 1997; Mullins et al., 1996; Nguyen et al., 1998). One point mutation, three deficiencies, and one rearrangement have been reported as bmp7/snailhouse mutations. snh, which has a mutation in the Bmp7 prodomain, causes C3 and C4 dorsalized phenotypes, whereas three deficiencies, snh, Df(LG11)snh, and Df(LG11)snh, and a rearrangement, snh, cause stronger phenotypes (Dick et al., 2000; Imai et al., 2000; Mullins et al., 1996; Schmid et al., 2000). Two bmp7/snailhouse antisense morpholinos, bmp7MO1 and bmp7-MO2, were injected separately into 1–4 cell-stage wild-type (TL) embryos. Although bmp7-MO1 is complementary to 11 to 125 of the bmp7 open reading frame, this MO has a low content of G 1 C (36%) and worked less efficiently (Table 1). Hence, we used bmp7-MO2, which produced dorsalized phenotypes at high frequency, for further analysis. Embryos injected with 840 pg bmp7-MO2 had C4-C5 dorsalized phenotypes (Fig. 2B; Table 1). These phenotypes are more similar to those of bmp7 deficiencies than to the snh point mutation, supporting the hypothesis that the deficiencies represent the null phenotype of bmp7. With lower doses (110–210 pg) of bmp7-MO2, weak dorsalized phenotypes were obtained (Fig. 2D–G; Table 1). Expression of krox20, a marker for hindbrain rhombomeres 3 and 5, was strongly expanded in embryos injected with 840 pg bmp7-MO2 (Fig. 2I), whereas ex-