Inhibition of ski A and ski B Gene Expression Ventralizes Zebraﬁsh Embryos

Dorsal–ventral (DV) patterning has been extensively studied in vertebrates. Signaling proteins identified in DV development include the bone morphogenetic proteins 2 and 4 (BMP2,4), members of the transforming growth factor (TGF)-b superfamily, which have ventralizing activities, and their antagonists Chordin, Noggin and Follistatin (Thomsen, 1997; Heasman, 1997). Interactions among these signaling proteins appear to establish gradients of BMP activity that are responsible for DV patterning. Twisted gastrulation is a conserved protein that functions with Chordin to antagonize BMP signaling (Ross et al., 2001), indicating the existence of multilayered regulating pathways that govern one of the most fundamental events in vertebrate embryogenesis. Smad proteins mediate the intracellular transmission of BMP signals. Smad proteins can be sorted into three distinct functional classes: receptor-regulated Smad proteins acting as positive mediators of TGF-b superfamily signaling (Smads 1, 2, 3, 5, and 8), global partners of receptor-regulated Smad proteins (Smad 4) and inhibitory Smads (6, 7, and 9) (Whitman, 1997; Kretzschmar and Massagué, 1998). In their inactive states, receptorregulated Smad proteins are cytoplasmic and form homotrimers that are activated by specific TGF-b receptors via phosphorylation of a conserved SSXS motif in their MH2 domains. After phosphorylation, receptor-regulated Smad homotrimers aggregate with trimers of Smad 4 and translocate to the nucleus, where they interact with their cofactors to regulate the transcription of target genes. Biochemical analysis indicates that Ski proteins act as transcriptional co-repressors through interaction with Smads -1, -2, -3, and -4 in cells (Luo et al., 1999; Akiyoshi et al., 1999; Xu et al., 2000; Wang et al., 2000). Altered expression of Ski can lead to oncogenesis (Li et al., 1986; Stavnezer et al., 1986). In a previous study, we showed that there are two paralogues of the ski gene family in zebrafish, skiA and skiB. Overexpression of either gene disrupted gastrulation and resulted in a dorsalized phenotype, suggesting that Ski proteins participate in the DV patterning (Kaufman et al., 2000). Here, we significantly extend the initial conclusions by using a “knockdown” strategy (Nasevicius and Ekker, 2000) to examine the roles of SkiA and SkiB during embryogenesis. Embryos injected with MO-skiA or MO-skiB have enlarged blood islands, expanded caudal somites, reduced or absent notochords, and little or no head structures after 24 h of development (Fig. 1 B–J), similar to the ventralized embryos described by Kishimoto et al. (1997). This confirms that SkiA and SkiB are involved in DV patterning in zebrafish. The level of ventralization was dose dependent and specific for each paralogue, as shown by secondary MOs directed against either skiA or skiB (Table 1). As expected (Nasevicius and Ekker, 2000), control MOs with four-base mismatches to either ski gene gave lower rates of ventralization when used at 60-ng levels (P , 0.0001 for both skiA and skiB) but not at the 10-ng levels (Table 1). However, both four-base mismatched MOs induced ventralization at significantly higher rates (P 5 0.0095) at 60 ng than a random (standard) MO that was used to control for general MO toxicity. At comparable doses, MO-skiB injection generated more severely ventralized embryos than MO-skiA (Table 2). Co-injection of two different MO oligos for the same ski gene synergistically blocked gene expression and caused higher rates and degrees of ventralization (Table 2). We note that at the higher doses of MOs and mRNAs there is considerable lethality. Both agents can be toxic, especially when they affect such a fundamental process as DV patterning. Note that when both mRNA and MOs are injected, some embryos (D/V) have regions