Dev Genes Evol (2008) 218:141–152 In situ hybridization and immunostaining of embryos A digoxigenin-labeled fragment of Tc-tiotsh was hybridized to whole-mount wild-type (Georgia-1) embryos as previous- ly described (Brown et al. 1994b). Embryos were secondar- ily immunostained with either α-Invected antibody (4D9) (Patel et al. 1989) or with α-Even-skipped antibody (2B8) (Patel et al. 1994), which cross-react with Tc-Engrailed and Tc-Even-skipped, respectively. Tc-spalt (Tc-sal) expression was assayed using the probe described by Tomoyasu et al. (2005). RNA interference (RNAi) dsRNA of two different, non-overlapping fragments of Tc- tiotsh was synthesized using the PCR template method described by Kennerdell and Carthew (2000). Briefly, primers were chosen to amplify a fragment of Tc-tiotsh (bases 1358– 1786 or 1848–2581 of GenBank sequence AF356647) and the T7 promoter sequence (TAATACGACTCACTATAGG) was added to the 5′ end of each primer. The resulting primers were used to amplify template for RNA synthesis. The resulting product was purified and 1 μg used as template for RNA synthesis using the Megascript T7 High Yield Transcription kit (Ambion). For embryonic RNAi, 0–2 h eggs were collected from wild-type beetles (GA-1) and injected with dsRNA (2–4 μg/ μl) as previously described by Brown et al. (1999). After several days, hatched larvae and unhatched eggs were treated with lactic acid/ethanol. Additional larvae were documented by SEM as described by Curtis et al. (2001). Injection into non-virgin adult females was performed by manually opening the elytra and wings of ether- anesthetized beetles and injecting 2 μg/μl dsRNA into the dorsal abdomen. Larval RNAi was performed as described by Tomoyasu and Denell (2004). Results Tribolium has a single teashirt family gene We cloned a member of the tsh gene family from the red flour beetle, Tribolium castaneum (see Materials and methods for details). Comparison of the cDNA sequence to the recently completed Tribolium genome sequence revealed three exons distributed over about 90 kb (Fig. 1). Most of this distance can be attributed to an approximately 86-kb intron between the first and second exons. Only three full-length Tsh family proteins have been described from insects: the Drosophila melanogaster paralogs Tsh and Tio, and the single member of the Tsh family in Anopheles gambiae, Ag-TioTsh (Fasano et al. 143 1991; Laugier et al. 2005). In addition, small fragments of tsh family genes have been amplified from several other insects (Herke et al. 2005; Peterson et al. 1999). Two conclusions have been drawn from these limited data. First, the two Drosophila genes seem to be the result of a recent duplication in the Drosophila lineage. Second, all of the Tsh family members from other insects appear to be more similar in sequence to Tio than to Tsh. In this paper, we present data that support and greatly strengthen these conclusions. We performed a tBLASTn search of several recently sequenced insect genomes for tsh family members (Table 1). The Tribolium castaneum gene that we cloned is the only member of the tsh family present in the genome sequence of this insect. Likewise, the Apis mellifera (honeybee) genome contains only a single member of this gene family. In contrast, all of the additional Drosophila species we searched (D. pseudoobscura, D. simulans, D. yakuba and D. virilis) have orthologs of both tio and tsh. Consistent with the situation in D. melanogaster (Laugier et al 2005), tio and tsh are closely linked (less than 250 kb apart) in all of the Drosophila species examined, suggesting that these genes likely arose from a relatively recent tandem duplica- tion event. As the Tribolium gene is related to both tio and tsh, we have named it Tc-tiotsh, after the convention established by Laugier et al. (2005) for the single Anopheles gene. We compared the predicted Tc-TioTsh protein to the other insect Tsh family members for which full-length predicted proteins are available. (Although, we were able to identify a tsh family gene in Apis, the 5′ portion of the transcription unit was apparently missing from the genome sequence.) Like tio and Ag-tiotsh (Laugier et al. 2005), Tc-tiotsh encodes four zinc fingers (Fig. 1), three of which are clustered near the middle of the protein (Fig. 2a), and one that is located near the C terminus (Fig. 2b). Besides the zinc finger regions, several additional short regions of sequence identity (shaded in Fig. 2 and data not shown) are present in all of the full-length predicted proteins. The three dipteran proteins are highly similar at their N termini, while Tio, Ag-TioTsh, and Tc-TioTsh share a C-terminal motif (in addition to the fourth zinc finger) that is absent in Tsh. Regions shared between the beetle and dipteran proteins have been conserved over a long phylogenetic distance, suggesting that they likely have important functions. To better understand the evolutionary history of the tsh gene family, we have performed phylogenetic analyses using conserved portions of the zinc-finger region (under- lined in Fig. 2) from the full-length insect Tsh family proteins that are currently available (Drosophila, Tribolium, and Anopheles). Both maximum likelihood (Fig. 3) and neighbor-joining (data not shown) methods produce trees in