Dev Genes Evol (2008) 218:141–152 Fig. 2 Multiple alignments of Teashirt family members. Ami- no acids identical or similar in at least two of the proteins are shaded black or gray, respec- tively. Tc Tribolium castaneum; Ag Anopheles gambiae, Dm Drosophila melanogaster; Mm Mus musculus; Dr Danio rerio. a Multiple alignment of the three clustered zinc fingers. Zinc fingers (ZF) 1–3 are indicated above the sequence. Regions underscored with asterisks were concatenated and subjected to phylogenetic analysis. b Multi- ple alignment of the C-terminal zinc finger region. Zinc finger 4 is delimited above the align- ment. Tsh lacks this zinc finger. c Distinct N-terminal motifs in dipteran Tsh family members (upper) and in Tribolium and vertebrates (lower) 145 spalt does not appear to repress Tc-tiotsh in Tribolium embryos In Drosophila, the limits of tsh expression are controlled either directly or indirectly by spalt-major (salm). In a salm mutant, the tsh expression domain expands both anteriorly and posteriorly (Roder et al. 1992). To assess whether the broader expression of Tc-tiotsh (compared to tsh) might reflect a difference in Tribolium spalt (Tc-sal) expression, we examined the embryonic expression pattern of the single Tribolium sal gene (Tomoyasu et al. 2005). We find that the Tc-sal expression domain (Fig. S3) significantly overlaps that of Tc-tiotsh, suggesting that Tc-sal does not repress Tc- tiotsh expression. It is possible that Tribolium has lost the regulatory interaction between these genes. Alternatively, regulation of tsh by sal may have evolved more recently, after the divergence of these lineages. Tc-tiotsh expression is reduced in embryos lacking Hox genes In Drosophila, some aspects of tsh expression are regulated by the Hox genes (Roder et al. 1992). We have examined expression of Tc-tiotsh in embryos homozygous for Df (HOMC), a deletion that removes most of the Tribolium Hox cluster. Tc-tiotsh expression is severely reduced in the ventral region of these embryos (Fig. 4f). These results