144 Dev Genes Evol (2008) 218:141–152 Fig. 1 Gene structure of the Tc-tiotsh gene and its Drosophila homologs. Exons are denoted by boxes and introns by thin lines. The coding region is shaded gray and zinc finger-encoding motifs are shown in black which the two Drosophila proteins cluster together, suggesting these genes were produced by duplication after the divergence of the mosquito and fly lineages. The branch lengths of the Drosophila proteins are much longer than those of Anopheles and Tribolium, suggesting rapid divergence of both genes after duplication. The mouse and human genomes each have three tsh family members (Caubit et al. 2000; Manfroid et al. 2004), which are much more similar to one another than they are to any of the insect proteins. Interestingly, vertebrate Tsh family proteins share an N-terminal motif (MPRRKQ) with the Tribolium protein (Fig. 2c), which is distinct from the N-terminal motif shared by the dipteran family members (Laugier et al. 2005). The conservation of this sequence in an insect and vertebrates suggests it has an important function and that it was probably present in the common ancestor of protostomes and deuterostomes. Taken together, these comparisons of tsh family mem- bers indicate that the ancestor of protostomes and deuter- ostomes had a single tsh family gene. We were unable to identify any tsh family members in the genomes of two non-bilaterian metazoans: the placozoan Trichoplax adhae- rens (http://genome.jgi-psf.org/Triad1/Triad1.home.html) and the cnidarian Nematostella vectensis (Putnam et al. 2007), suggesting this gene family may have arisen after the bilaterian/non-bilaterian split. Within the vertebrate lineage, duplication events (perhaps in association with whole genome duplications) produced at least three Table 1 Teashirt family genes in sequenced insect genomes paralogs. A duplication in the Drosophila lineage (after divergence from the mosquito lineage) resulted in the tsh and tio paralogs. Tc-tiotsh embryonic expression is not limited to the trunk We examined the embryonic expression pattern of Tc-tiotsh (Fig. 4) by in situ hybridization, using the posterior compartment marker Engrailed (Brown et al. 1994a; Patel et al. 1989) to determine the stage of embryonic develop- ment (segments are added sequentially) and provide a reference for the register of Tc-tiotsh expression. Tc-tiotsh expression develops in a dynamic, pair rule-like manner (Fig. S1 and S2), but eventually encompasses not only the trunk segments (thorax and abdomen), but also the gnathal segments and the labrum and intercalary segment in the procephalon (Fig. 4a). Tc-tiotsh expression in the thorax and the first abdominal segment appears somewhat stronger than in other regions (Fig. 4b). As appendages develop, Tc-tiotsh is expressed in the proximal portion of each leg (Fig. 4d). After germ-band elongation, Tc-tiotsh is expressed in the posterior region of the embryo. Initially, expression is seen in a patch on either side of the ventral midline (Fig. 4c), and later in six buds that extend from the invaginating hindgut (Fig. 4e). These structures are the primordia of the Malpighian tubules; they also express Tc-dachsund (Prpic et al. 2001) and Tc-Cut (data not shown). Organism Tribolium castaneum Apis mellifera Anopheles gambiae Drosophila simulans Drosophila yakuba Drosophila pseudoobscura Drosophila virilis Order Coleoptera Hymenoptera Diptera Diptera Diptera Diptera Diptera tsh family genes Genome Reference 1 1 1 2 (tio and tsh) 2 (tio and tsh) 2 (tio and tsh) 2 (tio and tsh) The Tribolium Genome Sequencing Consortium 2008 The Honeybee Genome Sequencing Consortium 2006 Holt et al. 2002 Drosophila 12 Genomes Consortium 2007 Drosophila 12 Genomes Consortium 2007 Richards et al. 2005 Drosophila 12 Genomes Consortium 2007 tBLASTn searches were used to determine the number of teashirt (tsh) family genes in several assembled insect genomes. All Drosophila species examined had orthologs of both tsh and tiptop (tio), while the non-drosophilid species each contained only one tsh family member.