142 Struhl 1983). When tsh function is also eliminated, the trunk segments are more severely affected in that denticle belts disappear and additional head-like cuticle is observed (Roder et al. 1992). Recent data suggests that tsh may play an even greater role in trunk development than was previously recognized. In the absence of tsh function, its paralog tiptop (tio) is ectopically expressed in the trunk region (Laugier et al. 2005). When tio and tsh functions are both absent, the trunk denticles are even more severely affected than in mutants that lack only tsh, suggesting that tio can partially substitute for tsh and reduce the effects of tsh mutations. Robertson and coworkers (2004) proposed the existence of a system of zinc-finger proteins that divides the Drosophila embryo into sections and limits the functional domains of Hox genes. They suggest that within this system tsh specifies the trunk region, while disconnected (disco) specifies the gnathal region. In the absence of tsh, disco (as well as its paralog disco-related) is ectopically expressed in the trunk, and there is some evidence that ectopic disco expression is responsible for at least some features of tsh mutants (e.g., loss of denticles and abnormal sensory neurons) that are thought to represent a switch to head/gnathal identity (Robertson et al. 2004). Manfroid et al. (2004) speculated that the trunk- specifying function of tsh family members might be conserved in vertebrates, as tsh genes from mice are expressed in the trunk region and can also rescue the phenotype of Drosophila tsh mutants when ectopically expressed. However, such conjecture requires a rather large phylogenetic leap, as it is not clear that the role of tsh in trunk specification is conserved even in other insects. tsh family genes of the firebrat, Thermobia domestica, and the milkweed bug, Oncopeltus fasciatus, are expressed pre- dominantly in the gnathos and thorax, arguing against a function in trunk specification (Herke et al. 2005; Peterson et al. 1999). Furthermore, when Herke et al. (2005) knocked down the function of the Oncopeltus tsh family gene by RNA interference (RNAi), they observed trans- formations of distal leg to antenna, but did not report any changes in trunk identity. Finally, our previous studies suggest that genetic specification of trunk identity in the red flour beetle, Tribolium castaneum, differs significantly from that in Drosophila. Removal of trunk Hox genes alone is sufficient to produce dramatic transformation of trunk to head identity (Brown et al. 2002). Antennae develop on all gnathal and trunk segments of embryos homozygous for a deficiency that removes the Tribolium orthologs of Deformed (Dfd), Scr, Antp, Ubx, and abd-A (Stuart et al. 1991). Thus, there is apparently no need to invoke the function of another gene for trunk identity. Peterson et al. (1999) predicted that either the Tribolium tsh gene has no function in trunk specification or that the expression of tsh is controlled by Dev Genes Evol (2008) 218:141–152 Hox genes. Taken together, these observations suggest that the trunk-specifying role of tsh observed in Drosophila might not be conserved in other insects. To better understand the evolution of tsh family gene function, we cloned and characterized a tsh homolog from Tribolium. This gene, Tc-tiotsh, is the sole member of this family in the Tribolium genome. It has a broad embryonic expression domain, encompassing not only the trunk region but much of the head as well. RNAi experiments revealed that Tc-tiotsh functions in several embryonic and imaginal tissues, but they did not suggest a conserved function in trunk specification. We discuss these results and reexamine the classification of tsh as a trunk-specifying gene in Drosophila. Materials and methods cDNA cloning A genomic fragment of the Tc-tiotsh gene was amplified using degenerate primers based on the conserved amino acid motifs VWLGKG and CKVCDK (first round) and ILKCMW and CKVCDK (second round). Additional, overlapping fragments were identified by RACE, using the 5′ and 3′ RACE Systems (GibcoBRL), by polymerase chain reaction (PCR) screening a Tribolium castaneum cDNA library as described previously (Nie et al. 2001), or by inverse PCR (Ochman et al. 1988). To confirm that these overlapping fragments represent a contiguous sequence, primers were designed near the ends of the putative contig, and reverse transcriptase (RT)-PCR was performed using total embryonic RNA (0–30 h of development) as template. The resulting PCR product was cloned and sequenced (GenBank accession number AF356647). Sequence analysis The nucleotide contig for Tc-tiotsh was assembled using the Seqman module of Lasergene (DNASTAR). To identify putative tsh family members in other genomes, we performed tBLASTn searches using Drosophila mela- nogaster Tsh and Tio as query sequences. Multiple align- ments were created with the AlignX module of Vector NTI 8 (InforMax). Minor manual adjustments were performed to maximize the alignments. The final output of the multiple alignment was created using BOXSHADE (avail- able at http://www.ch.embnet.org/software/BOX_form. html). Phylogenetic analyses were performed using MEGA version 3.1 (Kumar et al. 2004) or TreePuzzle (http:// bioweb.pasteur.fr/seqanal/interfaces/Puzzle.html), and Tree- View (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) was used to visualize the output.