150 relatively few adult effects of LOF tsh mutations have been reported. Other than the proximal leg defects that have been discussed above, the only other adult phenotypes reported are defects in wing posture (Soanes et al. 2001) and in structures that develop from the eye-antennal disc, namely, the eyes (Singh et al. 2002) and the maxillary palps (Bhojwani et al. 1997). This dearth of adult phenotypes could be a consequence of the pre-adult lethality of many tsh allele combinations. However, it might also be due to functional redundancy with tio. We examined tio expres- sion in Drosophila imaginal discs and found that it is indeed expressed in a pattern very similar to that of tsh (data not shown). Thus, loss of both tio and tsh during Drosophila imaginal development might produce a more severe phenotype. The principal question prompting our studies of the Tribolium Tc-tiotsh gene was whether the trunk-specifying function reported for tsh in Drosophila was conserved in other insects. However, insights from our work and other recent reports compel us to first reexamine whether tsh itself is really a trunk-specifying gene. The key observa- tions, which originally led to this conclusion, were the presence of head-like sclerites in embryos lacking tsh, and the positive correlation between expression of tsh and the presence of denticle belts (a trunk-specific feature) (de Zulueta et al. 1994; Fasano et al. 1991; Roder et al. 1992). The later discovery that tsh and disco form mutually exclusive domains in the trunk and head, respectively, seemed to support this view (Robertson et al. 2004). Below we synthesize recent studies that may lead to new interpretations of these observations. A number of mutant phenotypes in Drosophila, includ- ing those of tsh and some Hox genes, feature small patches of sclerotic tissue. The previous interpretation of such patches as ectopic head tissue has been called into question. Mace et al. (2005) showed that the sclerites that form in a segment lacking Hox gene expression are correlated with breaches in epidermal integrity and the resulting activation of a wound response pathway. If in fact, the sclerotized cuticle seen in tsh mutants is scar tissue rather than head cuticle, the interpretation of tsh as a trunk-specifying gene would be less convincing, but still possible. The case for tsh acting as a trunk specifier was apparently strengthened when Robertson et al. (2004) proposed the attractive hypothesis that a system of zinc finger proteins acted in conjunction with the Hox genes to specify body regions. Within this system, disco specified gnathal segments and tsh specified trunk segments. How- ever, the same laboratory recently reported that the conserved role of disco is to specify the medial portion of appendages, and suggested that the boundary between disco and tsh expression is actually an artifact of maggot morphology (Patel et al. 2007). That is, the reduced Dev Genes Evol (2008) 218:141–152 Drosophila larval head might lack proximal, tsh-expressing tissue (body wall) while retaining medial appendage tissue, whereas the trunk retains tsh-expressing tissue but has lost most appendage tissue. Taking this scenario a step further, ectopic expression of tsh might “restore” the body wall tissue of the head, creating larger, more complete segments. As the genes controlling segment formation also control denticle belt patterning, these enlarged head segments would have denticle belts as well. This interpretation is consistent with the observation that the additional denticle belts produced by ectopic tsh expression are interspersed with head-specific structures (de Zulueta et al. 1994). When considered in this light, perhaps tsh is no more a trunk identity gene than are other genes involved in segment formation and denticle patterning such as wingless, rhom- boid, or shavenbaby. On the other hand, tsh does seem to provide the spatial information that limits full segment formation to the trunk, and thus in Drosophila could be considered a trunk-specifying gene. In contrast, Tc-tiotsh does not appear to act in any way as a trunk identity gene. This is not to say that Tc-tiotsh has no role in the development of trunk segments, but rather that we find no evidence that it specifies trunk vs head fates. In embryos depleted of Tc-tiotsh the ventral portions of the labial and thoracic segments are reduced, but not visibly transformed. Interestingly, some of the labial abnormalities observed in Tc-tiotsh RNAi larvae are reminiscent of those produced by loss of function alleles of prothoraxless (Tc- Antp) (Shippy, unpublished data). Knockdown of Tc-tiotsh during imaginal development produces adults with pattern- ing defects in the thoracic and abdominal sclerites, including possible transformations of anterior abdominal sclerites to a more posterior identity. These phenotypes, together with the reduction in Tc-tiotsh expression seen in Df(HOMC) embryos, suggest that Tc-tiotsh is regulated to some extent by Hox genes and may carry out a subset of the functions of these genes in both the head and the trunk. Our studies of Tc-tiotsh indicate that trunk specification is not a universal function of tsh family members. It is possible that this function has been lost in Tribolium. However, expression pattern data from other insects and functional analysis in Oncopeltus argues against this conclusion. A more likely scenario is that tsh family members acquired a role in defining trunk at some point after the divergence of the coleopteran and dipteran lineages. Analysis of tsh function in additional holometab- olous insects should help pinpoint when this event occurred. Acknowledgments We thank Marcé Lorenzen for performing some of the embryonic RNAi injections, Sue Haas for helping characterize the embryonic RNAi phenotypes, and Deane Lehmann and Michelle Coleman for technical assistance. We are also grateful to members of the Denell and Brown laboratories for helpful discussions and to Kent