146 Fig. 3 Phylogenetic analysis of insect Teashirt family members. The two Drosophila paralogs cluster together in an unrooted tree produced by maximum likelihood analysis, suggesting that these genes resulted from a recent duplication in the Drosophila lineage. The bootstrap value of 100 indicates that the tree is very robust suggest that reduction of Tc-tiotsh expression might contribute to the transformations seen in Df(HOMC) embryos. However, our functional studies of Tc-tiotsh (presented below) reveal no evidence for this. Tc-tiotsh functions in the embryonic labial and thoracic segments We used RNAi to ascertain the probable LOF phenotype of Tc-tiotsh. Pupae injected with Tc-tiotsh dsRNA produced very few eggs upon reaching adulthood, and none of these progeny showed an abnormal phenotype. To circumvent the apparent effect of Tc-tiotsh on oogenesis, we injected Tc- tiotsh dsRNA either directly into eggs or into the abdomens of non-virgin adult females (egg production from these Dev Genes Evol (2008) 218:141–152 females quickly tapers off, further indicating an effect on oogenesis). Injection of Tc-tiotsh dsRNA by either technique produced embryos with defects in the labial and thoracic segments (Fig. 5, Table 2, and data not shown). During normal Tribolium embryonic development, the two labial appendages fuse at the ventral midline and migrate anteriorly to a position between the maxillary appendages (Fig. 5a). In moderately affected Tc-tiotsh RNAi larvae (Fig. 5b), the labial appendages are smaller than normal, and in the most severely affected embryos (Fig. 5c) these appendages fail to fuse. In all of these cases, the abnormal labial appendages fail to migrate to their normal position between the maxillary palps. These abnormalities produce secondary effects on the orientation of the other head appendages. Short legs were the most common feature seen in Tc- tiotsh RNAi larvae, being present in all larvae with labial defects, as well as in some larvae with apparently normal labial appendages (Table 2). The reduction in leg length stems from effects on the proximal leg segments. The coxa and trochanter are reduced and apparently fused, and the femur is slightly shortened (Fig. 5e). This phenotype is similar to that observed in the adult legs of hypomorphic Drosophila tsh mutants (Erkner et al. 1999) and thus may indicate a conserved role for tsh family genes in proximal limb development. We also noted that Tc-tiotsh RNAi larvae show a pronounced curvature of the head and thoracic segments, such that the head bends forward (Fig. 5e). This phenotype apparently results from reduction in length (anterior to posterior) of the ventral portions of the thoracic segments and posterior head (Fig. 5b,c,f). Fig. 4 Tc-tiotsh expression in Tribolium embryos. Engrailed expression is shown in brown, while Tc-tiotsh expression is in purple. Embryos are oriented with anterior to the left (except in panel d in which anterior is to the top). a During germband elongation, Tc-tiotsh is expressed in the gnathal and trunk segments as well as two procephalic regions (the intercalary segment [Ic] and the labrum [Lr]). b When germband elongation is completed, Tc-tiotsh expression is strongest in the thoracic (T1–T3) and first abdominal (A1) segments. c Slightly later, strong Tc-tiotsh expression appears in the posterior terminal region of the embryo (arrowhead). d Tc-tiotsh is expressed in only the proximal portion (arrow) of the developing legs. e In embryos undergoing germ- band retraction, Tc-tiotsh expression is present in the six Malpighian tubule primordia that evert from the hindgut. f In a Df(HOMC) homozygote lacking most Hox genes, Tc-tiotsh expression in the segmented portion of the germband is greatly reduced. In contrast, expression in the Malpighian tubule primordia remains strong