Dev Genes Evol (2008) 218:141–152 than those assayed. As additional insect Tsh family protein sequences become available, it will be possible to more accurately determine the point at which these N-terminal sequences began to diverge. Evolution of tsh family gene expression Although the sequences of other insect tsh family members are most similar to tio, their expression patterns (where known) seem to be more similar to tsh. The embryonic expression patterns of tsh and tio in Drosophila are strikingly different (Laugier et al. 2005). tsh is expressed throughout the ectoderm of trunk segments, while tio is expressed in only a few cells of the trunk ectoderm (as well as in the Malpighian tubules and parts of the CNS and gut). In tsh mutants, however, tio is more broadly expressed in the trunk, indicating that tsh represses tio expression in this region. Likewise, tsh is ectopically expressed in the stomodeal region when tio expression is knocked down by RNAi. The Tc-tiotsh expression pattern includes similiarities to both the tsh and tio expression patterns. The ectodermal expression in the segmented region of the germband is reminiscent of, although more extensive than, that of tsh. On the other hand, Tc-tiotsh is expressed in the Malpighian tubule primordia, similar to tio. The expression of tio near the stomodeum might also correspond to the expression of Tc-tiotsh in the labrum and intercalary segment, although it is difficult to compare expression patterns in the cephalic regions of these insects due to the derived structure of the Drosophila larval head. It is interesting that the Tc-tiotsh expression pattern approximates the sum of the tsh and tio patterns. This may indicate that the expression pattern of the ancestral gene that gave rise to tsh and tio had an expression pattern similar to Tc-tiotsh and that parts of its expression pattern (and perhaps its function) have been split between its daughters. Such division of labor resulting from comple- mentary loss of particular expression domains or functions is termed subfunctionalization and has been suggested to play a key role in the maintenance of duplicated genes (Force et al. 1999). Among other insects, expression patterns of tsh family members have been reported only for the firebrat, Thermo- bia domestica, and the milkweed bug, Oncopeltus fasciatus (Herke et al. 2005; Peterson et al. 1999). In both cases, these genes are expressed in the gnathal, thoracic, and first abdominal segments, with very weak (if any) expression in the remainder of the abdomen. Both insects also show expression in the posterior tip of the embryo (see below). Oncopeltus has additional expression in the procephalon. The leg expression pattern in firebrats has not been described, but Herke et al. (2005) reported that milkweed 149 bugs show expression throughout the legs, which fades from the distal tips later in development. Interestingly, all of the insect tsh family genes whose expression has been described are expressed in the post- abdominal region. However, these domains have been interpreted differently. In firebrats, expression in this region is reported to represent the anlagen of the cerci and the median caudal filament (appendages of the 11th abdominal segment) (Peterson et al. 1999). The posterior expression of tsh has been interpreted as the future anal opening (Fasano et al. 1991), and that of Oncopeltus tiptop is described only as the caudal region (Herke et al. 2005). Our results show that Tc-tiotsh is expressed in the Malpighian tubule primordia. Likewise, tio is expressed in the developing Malpighian tubules (Laugier et al. 2005). Thus, Malpighian tubule expression may have been part of the ancestral expression pattern of this gene family. It is possible that the posterior expression domains in other insects will, upon closer examination, be found to represent the developing Malpighian tubules as well. Evolution of tsh family function Tc-tiotsh is required for the proper development of the proximal portion of developing larval legs. Drosophila larvae lack external appendages, but tsh is expressed in the proximal regions of leg imaginal discs and hypomorphic tsh alleles cause proximal leg defects (Erkner et al. 1999). These results suggest that proximal leg expression and function were probably a feature of a tsh-like gene in the ancestor of beetles and flies. In contrast, Herke et al. (2005) reported that knockdown of Oncopeltus tiptop caused transformations of the distal legs to antennae. Based on this observation, they concluded that Oncopeltus tiptop acts as a distal leg selector gene, and suggested that tio or tsh might play a similar role in Drosophila. tsh expression is known to be restricted to the portion of the imaginal disc that will form the body wall and proximal leg segments (Gonzalez-Crespo and Morata 1996). Similarly, Tc-tiotsh is expressed in the proximal region of embryonic legs and leg imaginal discs (Fig. 4d and data not shown). We have also examined the expression of tio in Drosophila leg imaginal discs and found that expression is restricted to the body wall and proximal portion of the developing leg. Thus, it is highly unlikely that the “tiptop-like” distal leg-specifying function suggested by Herke et al. (2005) is performed by Tio, either alone or in combination with Tsh. We cannot rule out additional, internal functions for Tc- TioTsh. In particular, the expression of Tc-tiotsh in the Malpighian tubules is intriguing, as Drosophila tio (and possibly other homologs) show similar expression. The striking effects of larval Tc-tiotsh RNAi would not have been predicted based on Drosophila experiments, as