Molecular characterization of Cephalothorax, the Tribolium ortholog of Sex combs reduced.

Sex combs reduced (Scr), a Hox gene located in the Antennapedia complex of Drosophila melanogaster, is required for the proper development of the labial and first thoracic segments. The Tribolium castaneum genetically defined locus Cephalothorax (Cx) is a candidate Scr ortholog based on the location of Cx in the beetle Homeotic complex and mutant effects on the labial and first thoracic segments. To address this hypothesis, we have cloned and characterized the Tribolium ortholog of Scr (TcScr). The transcription unit is less complex and encodes a smaller protein than Scr. The predicted amino acid sequence of the Tribolium protein shares motifs with orthologous proteins from multiple species. In addition, we have analyzed the TcScr expression pattern during embryonic development. TcScr is expressed in parts of the maxillary, labial, and first thoracic segments in a pattern similar to but not identical to Scr. Furthermore, TcScr RNA interference results in a phenocopy of the Cephalothorax (Cx) mutant phenotype in which the labial palps are transformed into antennae and the head and first thoracic segment are fused. All of the available results indicate that Cx is the Tribolium ortholog of Scr.

Interactions of the Tribolium Sex combs reduced and proboscipedia orthologs in embryonic labial development.

The role of Hox genes in the development of insect gnathal appendages has been examined in three insects: the fruitfly, Drosophila melanogaster; the milkweed bug, Oncopeltus fasciatus; and the red flour beetle, Tribolium castaneum. In each of these organisms, the identity of the labium depends on the homeotic genes Sex combs reduced (Scr) and proboscipedia (pb). Loss of pb function in each of the three insects results in homeotic transformation of the labial appendages to legs. In contrast, loss of Scr function yields a different transformation in each species. Interestingly, mutations in Cephalothorax (Cx), the Tribolium ortholog of Scr, transform the labial appendages to antennae, a result seen in the other insects only when both pb and Scr are removed. We show here that the Tribolium labial appendages also develop as antennae in double mutants. Further, we demonstrate that expression of the Tribolium proboscipedia ortholog maxillopedia (mxp) is greatly reduced or absent in the labium of Cx mutant larvae. Thus, in the wild-type labial segment, Cx function is required (directly or indirectly) for mxp transcription. A similar interaction between Scr and pb during Drosophila embryogenesis has been described recently. Thus, this regulatory paradigm appears to be conserved at least within the Holometabola.

Ectopic gene expression and homeotic transformations in arthropods using recombinant Sindbis viruses.

BACKGROUND: The morphological diversity of arthropods makes them attractive subjects for studying the evolution of developmental mechanisms. Comparative analyses suggest that arthropod diversity has arisen largely as a result of changes in expression patterns of genes that control development. Direct analysis of how a particular gene functions in a given species during development is hindered by the lack of broadly applicable techniques for manipulating gene expression. RESULTS: We report that the Arbovirus Sindbis can be used to deliver high levels of gene expression in vivo in a number of non-host arthropod species without causing cytopathic effects in infected cells or impairing development. Using recombinant Sindbis virus, we investigated the function of the homeotic gene Ultrabithorax in the development of butterfly wings and beetle embryos. Ectopic Ultrabithorax expression in butterfly forewing imaginal discs was sufficient to cause the transformation of characteristic forewing properties in the adult, including scale morphology and pigmentation, to those of the hindwing. Expression of Ultrabithorax in beetle embryos outside of its endogenous expression domain affected normal development of the body wall cuticle and appendages. CONCLUSIONS: The homeotic genes have long been thought to play an important role in the diversification of arthropod appendages. Using recombinant Sindbis virus, we were able to investigate homeotic gene function in non-model arthropod species. We found that Ultrabithorax is sufficient to confer hindwing identity in butterflies and alter normal development of anterior structures in beetles. Recombinant Sindbis virus has broad potential as a tool for analyzing how the function of developmental genes has changed during the diversification of arthropods.

Woot, an active gypsy-class retrotransposon in the flour beetle, Tribolium castaneum, is associated with a recent mutation.

A recently isolated, lethal mutation of the homeotic Abdominal gene of the red flour beetle Tribolium castaneum is associated with an insertion of a novel retrotransposen into an intron. Sequence analysis indicates that this retrotransposon, named Woot, is a member of the gypsy family of mobile elements. Most strains of T. castaneum appear to harbor approximately 25-35 copies of Woot per genome. Woot is composed of long terminal repeats of unprecedented length (3.6 kb each), flanking an internal coding region 5.0 kb in length. For most copies of Woot, the internal region includes two open reading frames (ORFs) that correspond to the gag and pol genes of previously described retrotransposons and retroviruses. The copy of Woot inserted into Abdominal bears an apparent single frameshift mutation that separates the normal second ORF into two. Woot does not appear to generate infectious virions by the criterion that no envelop gene is discernible. The association of Woot with a recent mutation suggests that this retroelement is currently transpositionally active in at least some strains.