Robin E. Denell

Department of Agriculture facility about a mile from campus. He was trained as an insect toxicologist, but to aid in his research he had become a self-taught geneticist using the red flour beetle, Tribolium castaneum. Beeman told me about interesting data he had regarding the juxtaposition in Tribolium of the apparent orthologs of genes in the Antennapedia and bithorax complexes (Beeman 1987). He had heard me speak about the Drosophila complexes and recombinationally mapped putative orthologs in Tribolium. This interaction inspired my involvement, with many others, in the development of Tribolium as a genetic model system, and the use of this beetle for work on the evolution of developmental mechanisms (evo-devo). I will provide evidence that Tribolium now represents the third best (after Drosophila and Caenorhabditis) invertebrate for genetic and molecular studies. I will argue for the importance of genetically tractable insect systems other than Drosophila, especially given the derived nature of fly morphology. Although it is specialized in many ways, Tribolium is nevertheless relatively ancestral with regard to many morphological features and developmental events. Somewhat artificially, I am going to separate its growth as a model system from its contributions to evo-devo, where I will concentrate on studies of the Hox cluster.

In insects, the boundary between the embryonic head and thorax is formed by the dorsal ridge, a fused structure composed of portions of the maxillary and labial segments. However, the mechanisms that promote development of this unusual structure remain a mystery. In Drosophila, mutations in the Hox genes Sex combs reduced and Deformed have been reported to cause abnormal dorsal ridge formation, but the significance of these abnormalities is not clear. We have identified three mutant allele classes of Cephalothorax, the Tribolium castaneum (red flour beetle) ortholog of Sex combs reduced, each of which has a different effect on dorsal ridge development. By using Engrailed expression to monitor dorsal ridge development in these mutants, we demonstrate that Cephalothorax promotes the fusion and subsequent dorsolateral extension of the maxillary and labial Engrailed stripes (posterior compartments) during dorsal ridge formation. Molecular and genetic analysis of these alleles indicates that the N terminus of Cephalothorax is important for the fusion step, but is dispensable for Engrailed stripe extension. Thus, we find that specific regions of Cephalothorax are required for discrete steps in dorsal ridge formation.

We report here on the use of RNA interference (RNAi) to create pupal and adult loss-of-function phenotypes in the red flour beetle, Tribolium castaneum, by injection of double-stranded RNA (dsRNA) into late instar larvae (we refer to this method as larval RNAi). RNAi is well-established as a useful method to mimic loss-of-function phenotypes in many organisms including insects. However, with a few exceptions (such as in the fruit fly Drosophila melanogaster), RNAi analysis has usually been limited to studies of embryogenesis. Here we demonstrate that injection of green fluorescent protein (GFP) dsRNA into the larval body cavity can inhibit GFP expression beginning shortly after injection and continuing through pupal and adult stages. RNAi analysis of the Tc-achaete-scute-homolog (Tc-ASH) revealed that larval RNAi can induce morphological defects in adult beetles, and also that larval RNAi affects the entire body rather than being localized near the site of injection. The larval RNAi technique will be useful to analyze gene functions in post-embryonic development, giving us the opportunity to study the molecular basis of adult morphological diversity in various organisms.

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.

Summary: 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. genesis 30:12–20, 2001.

The last decade has seen a dramatic increase in interest in the extent to which morphological evolution depends on changes in regulatory pathways. Insects provide a fertile ground for study because of their diversity and our high level of understanding of the genetic regulation of development in Drosophila melanogaster. However, comparable genetic approaches are presently possible in only a small number of non‐Drosophilid insects. In a recent paper, Hughes and Kaufman(1) have used a new methodology, RNA interference, in the milkweed bug, Oncopeltus fasciatus, to phenocopy the effects of mutations in Hox genes. RNA interference involves the injection of double‐stranded RNA of the same sequence as the relevant mRNA resulting in a depletion of that transcript.(2) Hughes and Kaufman focused on the gnathal segments, which elaborate specialized appendages important to feeding. Their results indicate that gnathal adaptations in this bug are correlated with changes in Hox gene functions and interactions. BioEssays 23:379–382, 2001. © 2001 John Wiley & Sons, Inc.

The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

SUMMARY Null mutations in the Drosophila melanogaster homeotic gene proboscipedia (pb) cause transformation of the adult labial palps to legs. The similar phenotype produced by mutations in the Tribolium castaneum homeotic complex (HOMC) gene maxillopedia (mxp) has led to suggestions that the two genes may be orthologous. We have cloned the Tribolium ortholog of pb, which predicts a protein with a homeodomain identical to that of Drosophila Pb. The two proteins also share several additional regions of identity, including an N‐box, a motif unique to Pb orthologs. We have identified a frameshift mutation within Tribolium pb associated with an mxp null mutation, demonstrating that Tribolium pb corresponds to the mxp genetic locus. Thus, we will refer to the cloned gene as mxp. In addition, we have begun to construct a molecular map of the Tribolium HOMC. Two overlapping BAC clones which span the mxp locus also include the Tribolium labial ortholog (Tclabial ) and part of Tczerknüllt, indicating that the order of these genes in the HOMC is conserved between Drosophila and Tribolium.

Among insects, the genetic regulation of regional identities in the postoral head or gnathal segments (mandibular, maxillary, and labial) is best understood in the fly Drosophila melanogaster . In part, normal gnathal development depends on Deformed (Dfd) and Sex combs reduced (Scr) , genes in the split Drosophila homeotic complex. The gnathal segments of Dfd and Scr mutant larvae are abnormal but not homeotically transformed. In the red flour beetle, Tribolium castaneum , we have isolated loss-of-function mutations of the Deformed ortholog. Mutant larvae display a strong transformation of mandibular appendages to antennae. The maxillary appendages, normally composed of an endite and a telopodite, develop only the telopodite in mutant larvae. We previously reported that mutations in the beetle Scr and Antennapedia orthologs cause the labial and thoracic appendages, respectively, to be transformed to antennae. Moreover, a deficiency of most of the beetle homeotic complex causes all gnathal (as well as thoracic and abdominal) segments to develop antennae. These and other observations are consistent with the hypothesis that ancestral insect homeotic gene functions have been modified considerably during the evolution of the highly specialized maggot head. One of the ancestral homeobox genes that arose close to the root of the Eumetazoa appears to have given rise to Dfd, Scr , and the Antennapedia homeobox-class homeotic genes. Evidence from both Tribolium and Drosophila suggests that this ancestral gene served to repress anterior development as well as confer a trunk-specific identity.