Robin E. Denell

Larval RNAi in Tribolium (Coleoptera) for analyzing adult development

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.

Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects

Gene product distribution is often used to infer developmental similarities and differences in animals with evolutionarily diverse body plans. However, to address commonalties of developmental mechanisms, what is really needed is a method to assess and compare gene function in divergent organisms. This requires mutations eliminating gene function. Such mutations are often difficult to obtain, even in organisms amenable to genetic analysis. To address this issue we have investigated the use of double‐stranded RNA interference to phenocopy null mutations. We show that RNA interference can be used to phenocopy mutations of the Deformed orthologues in Drosophila and Tribolium. We discuss the possible use of this technique for comparisons of developmental mechanisms in organisms with differing ontogenies.

Genetic analysis of the homeotic gene complex (HOM-C) in the beetle Tribolium castaneum.

Our laboratories have undertaken both genetic and molecular studies of the homeotic gene complex (HOM-C) of the beetle Tribolium castaneum, and this paper discusses results from our genetic analyses. We describe here the adult phenotypes and complementation behavior of over 50 new mutations. Many of these homeotic phenotypes resemble those of Drosophila melanogaster, but few precisely parallel the segmental transformations seen in this fly. Analysis of putative loss-of-function mutations affecting the head and thorax suggests that the maxillopedia and Cephalothorax genes most closely resemble proboscipedia and Sex combs reduced of Drosophila. In the abdomen, putative loss-of-function alleles of Abdominal affect a domain corresponding to those of the combined abdominal-A and Abdominal-B genes of Drosophila. In contrast to the situation in flies, Abdominal loss-of-function variants in Tribolium cause anteriorward transformations in A3-A5a, but posteriorward transformations in A5p-A7. The implications of the differences in developmental strategies evolved in Tribolium vs Drosophila are discussed.

Repeated Co-options of Exoskeleton Formation during Wing-to-Elytron Evolution in Beetles

BACKGROUND The vast diversity in morphology of insect wings provides an excellent model to study morphological evolution. The best-described wing modification is the specification of halteres in Drosophila by a Hox-dependent mechanism, in which a Hox gene affects the expression of genes important for wing development to modify the resulting structure. We have previously shown that highly modified beetle elytra are Hox-free structures despite their divergent morphology, suggesting another mode of evolutionary modification. RESULTS To understand how elytra have evolved without Hox input, we have analyzed wing development in a coleopteran, Tribolium castaneum. Based on Drosophila mutant phenotypes, we first hypothesized that changes in the wing gene network might have contributed to elytral evolution. However, we found that the wing gene network defined in Drosophila is largely conserved in Tribolium and is also used to pattern the elytra. Instead, we found evidence that the exoskeleton formation has been co-opted downstream of the conserved wing gene network multiple times. We also show evidence that one of these co-options happened prior to the others, suggesting that repeated co-options may have strengthened an advantageous trait. In addition, we found that the Tribolium apterous genes are not only essential for exoskeletalization of the elytra but also are required for the proper identity of the hindwing-an unexpected role that we find to be conserved in Drosophila. CONCLUSIONS Our findings suggest that elytral evolution has been achieved by co-opting a beneficial trait several times while conserving the main framework of wing patterning genes.

Homoeosis in Drosophila: A description of the polycomb lethal syndrome☆

Adults heterozygous for dominant mutations at the haploabnormal Polycomb (Pc) locus display many homoeotic transformations. E. B. Lewis (1978) first described the cuticular morphology of lethal embryos homozygous for Polycomb mutant alleles, and suggested that the Pc+ gene product acts as a repressor of genes in the Bithorax gene complex. In the present work, we have further examined the Polycomb lethal syndrome by phase-contrast and scanning electron microscopy of whole mounts, and show that the phenotype is more complex than hitherto realized. Many cuticular features characteristic of more anterior body segments are partially or completely transformed to resemble those of more caudal segments; the posteriormost homoeotic features which develop are those of the 8th abdominal segment (rather than the 9th or 10th). Involution and dorsal closure of the head are usually incomplete, and the labium and dorsal head regularly develop cuticular features normally characteristic of abdominal segments. Not all homoeotic alterations are caudal, however, and embryos also display partial transformations of the 9th (or 10th) abdominal segment to 8th and of meso- and metathorax to prothorax. It should be emphasized that Polycomb mutations do not homoeotically transform segments as a whole; various cuticular markers differ considerably in their relative probability and extent of transformation, and a single segment may display features normally specific to several different segments. We suggest that these mutations result in an instability in the transmission of determined states.

Molecular characterization and embryonic expression of the even-skipped ortholog of Tribolium castaneum

In short germ insects, the procephalon and presumptive anterior segments comprise most of the embryonic rudiment which lengthens as posterior segments are added during development (Sander, K. (1976) Adv. Insect Physiol. 12, 125-238). The expression pattern of a grasshopper ortholog of the primary pair-rule gene even-skipped (eve) suggests that it is not relevant to segmentation in this short germ insect (Patel, N.H., Ball, E.E. and Goodman, C.S. (1992) Nature 357, 339-342). However in Drosophila, a long germ insect that forms all segments simultaneously, eve plays a vital role in segment formation (Nüsslein-Volhard, C., Wieschaus, E. and Klüding, H. (1984) Rouxs Arch. Dev. Biol. 193, 267-282). We have characterized the eve ortholog of the beetle Tribolium castaneum. The homeodomain sequence is highly conserved between beetle, fly, and grasshopper eve orthologs. Tc eve is expressed in stripes during segmentation, but in a pattern differing in some details from that of the fly gene. This pattern is coincident with that detected with a cross-reacting antibody (Patel, N.H., Condron, B.G. and Zinn, K. (1994) Nature 367, 429-434). Thus, an ancestral even-skipped gene appears to have evolved a role in segmentation in a common ancestor of flies and beetles. Unlike vertebrate orthologs but similar to eve, Tc eve is not linked to the homeotic complex.

Two orthodenticle-related genes in the short-germ beetle Tribolium castaneum

Abstract To investigate the molecular basis of head evolution, we searched for genes related to the Drosophila orthodenticle (otd) homeobox gene in the short-germ beetle Tribolium castaneum. Unexpectedly, we found that there are two otd-related genes in Tribolium, with predicted homeodomains highly similar to that of the single Drosophila gene. One of the two genes (Tc otd-1) is more related in both amino acid sequence and expression pattern to fruitfly otd. Tc otd-1 is expressed in a broad anterior stripe in the blastoderm embryo, suggesting a role in early head segmentation similar to that of the Drosophila gene. The second gene (Tc otd-2) is more similar in sequence to the otd-related genes isolated from different vertebrate species (the Otx gene family). Tc otd-2 is not transcribed in the blastoderm, but is expressed later in more limited subsets of cells in the anterior brain. Both Tribolium genes and the Drosophila gene are, unlike the vertebrate genes, also expressed at the developing ventral midline of the embryo. Our results are consistent with the idea that an otd/Otx gene specified anterior head structures in the last ancestor common to arthropods and vertebrates. Within the arthropod lineage, we propose that this gene acquired a function in cells at the developing midline prior to the duplication that generated the two Tribolium genes.

The Tribolium decapentaplegic gene is similar in sequence, structure, and expression to the Drosophila dpp gene.

Abstract We are characterizing members of the Transforming Growth Factor-β (TGF-β) superfamily in the red flour beetle, Tribolium castaneum, in order to examine the evolutionary conservation of the structure and function of TGF-β-like genes during insect development. A decapentaplegic-like gene of the TGF-β superfamily was isolated in Tribolium (Tc dpp) that is similar in sequence, organization, and expression to the Drosophila melanogaster dpp gene (Dm dpp). Conserved features include a high degree of sequence similarity in both the pro-domain and mature domains of the encoded polypeptide. In addition, the position of an intron within the protein-coding region is conserved in Tc dpp, Dm dpp, and two bone morphogenetic protein genes of the TGF-β superfamily in humans, BMP2 and BMP4. Consensus binding sites for the dorsal transcription factor are found within this intron in Tc dpp similar to the intronic location of several dorsal binding sites in Dm dpp. During embryogenesis, Tc dpp is expressed in an anterior cap of serosa cells at the blastoderm stage, in the dorsal ectoderm at the lateral edges of the developing and extended germ band, and in the distal tips of developing embryonic appendages. Several aspects of embryonic expression, similar in both flies and beetles, suggest conserved roles for dpp in cellular communication during the development of these distantly related insects.

Developmental studies of lethality associated with the Antennapedia gene complex in Drosophila melanogaster

Abstract A number of dominant homoeotic mutations are localized to the proximal right arm of chromosome 3 of Drosophila melanogaster and are thought to represent members of a gene complex that controls normal determinative decisions in the head and thorax. We have designated this complex the Antennapedia gene complex (ANT-C). Developmental studies were done to investigate the nature of the lethality associated with members of two of the complementation groups within ANT-C. The first complementation group, represented by the mutant Multiple Sex Combs (Msc) is characterized by embryonic lethality when heterozygous with a deletion of the ANT-C. The second complementation group consists of Antennapedia (Antp), Antennapedia-Extra Sex Combs (AntpScx), and the lethals recovered as revertants of AntpNs. When heterozygous for a deletion of the ANT-C or in heterozygous condition with each other, the members of this group show effective lethal phases spanning from embryo-larval boundary to late larval stages. Wakimoto and Kaufman (1981) show that the Antp+ gene acts to establish normal determinative states in the thorax. In the present work, transplantation of eye-antennal disks from lethal individuals heterozygous for two different AntpNs revertant chromosomes into wild-type hosts allowed the assessment of the function of the Antp+ allele in the antenna. Since these transplants formed only antennal structures and showed no evidence of the antennal → leg transformation seen in AntpNs controls, we conclude that the wild-type function of the Antp locus is not necessary for the establishment and/or maintenance of the antennal determined state. We suggest that regulatory mechanisms associated with the Antp+ structural gene normally function both to allow its expression in the thorax and to repress it in the antenna.

piggyBac‐based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids

We describe an efficient method for generating new piggyBac insertions in the germline of F1 hybrid Tribolium castaneum derived from crosses between transgenic helper and donor strains. Helper strains carried single Minos elements encoding piggyBac transposase. The donor strain carried a single piggyBac element inserted into an actin gene, expanding the eye‐specific, 3xP3‐EGFP (enhanced green fluorescent protein) reporter expression domain to include muscle. Remobilization of the donor element is accompanied by loss of muscle fluorescence but retention of eye fluorescence. In a pilot screen, the piggyBac donor was remobilized in 84% of the hybrid crosses, generating hundreds of new lethal, enhancer‐trap, semisterile and other insertions. The jumpstarter system described herein makes genome‐wide, saturation insertional mutagenesis a realistic goal in this coleopteran species.