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 F(1) 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.

piggyBac-mediated germline transformation in the beetle Tribolium castaneum.

The lepidopteran transposable element piggyBac can mediate germline insertions in at least four insect orders. It therefore shows promise as a broad-spectrum transformation vector, but applications such as enhancer trapping and transposon-tag mutagenesis are still lacking. We created, cloned, sequenced and genetically mapped a set of piggyBac insertions in the red flour beetle, Tribolium castaneum. Transpositions were precise, and specifically targeted the canonical TTAA recognition sequence. We detected several novel reporter-expression domains, indicating that piggyBac could be used to identify enhancer regions. We also demonstrated that a primary insertion of a non-autonomous element can be efficiently remobilized to non-homologous chromosomes by injection of an immobile helper element into embryos harbouring the primary insertion. These developments suggest potential for more sophisticated methods of piggyBac-mediated genome manipulation.

Transgene expression from the Tribolium castaneum Polyubiquitin promoter.

The highly conserved Ubiquitin proteins are expressed from genes with strong, constitutively active promoters in many species, making these promoters attractive candidates for use in driving transgene expression. Here we report the cloning and characterization of the Tribolium castaneum Polyubiquitin (TcPUb) gene. We placed the TcPUb promoter upstream of the coding region of the T. castaneum eye-colour gene Tc vermilion (Tcv) and injected this construct into embryos from a Tcv-deficient strain. Transient expression of Tcv during embryogenesis resulted in complete rescue of the larval mutant phenotype. We then incorporated the TcPUb-Tcv chimera into a piggyBac donor. Resulting germline transformants were easily recognized by rescue of eye pigmentation, illustrating the potential of the TcPUb promoter for use in driving transgene expression.

Molecular characterization of Tclabial and the 3' end of the Tribolium homeotic complex.

The homeotic selector gene labial is located at the 3' end of the Antennapedia complex (ANTC) and is required for proper head development in Drosophila. We have cloned and characterized the Tribolium ortholog of labial, Tclabial (Tclab). Similar to Drosophila labial, Tclab contains a single large intron upstream of the homeobox. In contrast, Tclab lacks an intron within the homeobox. The Tribolium ortholog of chaoptic, Tcchaoptic, transcribed from the opposite strand, is located immediately downstream of the homeotic complex, and its 3'UTR overlaps that of Tclab by 50 nucleotides. We have also sequenced the 13.5 kb interval between Tclab and maxillopedia (the Tribolium ortholog of Drosophila proboscipedia). In contrast to Drosophila, there is not a cluster of cuticle genes in this region. Finally, we have examined the expression of Tclab transcripts in Tribolium embryos. As previously described for Drosophila and other insects, the expression of Tclab is specific to the intercalary segment.

Comparative insect developmental genetics: phenotypes without mutants.

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 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. 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.

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.

Analysis of maxillopedia expression pattern and larval cuticular phenotype in wild-type and mutant tribolium.

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.

Implications of the Tribolium Deformed mutant phenotype for the evolution of Hox gene function.

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.

Maxillopedia is the Tribolium ortholog of proboscipedia.

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.

Molecular and genetic analysis of the Tribolium Ultrabithorax ortholog, Ultrathorax.

Ultrabithorax (Ubx) is essential for the proper patterning of the posterior thorax and anterior abdomen in Drosophila. The Coleoptera and Diptera differ in the organization and structure of their thorax and anterior abdomen. Changes in the regulation of Ubx and/or its downstream target genes are predicted to underlie these altered morphologies. We exploited the feasibility of genetic analysis in the red flour beetle, Tribolium castaneum, to examine the role of its Ubx ortholog in development. We analyzed genomic and cDNA clones that predict a polypeptide with nearly 100% identity with the Drosophila Ubx gene in the homeodomain and flanking sequences. Southern blot analysis indicates that these clones represent DNA sequences within the Homeotic complex (HOM-C) of Tribolium. Phenotypic analysis of mutant variants of the Ultrathorax (Utx) gene, and its location within the beetle HOM-C, strongly supports Utx being the Tribolium ortholog of Ubx. The embryonic expression pattern of Ubx-homologous transcripts coincides with the phenotypes associated with Utx mutations, providing support that the Ubx-homologous cloned DNA represents the Utx locus. By mid-germband extension Utx transcripts are expressed in a pattern similar to Ubx in Drosophila. However, during early germband formation Utx transcripts differ in both spatial and temporal progression. Utx expression is initially detected in parasegments 4 and 5 (T1p-T3a) as they are established during early germband formation. This is the first report of the wild-type parasegmental expression of an insect Ubx ortholog extending through parasegment 4. The earlier and more anterior expression in the thorax may underlie the modification of the Coleopteran thorax.

Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant.

We have analyzed the Tribolium castaneum ortholog of the Drosophila homeotic gene Deformed (Dfd) and determined its expression pattern during embryogenesis in this beetle. Tc Deformed (Tc Dfd) is expressed in the blastoderm and the condensing germ rudiment in a region that gives rise to gnathal segments. During germ band extension Tc Dfd is expressed in the mandibular and maxillary segments, their appendages, and the dorsal ridge. Comparison of insect Dfd protein sequences reveals several highly conserved regions. To determine whether common molecular features reflect conserved regulatory functions we used the Gal4 system to express the Tribolium protein in Drosophila embryos. When Tc Dfd is expressed throughout embryonic ectoderm under the control of P69B, the beetle protein autoregulates the endogenous Dfd gene. In addition, the Drosophila proboscipedia gene (a normal target of Dfd) is ectopically activated in the antennal and thoracic segments. We also compared the ability of the beetle and fly proteins to rescue defects in Dfd- mutants by expressing each throughout the embryonic during embryogenesis. Both proteins rescued Dfd- defects to the same extent in that they each restore the development of mouth hooks and cirri, as well as cause gain-of-function abnormalities of posterior mouth parts. As before, pb was ectopically activated in the antennal segment. This is the first demonstration of the ability of a heterologous homeotic selector protein to directly regulate a target gene independent of an endogenous Drosophila autoregulatory loop.

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.

Molecular characterization of the Tribolium abdominal-A ortholog and implications for the products of the Drosophila gene.

The Drosophila homeotic selector gene abdominal-A is important for determinative decisions in the anterior abdomen. Insects vary considerably with respect to abdominal morphology, and changes in the function of homeotic selector genes and/or downstream genes under their control presumably have been important to the evolution of these differences. Mutations in Abdominal, the Tribolium ortholog of abdominal-A, have been described, and have more posterior homeotic transformations than do Drosophila variants. Here we present the organization of the Abdominal gene and the sequences of its predicted proteins, the first such report for a non-Drosophilid insect. Two predicted proteins share N-terminal sequences with those proposed to be synthesized by the Drosophila ortholog. In addition, we describe the distribution of Abdominal transcripts during embryogenesis. The Tribolium expression pattern closely resembles that of Drosophila, and does not account for the differences in mutant phenotypes.

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) Roux's 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.

Class 3 Hox genes in insects and the origin of zen.

We have cloned, from a beetle and a locust, genes that are homologous to the class 3 Hox genes of vertebrates. Outside the homeobox they share sequence motifs with the Drosophila zerknüllt (zen) and z2 genes, and like zen, are expressed only in extraembryonic membranes. We conclude that the zen genes of Drosophila derive from a Hox class 3 sequence that formed part of the common ancestral Hox cluster, but that in insects this (Hox) gene has lost its role in patterning the anterio-posterior axis of the embryo, and acquired a new function. In the lineage leading to Drosophila, the zen genes have diverged particularly rapidly.

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.

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

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.

We are characterizing members of the Transforming Growth Factor-ß (TGF-ß) superfamily in the red flour beetle, Tribolium castaneum, in order to examine the evolutionary conservation of the structure and function of TGF-ß-like genes during insect development. A decapentaplegic-like gene of the TGF-ß 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-ß 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.