RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

RESUMEN

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.

RESUMEN

The genetic control of embryonic organization is far better understood for the fruit fly Drosophila melanogaster than for any other metazoan. A gene hierarchy acts during oogenesis and embryogenesis to regulate the establishment of segmentation along the anterior-posterior axis, and homeotic selector genes define developmental commitments within each parasegmental unit delineated. One of the most intensively studied Drosophila segmentation genes is fushi tarazu (ftz), a pair-rule gene expressed in stripes that is important for the establishment of the parasegmental boundaries. Although ftz is flanked by homeotic selector genes conserved throughout the metazoa, there is no evidence that it was part of the ancestral homeotic complex, and it has been unclear when the gene arose and acquired a role in segmentation. We show here that the beetle Tribolium castaneum has a ftz homolog located in its Homeotic complex and expressed in a pair-rule fashion, albeit in a register differing from that of the fly gene. These and other observations demonstrate that a ftz gene preexisted the radiation of holometabolous insects and suggest that it has a role in beetle embryogenesis which differs somewhat from that described in flies.

Asunto(s)

RESUMEN

Many of the traditional experimental advantages of insects recommend their use in studies of gravitational and space biology. The fruit fly, Drosophila melanogaster, is an obvious choice for studies of the developmental significance of gravity vectors because of the unparalleled description of regulatory mechanisms controlling oogenesis and embryogenesis. However, we demonstrate that Drosophila could not survive the conditions mandated for particular flight opportunities on the Space Shuttle. With the exception of Drosophila, the red flour beetle, Tribolium castaneum, is the insect best characterized with respect to molecular embryology and most frequently utilized for past space flights. We show that Tribolium is dramatically more resistant to confinement in small sealed volumes. In preparation for flight experiments we characterize the course and timing of the onset of oogenesis in newly eclosed adult females. Finally, we present results from two shuttle flights which indicate that a number of aspects of the development and function of the female reproductive system are not demonstrably sensitive to microgravity. Available information supports the utility of this insect for future studies of gravitational biology.

Asunto(s)

RESUMEN

The power of genetic analysis possible with the fruit fly, Drosophila melanogaster, has yielded a detailed understanding of pattern formation controlled by homeotic and segmentation genes in early embryogenesis. We are studying the genetic regulation of embryogenesis in the red flour beetle, Tribolium castaneum. The dynamic process of germ rudiment formation and sequential segmentation exhibited by Tribolium provides a context different than Drosophila within which to assess the function of homeotic and segmentation gene homologs. Our analyses of the genes in the HOM-C suggest many similarities in structure and function with the well-characterized Drosophila genes. Abdominal resembles its Drosophila homolog abdominal-A in functioning to establish segmental identities in the abdomen, such that in each case mutations result in homeotic transformations to PS6. Although the anterior functional boundary of abdominal-A homologs is precisely conserved, the domain within which Abdominal is important extends more posterior than that of abdominal-A. The final expression pattern of the segmentation gene engrailed in Tribolium is identical to Drosophila, suggesting that these homologs are involved in a conserved developmental process. However, as expected the development of that pattern is different; engrailed stripes anticipate the formation of each new segment as they appear sequentially in the elongating germ band. Although the grasshopper even-skipped and fushi tarazu homologs are not apparently important in segmentation, the expression patterns of the Tribolium homologs strongly suggest that they have gained a role in segmentation in the lineage leading to beetles and flies. Nevertheless, differences between Tribolium and Drosophila in the dynamics of even-skipped expression and the fushi tarazu mutant phenotype indicate divergence in the regulation and roles of these genes.

Asunto(s)

RESUMEN

We have cloned and sequenced the single Tribolium homolog of the Drosophila engrailed gene. The predicted protein contains a homeobox and several domains conserved among all engrailed genes identified to date. In addition it contains several features specific to the invected homologs of Bombyx and Drosophila, indicating that these features most likely were present in the ancestral gene in the common ancestor of holometobolous insects. We used the cross-reacting monoclonal antibody, 4D9, to follow the expression of the Engrailed protein during segmentation in Tribolium embryos. As in other insects, Engrailed accumulates in the nuclei of cells along the posterior margin of each segment. The first Engrailed stripe appears as the embryonic rudiment condenses. Then as the rudiment elongates into a germ band, Engrailed stripes appear in an anterior to posterior progression, just prior to morphological evidence of the formation of each segment. As in Drosophila (a long germ insect), expression of engrailed in Tribolium (classified as a short germ insect) is preceded by the expression of several homologous segmentation genes, suggesting that similar genetic regulatory mechanisms are shared by diverse developmental types.

Asunto(s)

RESUMEN

The powerful combination of genetic, developmental and molecular approaches possible with the fruit fly, Drosophila melanogaster, has led to a profound understanding of the genetic control of early developmental events. However, Drosophila is a highly specialized long germ insect, and the mechanisms controlling its early development may not be typical of insects or Arthropods in general. The beetle, Tribolium castaneum, offers a similar opportunity to integrate high resolution genetic analysis with the developmental/molecular approaches currently used in other organisms. Early results document significant differences between insect orders in the functions of genes responsible for establishing developmental commitments.

Asunto(s)

RESUMEN

The Abdominal gene is a member of the single homeotic complex of the beetle, Tribolium castaneum. An integrated developmental genetic and molecular analysis shows that Abdominal is homologous to the abdominal-A gene of the bithorax complex of Drosophila. abdominal-A mutant embryos display strong homeotic transformations of the anterior abdomen (parasegments 7-9) to PS6, whereas developmental commitments in the posterior abdomen depend primarily on Abdominal-B. In beetle embryos lacking Abdominal function, parasegments throughout the abdomen are transformed to PS6. This observation demonstrates the general functional significance of parasegmental expression among insects and shows that the control of determinative decisions in the posterior abdomen by homeotic selector genes has undergone considerable evolutionary modification.

Asunto(s)