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.     

Genetic Control of Early Embryogenesis in the Red Flour Beetle, Tribolium castaneum

SYNOPSIS. The power of genetic analysis possible with the fruitfly, Drosophila melanogaster, has yielded a detailed understandingof pattern formation controlled by homeotic and segmentationgenes in early embryogenesis. We are studying the genetic regulationof embryogenesis in the red flour beetle, Tribolium castaneum.The dynamic process of germ rudiment formation and sequentialsegmentation exhibited by Tribolium provides a context differentthan Drosophila within which to assess the function of homeoticand segmentation gene homologs. Our analyses of the genes inthe HOM-C suggest many similarities in structure and functionwith the well-characterized Drosophila genes. Abdominal resemblesits Drosophila homolog abdominal-A in functioning to establishsegmental identities in the abdomen, such that in each casemutations result in homeotic transformations to PS6. Althoughthe anterior functional boundary of abdominal-A homologs isprecisely conserved, the domain within which Abdominal is importantextends more posterior than that of abdominal-A. The final expression pattern of the segmentation gene engrailedin Tribolium is identical to Drosophila, suggesting that thesehomologs 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 segmentas they appear sequentially in the elongating germ band. Althoughthe grasshopper even-skipped and fushi tarazu homologs are notapparently important in segmentation, the expression patternsof the Tribolium homologs strongly suggest that they have gaineda role in segmentation in the lineage leading to beetles andflies. Nevertheless, differences between Tribolium and Drosophilain the dynamics of even-skipped expression and the fushi tarazumutant phenotype indicate divergence in the regulation and rolesof these genes.     

Divergent segmentation mechanism in the short germ insect Tribolium revealed by giant expression and function

Segmentation is well understood in Drosophila, where all segments are determined at the blastoderm stage. In the flour beetle Tribolium castaneum, as in most insects, the posterior segments are added at later stages from a posteriorly located growth zone, suggesting that formation of these segments may rely on a different mechanism. Nevertheless, the expression and function of many segmentation genes seem conserved between Tribolium and Drosophila. We have cloned the Tribolium ortholog of the abdominal gap gene giant. As in Drosophila, Tribolium giant is expressed in two primary domains, one each in the head and trunk. Although the position of the anterior domain is conserved, the posterior domain is located at least four segments anterior to that of Drosophila. Knockdown phenotypes generated with morpholino oligonucleotides, as well as embryonic and parental RNA interference, indicate that giant is required for segment formation and identity also in Tribolium. In giant-depleted embryos, the maxillary and labial segment primordia are normally formed but assume thoracic identity. The segmentation process is disrupted only in postgnathal metamers. Unlike Drosophila, segmentation defects are not restricted to a limited domain but extend to all thoracic and abdominal segments, many of which are specified long after giant expression has ceased. These data show that giant in Tribolium does not function as in Drosophila, and suggest that posterior gap genes underwent major regulatory and functional changes during the evolution from short to long germ embryogenesis.     

Functional Differences between Ultrabithorax Protein Isoforms in Drosophila Melanogaster: Evidence from Elimination, Substitution and Ectopic Expression of Specific Isoforms

The homeotic selector gene Ultrabithorax (Ubx) specifies regional identities in multiple tissues within the thorax and abdomen of Drosophila melanogaster. Ubx encodes a family of six developmentally specific homeodomain protein isoforms translated from alternatively spliced mRNAs. The mutant allele Ubx(195) contains a stop codon in exon mII, one of three differential elements, and consequently produces functional UBX protein only from mRNAs of type IVa and IVb, which are expressed mainly in the central nervous system. Although it retains activity for other processes, Ubx(195) behaves like a null allele with respect to development of the peripheral nervous system, indicating that UBX-IVa and IVb alone do not contribute detectable Ubx function for this tissue. The mutant allele Ubx(MX17) contains an inversion of exon mII. We find that this allele only produces mRNAs of type IVa, but the expression pattern of the resulting UBX-IVa protein is indistinguishable from that of total UBX protein expression in wild-type embryos. The phenotype of homozygous Ubx(MX17) embryos indicates that UBX-IVa cannot substitute functionally for other isoforms to promote normal development of the peripheral nervous system. This functional limitation is confirmed by a detailed analysis of the peripheral nervous system in embryos that express specific UBX isoforms ectopically under control of a heat shock promoter. Additional observations suggest that UBX isoforms also differ in their ability to function in other tissues.     

Kruppel is a gap gene in the intermediate germband insect Oncopeltus fasciatus and is required for development of both blastoderm and germband-derived segments

Segmentation in long germband insects such as Drosophila occurs essentially simultaneously across the entire body. A cascade of segmentation genes patterns the embryo along its anterior-posterior axis via subdivision of the blastoderm. This is in contrast to short and intermediate germband modes of segmentation where the anterior segments are formed during the blastoderm stage and the remaining posterior segments arise at later stages from a posterior growth zone. The biphasic character of segment generation in short and intermediate germ insects implies that different formative mechanisms may be operating in blastoderm-derived and germband-derived segments. In Drosophila, the gap gene Krüppel is required for proper formation of the central portion of the embryo. This domain of Krüppel activity in Drosophila corresponds to a region that in short and intermediate germband insects spans both blastoderm and germband-derived segments. We have cloned the Krüppel homolog from the milkweed bug, Oncopeltus fasciatus (Hemiptera, Lygaeidae), an intermediate germband insect. We find that Oncopeltus Krüppel is expressed in a gap-like domain in the thorax during the blastoderm and germband stages of embryogenesis. In order to investigate the function of Krüppel in Oncopeltus segmentation, we generated knockdown phenotypes using RNAi. Loss of Krüppel activity in Oncopeltus results in a large gap phenotype, with loss of the mesothoracic through fourth abdominal segments. Additionally, we find that Krüppel is required to suppress both anterior and posterior Hox gene expression in the central portion of the germband. Our results show that Krüppel is required for both blastoderm-derived and germband-derived segments and indicate that Krüppel function is largely conserved in Oncopeltus and Drosophila despite their divergent embryogenesis.     

The embryonic expression pattern of labial, posterior homeotic complex genes and the teashirt homologue in an apterygote insect

 During embryogenesis of the fruit fly, Drosophila melanogaster, the homeotic genes are required to specify proper cell fates along the anterior-posterior axis of the embryo. We cloned partial cDNAs of homologues of the Drosophila homeotic gene teashirt and five of the homeotic-complex (HOM-C) genes from the thysanuran insect, Thermobia domestica, and assayed their embryonic expression patterns. The HOM-C genes we examined were labial, Antennapedia, Ultrabithorax, abdominal-A and Abdominal-B. As the expression pattern of these HOM-C genes is largely conserved among insects and as Thermobia is a member of a phylogenetically basal order of insects, we were able to infer their ancestral expression patterns in insects. We compare the expression patterns of the Thermobia HOM-C genes with their expression in Drosophila and other insects and discuss the potential roles these genes may have played in insect evolution. Interestingly, the teashirt homologue shows greater variability between Thermobia and Drosophila than any of the HOM-C genes. In particular, teashirt is not expressed strongly in the Thermobia abdomen, unlike Drosophila teashirt. We propose that teashirt expression has expanded posteriorly in Drosophila and contributed to a homogenization of the Drosophila larval thorax and abdomen. Received: 23 July 1998 / Accepted: 1 November 1998     

Maxillopedia is the Tribolium ortholog of proboscipedia

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.     

Homeotic gene expression in the locust Schistocerca: An antibody that detects conserved epitopes in ultrabithorax and abdominal-A proteins

To investigate what role homeotic genes may play in morphological evolution, we are comparing homeotic gene expression in two very different insects, Drosophila (Diptera) and Schistocerca (Orthoptera). In this paper we describe a monoclonal antibody, FP6.87, that recognizes the products of both the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes in Drosophila, via an epitope common to the carboxy terminal region of these two proteins. This antibody recognizes nuclear antigens present in the posterior thorax and abdomen of Schistocerca. We infer that it recognizes the Schistocerca homolog of UBX protein, and probably also of ABD-A. As the distribution of Schistocerca ABD-A protein is already known, we can use this reagent to map the expression of Schistocerca UBX in the thorax and anterior abdomen, where ABD-A is not expressed. Both the general domain, and many of the details, of UBX exp ression are remarkably conserved compared with Drosophila. Thus UBX expression extends back from T2 in the ectoderm (including the CNS), but only from A1 in the mesoderm. As noted for other bithorax complex genes in Schistocerca, expression begins in the abdomen, at or shortly before the time of segmentation. It only later spreads anteriorly to the thorax. For much of embryogene-sis, the expression of UBX in the thoracic epidermis is largely restricted to the T3 limb. Inthis limb, UBX is strikingly regulated, in a complex pattern that reflects limb segmentation. Reviewing these and earlier observations, we conclude that evolutionary changes affect both the precise regulation of homeotic genes within segments, and probably also the spectrum of downstream genes that respond to homeotic gene expression in a given tissue. Overall domains of homeotic gene expression appear to be well conserved between different insect groups, though a change in the extent and timing of homeotic gene expression may underlie the modification of the posterior abdomen in different insect groups. © 1994 Wiley-Liss, Inc.