Tc-knirps plays different roles in the specification of antennal and mandibular parasegment boundaries and is regulated by a pair-rule gene in the beetle Tribolium castaneum

Background: The Drosophila larval head is evolutionarily derived at the genetic and morphological level. In the beetle Tribolium castaneum, development of the larval head more closely resembles the ancestral arthropod condition. Unlike in Drosophila, a knirps homologue (Tc-kni) is required for development of the antennae and mandibles. However, published Tc-kni data are restricted to cuticle phenotypes and Tc-even-skipped and Tc-wingless stainings in knockdown embryos. Hence, it has remained unclear whether the entire antennal and mandibular segments depend on Tc-kni function, and whether the intervening intercalary segment is formed completely. We address these questions with a detailed examination of Tc-kni function. Results: By examining the expression of marker genes in RNAi embryos, we show that Tc-kni is required only for the formation of the posterior parts of the antennal and mandibular segments (i.e. the parasegmental boundaries). Moreover, we find that the role of Tc-kni is distinct in these segments: Tc-kni is required for the initiation of the antennal parasegment boundary, but only for the maintenance of the mandibular parasegmental boundary. Surprisingly, Tc-kni controls the timing of expression of the Hox gene Tc-labial in the intercalary segment, although this segment does form in the absence of Tc-kni function. Unexpectedly, we find that the pair-rule gene Tc-even-skipped helps set the posterior boundary of Tc-kni expression in the mandible. Using the mutant antennaless, a likely regulatory Null mutation at the Tc-kni locus, we provide evidence that our RNAi studies represent a Null situation. Conclusions: Tc-kni is required for the initiation of the antennal and the maintenance of the mandibular parasegmental boundaries. Tc-kni is not required for specification of the anterior regions of these segments, nor the intervening intercalary segment, confirming that Tc-kni is not a canonical 'gap-gene'. Our finding that a gap gene orthologue is regulated by a pair rule gene adds to the view that the segmentation gene hierarchies differ between Tribolium and Drosophila upstream of the pair rule gene level. In Tribolium, as in Drosophila, head and trunk segmentation gene networks cooperate to pattern the mandibular segment, albeit involving Tc-kni as novel component

A conserved function of the zinc finger transcription factor Sp8/9 in allometric appendage growth in the milkweed bug Oncopeltus fasciatus

The genes encoding the closely related zinc finger transcription factors Buttonhead (Btd) and D-Sp1 are expressed in the developing limb primordia of Drosophila melanogaster and are required for normal growth of the legs. The D-Sp1 homolog of the red flour beetle Tribolium castaneum, Sp8 (appropriately termed Sp8/9), is also required for the proper growth of the leg segments. Here we report on the isolation and functional study of the Sp8/9 gene from the milkweed bug Oncopeltus fasciatus. We show that Sp8/9 is expressed in the developing appendages throughout development and that the downregulation of Sp8/9 via RNAi leads to antennae, rostrum, and legs with shortened and fused segments. This supports a conserved role of Sp8/9 in allometric leg segment growth. However, all leg segments including the claws are present and the expression of the leg genes Distal-less, dachshund, and homothorax are proportionally normal, thus providing no evidence for a role of Sp8/9 in appendage specification

Development of the Bi-Partite Gal4-UAS System in the African Malaria Mosquito, Anopheles gambiae

Functional genetic analysis in Anopheles gambiae would be greatly improved by the development of a binary expression system, which would allow the more rapid and flexible characterisation of genes influencing disease transmission, including those involved in insecticide resistance, parasite interaction, host and mate seeking behaviour. The Gal4-UAS system, widely used in Drosophila melanogaster functional genetics, has been significantly modified to achieve robust application in several different species. Towards this end, previous work generated a series of modified Gal4 constructs that were up to 20 fold more active than the native gene in An. gambiae cells. To examine the Gal4-UAS system in vivo, transgenic An. gambiae driver lines carrying a modified Gal4 gene under the control of the carboxypeptidase promoter, and responder lines carrying UAS regulated luciferase and eYFP reporter genes have been created. Crossing of the Gal4 and UAS lines resulted in progeny that expressed both reporters in the expected midgut specific pattern. Although there was minor variation in reporter gene activity between the different crosses examined, the tissue specific expression pattern was consistent regardless of the genomic location of the transgene cassettes. The results show that the modified Gal4-UAS system can be used to successfully activate expression of transgenes in a robust and tissue specific manner in Anopheles gambiae. The midgut driver and dual reporter responder constructs are the first to be developed and tested successfully in transgenic An. gambiae and provide the basis for further advancement of the system in this and other insect species

Unique establishment of procephalic head segments is supported by the identification of cis-regulatory elements driving segment-specific segment polarity gene expression in Drosophila

Anterior head segmentation is governed by different regulatory mechanisms than those that control trunk segmentation in Drosophila. For segment polarity genes, both initial mode of activation as well as cross-regulatory interactions among them differ from the typical genetic circuitry in the trunk and are unique for each of the procephalic segments. In order to better understand the segment-specific gene network responsible for the procephalic expression of the earliest active segment polarity genes wingless and hedgehog, we started to identify and analyze cis-regulatory DNA elements of these genes. For hedgehog, we could identify a cis-regulatory element, ic-CRE, that mediates expression specifically in the posterior part of the intercalary segment and requires promoter-specific interaction for its function. The intercalary stripe is the last part of the metameric hedgehog expression pattern that appears during embryonic development, which probably reflects the late and distinct establishment of this segment. The identification of a cis-regulatory element that is specific for one head segment supports the mutant-based observation that the expression of segment polarity genes is governed by a unique gene network in each of the procephalic segments. This provides further indication that the anterior-most head segments represent primary segments, which are set up independently, in contrast to the secondary segments of the trunk, which resemble true repetitive units

Candidate Gene Screen in the Red Flour Beetle Tribolium Reveals Six3 as Ancient Regulator of Anterior Median Head and Central Complex Development

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly