Hairy stripes in Tribolium are generated during blastoderm and germ band extension, but a direct role for Tc-h in trunk segmentation was not found. We have studied here several aspects of hairy function and expression in Tribolium, to further elucidate its role. First, we show that there is no functional redundancy with other hairy paralogues in Tribolium. Second, we cloned the hairy orthologue from Tribolium confusum and show that its expression mimics that of Tribolium castaneum, implying that stripe expression should be functional in some way. Third, we show that the dynamics of stripe formation in the growth zone is not compatible with an oscillatory mechanism comparable to the one driving the expression of hairy homologues in vertebrates. Fourth, we use parental RNAi experiments to study Tc-h function and we find that mandible and labium are particularly sensitive to loss of Tc-h, reminiscent of a pair-rule function in the head region. In addition, lack of Tc-h leads to cell death in the gnathal region at later embryonic stages, resulting in a detachment of the head. Cell death patterns are also altered in the midline. Finally, we have analysed the effect of Tc-h knockdown on two of the target genes of hairy in Drosophila, namely fushi tarazu and paired. We find that the trunk expression of Tc-h is required to regulate Tc-ftz, although Tc-ftz is itself also not required for trunk segmentation in Tribolium. Our results imply that there is considerable divergence in hairy function between Tribolium and Drosophila.

Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.

The gap gene hunchback in Drosophila acts during syncytial blastoderm stage via a short-range gradient and concentration-dependent activation or repression of target genes. Orthologues of hunchback can be easily found in other insects, but it has been unclear how well its functions are conserved. The segmentation process in most insect embryos occurs under cellular conditions, which should not allow the formation of diffusion-controlled transcription factor gradients. We have studied here in detail the function of hunchback in the short germ embryo of Tribolium using parental RNAi and interaction with possible target genes. We find that hunchback is a major regulator of the trunk gap genes and Hox genes in Tribolium, but may only indirectly be required to regulate other segmentation genes. The core function of hunchback appears to be the setting of the Ultrabithorax expression border via a repression effect, and the activation of the Krüppel expression domain. These regulatory effects are likely to be direct and are conserved between Drosophila and Tribolium. We find no evidence for a classical gap phenotype in the form of loss of segments in the region of expression of hunchback. However, the phenotypic effects in Tribolium are highly comparable with those found for other short germ embryos, i.e. the core functions of hunchback in Tribolium appear to be the same in these other insects, although they are evolutionarily more distant to Tribolium, than Tribolium is to Drosophila. These results allow the disentanglement of the conserved role of hunchback in insects from the derived features that have been acquired in the lineage towards Drosophila. Given that the gap phenotype appears to occur only in long germ embryos and that the main role of hunchback appears to be the regionalization of the embryo, it may be appropriate to revive an alternative name for the class of gap genes, namely 'cardinal genes'.

Segmentation genes in insects are required for generating the subdivisions of the early embryo. We describe here a new member of the gap family of segmentation genes in the flour beetle Tribolium, mille-pattes (mlpt). mlpt knockdown leads to transformation of the abdominal segments into thoracic segments, providing embryos with up to ten pairs of legs. We show that there are crossregulatory interactions between mlpt and the known gap genes in Tribolium, suggesting that mlpt is itself a gap gene. The mlpt gene reveals an unusual structure, as it encodes a polycistronic mRNA that codes for four peptides. mlpt appears to be the prototype of this previously unknown gene structure in eukaryotes, as we find homologous genes with the same polycistronic arrangement in other insect genomes as well.