The gap genes encode transcription factors that play a central role in the process of segment patterning in Drosophila. Specifically, they interact to form the well-characterised ‘gap gene network’, which directs the formation of segment boundaries (through regulation of pair-rule genes), and the subsequent diversification of segments (through regulation of Hox genes). Although homologues of the gap genes play important roles in segment patterning in many insects, there is as yet no clear understanding of the network as a whole outside of Drosophila. In particular, the existence of a ‘gap gene gap’ (a region of the axis that expresses no gap genes) in embryos of the beetle Tribolium castaneum may indicate the existence of additional, as yet unidentified gap genes in this species.

In this work, I investigate the hypothesis that the neuroblast timer genes nubbin (nub) and castor (cas) may act as components of the gap gene network in Tribolium. I first utilise Hybridisation Chain Reaction in situ hybridisation to produce a comprehensive description of the dynamics of gap gene expression in Tribolium, concluding that, although the non-canonical gap genes Tc-mille-pattes and Tc-shavenbaby are expressed in the gap gene gap, their role may be distinct from that of the canonical gap genes, and that there are likely to be other, unknown gap genes expressed alongside them. I show that the four genes of the neuroblast timer series (hunchback, Krüppel, nub and cas) are expressed sequentially in the segment addition zone, with the result that nub and cas are expressed in the gap gene gap. Knocking down the expression of nub, but not cas, using RNAi results in weak homeotic transformations of the first abdominal segment towards a thoracic fate. Finally, I show that this phenotype is dramatically increased in severity and penetrance when Tc-nub is knocked down in addition to the trunk gap genes Tc-gt and/or Tc-kni. In triple knockdowns, all abdominal segments are transformed into thoracic segments due to a posterior expansion of the thoracic gap gene Tc- Kr and subsequent loss of expression of the abdominal Hox genes Tc-abdominal A and Tc- Ultrabithorax. These data indicate that Tc-nub, Tc-gt and Tc-kni act redundantly to repress the expression of Tc-Kr in the abdomen, and that both Tc-nub and Tc-kni should therefore be considered as components of the gap gene network in Tribolium.

My work strengthens the hypothesis that the gap gene network may have ancient evolutionary ties to the neuroblast timer network, and promotes a more ‘modular’ view of the gap gene network. I hope that this will inform future studies that aim to unravel the developmental role of the gap genes in sequentially segmenting arthropods, and the evolutionary origins of the gap gene network.