Investigating the Hes/Hey and odd family genes for roles in sequential segment patterning in the red flour beetle, Tribolium castaneum

Body segmentation is a defining characteristic of arthropods, however, the mechanism by which segments are patterned during development has diverged greatly during evolution. The ancestral mode of patterning, in which segments are added sequentially to the posterior of the embryo during axial elongation, relies on a clock and wavefront mechanism that remains poorly understood and variable in nature between different species. Using the sequentially segmented beetle, Tribolium castaneum, I attempt to address some of the underlying complexities of this system and improve our understanding of the evolutionary processes responsible for this diversity in patterning mechanisms across arthropods. Driven by recent data from our lab that question the existing Tribolium segmentation clock circuit, I investigate candidates from the Hes/Hey and odd-related gene families for possible roles in primary segment patterning. Phylogenetic analyses demonstrate a strong potential for redundancy and developmental system drift within both gene families and provide evidence for concerted evolution among the arthropod odd-family genes. I offer a revised understanding of Tribolium odd-family gene expression patterns which, while inconsistent with roles in primary segment patterning, suggest Tc-sister-of-bowel and Tc-drumstick may be involved in the late segmentation network. A screen of Tribolium Hes/Hey gene expression identifies a novel segmentation clock candidate, Tcextramacrochaetae (Tc-emc), while multiplex in situ hybridisations provide evidence linking oscillations of Tc-hairy, Tc-deadpan and Tc-emc to genes from the primary clock circuit (Tc-even-skipped, Tc-odd-skipped, Tc-runt). Although segmentation roles for Tcdeadpan were not identified in this study, knockdown of Tc-hairy in Tc-odd-null mutants indicates some redundancy in thoracic segment patterning, while Tc-emc knockdowns generate phenotypes consistent with a key role in abdominal segment patterning. As these genes are commonly associated with Notch signalling, these findings renew the discussion of Notch function in Tribolium segmentation while also highlighting changes to the network that occur across different stages of segment addition.