Comparative studies on the role of Egfr, Wingless and Decapentaplegic signalling in leg development in the red flour beetle Tribolium castaneum

Abstract

Leg development and its underlying genetic and molececular mechanisms are best understood in the prime insect model Drosophila melanogaster and this knowledge can serve as a first guide to study leg development in other arthropod species. The pattering of the proximo-distal axis in the leg is guided by a hierarchic gene cascade and the morphogens Wingless (Wg) and Decapentaplegic (Dpp) are the key factors which control the expression of proximo-distal patterning genes at the next level of the cascade. Beside its function in distal development, wg is also crucial for the allocation of the the limb primordia and specification of ventral cell fate in the leg. The development of the leg in Drosophila melanogaster presents a highly derived mode of arthropod leg development and differs from other insects and arthropod groups, where the limbs are direct outgrowths of the embryonic body wall. Recent work provides evidence against a conservation of the Wg/Dpp leg pattering system. Although the expression pattern of wg is highly conserved in arthropods, functional studies in the insects Oncopeltus and Gryllus could not support a role of wg in leg development of these species. At least, in the coleopteran Tribolium castaneum a role of wg in leg allocation could be proven. Also the role of dpp in leg development has been questioned. The expression of dpp is not conserved between Drosophila and other arthropod species and indicates a non-conserved role of dpp in limb development of arthropods. In the first part of this thesis, I present a functional analysis of wg and dpp in leg development in the red flour beetle Tribolium castaneum. This analysis has been performed via staggered stage-specific embryonic RNAi and different gene expression studies in RNAi embryos. This analysis revealed that the function of wg is similar between Drosophila and Tribolium. Wg is essential in leg allocation and distal leg development during early development as well as during late development in establishment of ventral cell fate in the legs. I n addition, I could demonstrate, that dpp is indeed required for distal leg development in Tribolium but lacks a function in establishment dorsal cell fate in the legs. Despite of these results in Tribolium there is increasing evidence that in other arthropod groups the Wg/Dpp system is not that crucial in leg axis pattering as in Drosophila. Instead EGFR signalling, which in Drosophila is needed in late development for pattering the tarsal region, might have a more decisive role in proximo-distal leg formation in other arthropod species. Therefore, in the second part of my thesis, I present a functional analysis of the Egf receptor, its ligand spitz and the transcription factor pointed in leg development in Tribolium castaneum. This functional analysis revealed, that Egfr signalling fulfills a more complex role in pattering and formation of the legs in Tribolium as it is known in Drosophila. I could demonstrate that the requirement of Egfr signalling is not restricted to the distal part of the leg but also includes pattering and formation of the medial parts of the leg. Interestingly, my results strongly suggest that Egfr signalling in the medial leg is not mediated via pointed as in the distal portion of the leg but a different, not yet identified transcription factor. In summary, in this thesis I could show that the beetle Tribolium castaneum likely represents an intermediate evolutionary state as far as the proximal-distal pattering mechanisms in the legs are concerned. Although the Wg/Dpp system is already the predominant system in leg development in Tribolium, Egfr signalling has an important and more complex role than in Drosophila.