Functional analysis of novel head patterning genes
Final Report Abstract
It has remained unclear what genes are involved in the formation of the head and the brain of insects. Unfortunately, for technical reasons it has been difficult to study this process in the main model organism for insect genetics, the fly Drosophila melanogaster. Therefore, we have been using the red flour beetle Tribolium castaneum as model to study this process. Based on knowledge previously gained in the fly and in vertebrate brain patterning, we had found a number of genes that are involved in beetle head patterning. However, even after screening all the candidate genes we were still missing important players. Therefore, we mined the iBeetle screen for phenotypes affecting the head cuticle. Indeed, we were able to find about nine novel genes required for this process. Unexpectedly, when studying one candidate of head patterning we found that the knockdown of the same gene can lead to different phenotypes in different beetle strains. A similar phenomenon had been known from mutants where the genetic background influences the phenotype. However, this phenomenon had not been described for RNAi experiments so far. Further, we found and analyzed a selection of new head patterning genes: First, we were able to show that the Notch pathway is involved in the formation of the labrum, which is an appendage like structure anterior to the mouth opening. Specifically, it activates cell division, which is required for outgrowth and concomitant patterning. Interestingly, the analysis of the genetic interactions showed that this pathway is at a much more upstream position in the respective network than in other appendages giving insight into the evolution of genetic networks. Second, with Tc-foxQ2 we found a novel gene at the top level of the patterning network. Tc-foxQ2 is required for the expression of Tc-six3, which is the most upstream component of the network known so far. Interestingly, this interaction is mutual such that these two genes form a regulatory module at the top level of head formation. Together, these genes regulate a large number of downstream genes, which are required for subdividing the head into the different substructures. Knockdown of the function of Tc-foxQ2 leads to loss of the labrum and the central complex, which is an important brain structure of insects. In the future, we will study the role of Tc-foxQ2 in central complex formation. Third, the Klingler lab found two genes required for giving the freshly laid egg the proper anteriorposterior polarity. Our joint analysis revealed that when the function of these genes was knocked out, the larvae developed a mirror image abdomen instead of the head. It came as a big surprise that the gene Tc-germ cell-less was required for anterior patterning because this gene is active only at the posterior pole in flies where it is required for germ cell development. The other gene is Tchomeobrain, for which no functional data was known in the fly. We showed that Tc-germ cell-less is required in the mother for expression and probably also the transport of Tc-axin into the developing oocyte. Ultimately, this leads to zygotic expression of a number of anterior genes like Tc-zen1 and Tchomeobrain. Tc-homeobrain acts together with Tc-zen1 to repress the formation of an abdomen at the anterior pole to allow anterior development. We also found that duplication of anterior structures can be initiated by knocking down both WNT signaling and Tc-caudal. In summary, this work revealed two unexpected components of axis formation in insects that would not have been found based on the candidate gene approach.
Publications
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RNAi phenotypes are influenced by the genetic background of the injected strain. BMC Genomics. 2013;14:5
Kitzmann P, Schwirz J, Schmitt-Engel C, Bucher G
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Notch signaling induces cell proliferation in the labrum in a regulatory network different from the thoracic legs. Dev. Biol. 2015;408:164–77
Siemanowski J, Richter T, Dao VA, Bucher G
