Evolution of the insect neuropeptidergic system: function of the prohormone convertases PC1/3 and PC2 in the beetle Tribolium castaneum
Final Report Abstract
Hormonal signalling through neuropeptides is used by vertebrates such as humans as well as by invertebrates like insects to regulate a range of biological processes. The maturation of neuropeptides requires evolutionary conserved processing enzymes like prohormone convertases (PC1/3 and PC2) and carboxypeptidases (CPE and CPD/Svr). Insects can serve models to understand molecular principles. Furthermore, insects are extremely diverse and different species use different principles of development, growth, and reproduction which all are processes that are under endocrine control. The fly Drosophila melanogaster is the best studied insect model for neuropeptide signalling and processing. However, the neuropeptidergic system of the fly is rather untypical for insects as flies have lost the otherwise highly conserved processing enzymes PC1/3 and CPE. Therefore, we studied function and expression of these genes in another insect model, the beetle Tribolium castaneum which has a more conservative genomic complement of neuroendocrine processing genes. First, we analysed shared and individual functions of the paralogous gene pairs PC1/3 and PC2 as well as CPE and CPD/svr. Interestingly we found that the genes that were lost in Drosophila have essential functions in Tribolium. RNAi-knockdown of PC1/3 at the larval stage leads to an inhibition of larval growth and the injected animals do not develop into pupae. Eggs from females treated with RNAi targeting CPE, the carboxypeptidase that is lost in Drosophila, die during embryogenesis. This contrasts with knockdown of Tribolium CPD/svr, which does not cause any severe phenotype. These finding show that in this other insect taxon, there is no redundancy of gene function of neuroendocrine pathway genes that were lost in flies. Focussing on the Tribolium prohormone convertases PC2 and PC1/3 we further asked how they differ in terms of cell specific expression and neuropeptide targets. To approach this question, we have used CRISPR-Cas9 induced homology directed repair to create two bicistronic reporter lines. A fluorescent reporter was attached to the 3’ end of the coding sequence but separated by the self-cleaving 2A-peptide to produce two independent proteins. Although in Tribolium insertion of large fragments using homology directed repair still suffers from a low efficiency we were successful at creating reporter lines for both genes. We can now visualize expression within the nervous system as well as in other organs as for example the gut and detect co-expression of the prohormone convertases with specific neuropeptides. We also aimed to identify transcription factors that regulate the expression of neuropeptide pathway genes as key markers for peptidergic cells, or more broadly, regulate processes that are influenced by different hormonal systems. Whilst with our methods we could not identify direct regulators of the neuropeptide pathway we identified pdm3 as factor that limits the production of eggs in females through the interaction with ecdysteroid and juevenile hormone metabolism. In conclusion we could successfully resolve the essential functional roles of conserved neuroendocrine pathway genes and successfully create two imaging lines that enable ongoing work on neuroendocrine specification and on cell-type specific function of prohormone convertases. We could also for the first time show that pdm3 influences hormone metabolism to limit female fertility.
