Wnt signalling in the hydrozoan Hydractinia echinata
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Canonical Wnt signalling controls many developmental processes in metazoans including primary axis formation in embryonic development and stem cell decision making. The evolutionary history of Wnt signalling and its ancestral role have been of great interest but are not well understood. During my study I examined Wnt signalling in Hydractinia echinata, a member of the basal metazoan phylum Cnidaria. To date, only two Wnt ligands had been described in this animal, Wnt3 and Wnt5A. However, it was expected that additional Wnt genes should be encoded by this animal's genome, as past studies carried out in other cnidarians revealed genes belonging to nearly all known Wnt subfamilies. I identified 9 additional Wnt genes in Hydractinia and analysed their coding sequences and their expression pattern throughout the entire life cycle of this animal. Phylogenetic analysis of all Hydractinia predicted Wnt proteins, together with other published and unpublished sequences from cnidarians and bilaterians, was performed by Dr. Paulyn Cartwright. The resulting phylogenetic trees contradict the widely accepted hypothesis of pre-bilaterian Wnt subfamily divergence. Our data is consistent with lineage-specific ramification of a small number of ancestral Wnt homologues. In situ hybridisation of the novel Hydractinia genes revealed non-conserved expression pattern of Wnt genes even within the Hydrozoa. I identified three genes as likely candidates to be associated with Hydractinia stem cell functioning. The genes, Wnt2, 11B and -16 were the only ones that were expressed in the correct anatomical region, the stolons. Furthermore, these genes were also the only ones whose expression was altered following ectopic activation of canonical Wnt signalling, suggesting that they might be functioning either in positive or negative feedback loops. Finally, I have established new protocols for analysing and sorting Hydractinia cell suspensions by flow cytometry and fluorescence activated cell sorting (FACS). These protocols open up new directions of research and enable qualitative and quantitative analyses of specific genetic manipulations in Hydractinia.