An analysis of wnt signalling molecules in Xenopus pronephros development

The aim of the project was to characterise known wnt signalling molecules during the early pronephros patterning using novel and effective bioassays in Xenopus embryos. Anterior somites have been shown previously to have unique biological activity to induce pronephros formation in Xenopus embryos (Seufert et al., 1999). A molecular approach consisted of analysing the expression of canonical wnts (wnt6, wnt7b, wnt8, wnt9a and wnt9b) and the non-canonical wnts (wnt4, wnt5a, wnt11 and wnt11b) genes in isolated pronephric anlagen and pronephros from stage 12.5 to stage 35 and in anterior and posterior somites. This allowed the identification of potential candidate wnt genes which could act as pronephric inducers. Their potential to induce pronephros was tested in vitro using the Holtfreter sandwich culture pronephrogenesis assay, which consists of unspecified intermediate mesoderm cultured inside two animal caps over-expressing the wnt molecule of interest. Results suggest that the canonical wnt molecules (wnt6 and wnt8) are capable of modifying the intermediate mesoderm leading to formation of somite and neural tissue that in turn, can secondarily induce pronephric tubule formation. By contrast, signals of the non-canonical wnt11 and wnt11b are sufficient to directly specify the intermediate mesoderm to become kidney. In vivo, the role of the canonical wnt6, non-canonical wnt11b and the closely related wnt11 gene was investigated by gain and loss-of-function experiments. Results suggest that mis-expression of these genes disturb the normal formation of the pronephric tubules and suggest that both canonical and non-canonical wnt molecules are required for formation of functional pronephros. This thesis also reports the identification of the novel Xlwnt9a and Xlwnt9b genes, their temporal and spatial expression in both X. laevis and X. tropicalis embryos and dissected adult organs and analyses the effects of Xtwnt9b mis-expression on X. laevis pronephros development