Dact genes in mouse kidney development

Abstract

Mammalian kidney development proceeds through a series of interactions among metanephric mesenchyme, ureteric bud, extracellular matrix, growth factors and various other signalling molecules. These complex, but well integrated, networks control cell proliferation, differentiation, migration and survival and thus orchestrate kidney development. More and more molecules in these networks have been identified since the past few years. Therefore, it is crucial to explore their functions and the mechanisms by which they work to fill the research gaps. DACTs have recently been reported as pathway-specific regulators in WNT signalling, known to be important in kidney development, but their expressions and functions in mammalian kidneys are yet to be elucidated. The aims of this thesis are to describe the expression patterns of these two new genes, Dact1 and Dact2, in mouse embryonic kidneys and to further investigate their functional roles by applying RNAi to cell culture-based models. The first goal of this thesis is to establish the temporospatial expression patterns of Dact1 and Dact2 in kidneys by conventional end-point RT-PCR, quantitative real time PCR and RNA in situ hybridisation. Based on the expression patterns and preliminary observations in cell cultures, I hypothesize that Dact1 regulates cell proliferation while Dact2 governs cell migration. Experiments including siRNA transfection, BrdU proliferation assay, generation of stable cell lines expressing Dact2 shRNA and wound assay, are designed to test these hypotheses and the results may offer implications of functional roles of both molecules in kidney development. The results obtained are as follows. • Dact1 and Dact2 show different temporal expression patterns in mouse kidneys. In adult kidneys, Dact1 is greatly downregulated while Dact2 is still expressed at a comparable level to that at E14.5. • Dact1 is initially expressed in metanephric mesenchyme and, as development proceeds, shows a characteristic pattern of renal stroma whilst Dact2 is exclusively expressed in ureteric buds throughout embryonic stages. • Dact1 and Dact2 expressions are regulated by known regulators of kidney development including retinoic acid and chlorate. • Silencing of Dact1 facilitates proliferation of embryonic cells. • Silencing of Dact2 hinders migration of renal collecting duct cells. Taken together, I have characterised temporospatial expression patterns of Dact1 and Dact2 in kidneys and provided evidences of functional roles of both novel molecules in cell cultures. Based on this thesis, further studies on Dact1 and Dact2 using either in vitro or in vivo mammalian kidney models will offer more insights into their functions and regulations in renal organogenesis.