Identification and functional analysis of pronephric development genes in Xenopus laevis
The formation of the kidney in vertebrates proceeds through a succession of up to three structures, the pronephros, metanephros and mesonephros. This project aimed to identify a suitable target gene involved in the development of the pronephros and study its role in development. By analysing the functional role of a target gene. this thesis ultimately aims to further elucidate the developmental regulation of pronephric organogenesis. The candidate target genes were identified from 3 sources. Firstly. a Xenopus laevis stage 13 cDNA library was screened using a probe made from retinoic acid and activin A treated animal caps. Secondly. four UniGene cluster genes were investigated. after initial analysis indicated they were highly pronephros specific (Personal correspondence Pollet, N). Finally, data mining identified Xenopus homologues of important kidney development genes identified in higher vertebrates or newly identified genes expressed in the pronephros. On the basis of preliminary data, Darmin r and Pod I were taken forward for functional studies. Pod I, a basic-helix-loop-helix transcription factor, was found to be expressed from pronephric initiation with enhanced expression in the pronephros. The expression patterns of Pod I strongly matched that of the mouse homologue, implying a conserved evolutionary role. Pronephros targeted over-expression resulted in the reduction of glomus tissue, with no alteration in pronephric tubule or duct morphology. In addition, targeted knock-down resulted in the absence of cells in the glomus region. Preliminary analysis of Darmin r, a cytosolic non-specific dipeptidase. indicated a role later in pronephric tubule development. Targeted over-expression experiments disrupted tubule morphology and reduced the size of the pronephric anlagen. Initial promising data clearly placed Pod I and Darmin r as important pronephric development genes.