The Wilms' tumour gene, WT1, in the development of the mouse embryo and its kidney

Wilms' tumour (WT), a renal tumour of early childhood, arises as a result of uncontrolled proliferation in the stem cells of the kidney due to an anomaly in their developmental pathway. A strong genetic element is known to be involved in its genesis, with more than one gene being implicated. The first of these, WT1, has recently been isolated and shown to be expressed in a specific pattern in both the developing human kidney and the tumour itself. The purpose of the work presented in this thesis was to examine the role that this gene plays in mouse embryogenesis, a system that lends itself to experimentation more readily than the human. Here, the expression of the mouse homologue of WT1 was examined in three complimentary systems; the developing mouse embryo, the kidney as it formed both in vivo and in vitro and finally, in a possible mouse model for WT.A comprehensive study of the expression pattern of WT1 during development was undertaken, using in situ mRNA hybridisation. Expression was first apparent in a small area of the lateral mesoderm in the 9 day embryo. Within 12 hours this had increased markedly, with both the lining of the whole coelomic cavity and the early urogenital ridge being labelled. As development proceeded, expression was initiated in a limited set of tissues which included the metanephros, the mesothelium, the gonads, the spleen, the developing body -wall musculature, the spinal cord and the brain. Expression was present in 15 day embryos but markedly reduced by 19 days with labelling being restricted to the kidney.The expression of WT1 in cultured kidney rudiments was then examined and found to be consistent with that observed in vivo. The gene was expressed at a low level in condensed mesenchyme, with a much higher level being detected in the developing renal corpuscle. Using the transfilter system of organ culture, it was shown that WT1 was expressed in the cap of condensed metanephric mesenchyme prior to induction and that this level increased after induction, a result confirmed using very early 11 day embryos.The mouse model of WT involved placing embryonic kidneys under the capsule of adult kidney or testis from 3 strains of mice, with the growths being recovered after a defined period. In contradiction of the published data, two distinct morphologies were observed, neither of which resembled classic WT. Both types of morphology were examined with WTI and antibodies to developmental markers and it was found that the degree of differentiation was considerably greater in the transplanted tissue than would be expected for WT.The results as a whole substantiate the importance of WT1 in tissue undergoing a mesenchymeto- epithelial transition, 'particularly during nephrogenesis. The functional significance of WT1 transcription in tissues outwith this group is less clear. Skeletal muscle is sometimes observed as a component of WT and it is intriguing that the gene is expressed transiently during a stage of muscle development. The data from the mouse points to a key role for WT1 in the initiation of the cascade of differentiation necessary for normal kidney development. The possible reasons for the failure of the mouse model to produce the Wilms' phenotype are discussed

Hitch-hiking from HRAS1 to the WAGR locus with CMGT markers.

The clinical association of Wilms' tumour with aniridia, genitourinary abnormalities and mental retardation (WAGR syndrome) is characterised cytogenetically by variable length, constitutional deletion of the short arm of chromosome 11, which always includes at least part of band 11p13. HRAS1-selected chromosome mediated gene transfer (CMGT) generated a transformant, E65-6, in which the only human genes retained map either to band 11p13 or, with HRAS1, in the region 11p15.4-pter. Human recombinants isolated from E65-6 were mapped to a panel of five WAGR deletion hybrids and two clinically related translocations. We show that E65-6 is enriched congruent to 400-fold for 11p15.4-pter markers and congruent to 200-fold for 11p13 markers. 'Hitch-hiking' from HRAS1 with CMGT markers has allowed us to define seven discrete intervals which subtend band 11p13. Both associated translocations co-locate within the smallest region of overlap for the WAGR locus, which has been redefined by identifying a new interval closer than FSHB