Human Molecular Genetics Advance Access published online on November 25, 2003
Human Molecular Genetics, doi:10.1093/hmg/ddh015
© 2003 by Oxford University Press
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1 Dept. Anatomy, Edinburgh University, Edinburgh, EH8 9AG, UK
* To whom correspondence should be addressed. E-mail: Nick.Hastie{at}hgu.mrc.ac.uk.
Wt1 is a tumour suppressor gene, mutation of which is a cause of Wilms' tumour, a childhood renal nephroblastoma. Wt1 is expressed in a rich pattern during renal development suggesting that it acts at three stages; determination of the kidney area, the differentiation of nephrons, and maturation of glomeruli. Wt1-/- mice confirm that Wt1 is essential for the inception of kidney development; cells that ought to form kidneys die by apoptosis instead. Specific human WT1 mutations cause defects of glomerular maturation (Denys-Drash and Frasier syndromes), providing circumstantial evidence for action of Wt1 during glomerular maturation. There is, however, no genetic evidence for a function during nephron differentiation because this stage never reached in Wt1-/- mice. We have therefore developed a novel technique, based on small interfering RNA (siRNA), to repress the expression of Wt1 and other specific genes at different stages of kidney development in culture. We find that early repression of Wt1 phenocopies the Wt1-/- mouse, but later repression prevents cells differentiating into nephrons and causes them instead to proliferate abnormally, possibly mimicking aspects of Wilms' tumour. In line with established hypotheses about genetic pathways that control kidney development, we find that repressing Pax2 using siRNAs represses Wt1 expression and blocks both bud growth and nephron differentiation, but that repressing Wnt4 blocks nephron differentiation without affecting Wt1 expression. As well as illuminating previously inaccessible aspects of Wt1 biology, our results suggest that siRNA in organ culture will be a powerful method for analysing other developmental pathways and testing the effects of stage-specific loss of tumour suppressor genes.
Article
Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation
2 Comparative and Developmental Genetics, MRC Human Genetics Unit, Western General Hospital, Crewe Rd., Edinburgh EH4 2XU, United Kingdom; Centre for Research in Biomedicine, University of the West of England, Bristol BS16 1QY, UK
3 Comparative and Developmental Genetics, MRC Human Genetics Unit, Western General Hospital, Crewe Rd., Edinburgh EH4 2XU, United Kingdom
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