Evolutionary comparisons frequently pinpoint crucial parts of a protein but, even within coding regions, nucleotides can do more than determine amino acid sequence. One highly conserved feature of the Wilms' tumour suppressor gene, WT1, is the potential, following alternative pre-mRNA splicing, to insert three amino acids (KTS) between the third and fourth zinc fingers. The nucleotides at this position simultaneously define amino acids and the alternative splice site. At the protein level this insertion influences DNA binding affinity and specificity, protein-protein interactions and subnuclear localization. Mutations within the +/-KTS splice junction lead to severe urogenital developmental abnormalities such as Frasier syndrome, indicating that the isoform ratio is critical for wild-type function. Using a series of site-directed mutations in both the genomic and cDNA context, the nucleotide-amino acid relationship was investigated. Mutational analysis within the cDNA suggests that the precise amino acids inserted may not be critical, but rather the disruption of the zinc finger structure alone may be sufficient to generate proteins with different in vitro properties. However, analysis within the genomic context suggests that the precise structure of the splice junction is crucial in retaining the balance between the isoforms, and this may account for the high nucleo-tide conservation of this unusual gene structure from fish to mammals.

Wilms' tumor is a childhood kidney tumor that is a striking example of the way that cancer may arise through development gone awry. A proportion of these tumors develop as a result of the loss of function mutations in the Wilms' tumor suppressor gene, WT1. Inherited mutations in the WT1 gene can lead to childhood kidney cancer, severe gonadal dysplasia, and life-threatening hypertension. Knockouts show that the gene is essential for the early stages of kidney and gonad formation. These tissues are completely absent in null mice. The WT1 gene encodes numerous protein isoforms, all of which share four zinc fingers. There is a large body of evidence supporting the notion that WT1 is a transcription factor, particularly a transcriptional repressor. Recently, however, we obtained evidence that WT1 colocalizes and is physically associated with splice factors. What is more, one alternative splice isoform of WT1 containing three amino acids, Lys-Thr-Ser (KTS; inserted between zinc fingers 3 and 4) is preferentially associated with splice factors, whereas the other alternative splice version, lacking these three amino acids, preferentially associates with the transcriptional apparatus. Both genetic and evolutionary considerations suggest that these two different forms of the protein have different functions. We will discuss recent evidence to further implicate WT1 in splicing. Our results raise the possibility that regulation of splicing is a crucial factor in the development of the genitourinary system, and that tumors may arise through aberrant splicing. To pursue the regulation and function of WT1 in whole animals, we have been introducing the human gene and large flanking regions cloned in yeast artificial chromosomes directly into mice. These studies have allowed us to dissect the function of WT1 at late as well as at early stages in organogenesis and to identify new sites and surprising new potential functions for the gene.

WT1 is essential for normal kidney development, and genetic alterations are associated with Wilms' tumor, Denys Drash (DDS), and Frasier syndromes. Although generally considered a transcription factor this study has revealed that WT1 interacts with an essential splicing factor, U2AF65, and associates with the splicing machinery. WT1 is alternatively spliced and isoforms that include three amino acids, KTS, show stronger interaction with U2AF65 in vitro and better colocalization with splicing factors in vivo. Interestingly a mutation associated with DDS enhanced both -KTS WT1 binding to U2AF65 and splicing-factor colocalization. These data illustrate the functional importance of WT1 isoforms and suggest that WT1 plays a role in pre-mRNA splicing.

This is a review of recent work based on an in vitro model which has allowed us to investigate the postnatal maturation of renal epithelial cells. Renal proximal tubule cells from 8- to 40-day-old Sprague-Dawley rats were studied after 48 h of primary culture. The regulation of intracellular pH (pH(i)) was measured by quantitative fluorescence microscopy using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Recordings were made under basal conditions and after imposing a cytoplasmic alkalosis or acidosis using 15 mM ammonium salt. The ability of the cells to recover from both acidosis and alkalosis improved during post-natal maturation. The improvement in recovery from intracellular acidosis could be entirely accounted for by an increase in the rate of Na+/H+ exchange. The capacity for Na+/H+ exchange was independent of the cellular growth rate, but depended on cellular differentiation. A developmental increase in the activity of Cl-/HCO3- exchange between 12 and 14 days of age was also demonstrated. No developmental change was seen in either steady-state pH(i) (7.27-7.35) or in cytoplasmic buffer capacity (37.6-44.4 mM/pH(i)). The characteristics of transporter maturation revealed by these experiments are very similar to those observed in isolated perfused proximal tubules of developing rabbits.