In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras tm⌬4A/؉ mice produced viable K-ras tm⌬4A/tm⌬4A offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras tm⌬4A/tm⌬4A mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola ؋ C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H-and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.

The Wilms tumor-suppressor gene, WT1, plays a key role in urogenital development, and WT1 dysfunction is implicated in both neoplastic (Wilms tumor, mesothelioma, leukemias, and breast cancer) and nonneoplastic (glomerulosclerosis) disease. The analysis of diseases linked specifically with WT1 mutations, such as Denys-Drash syndrome (DDS), can provide valuable insight concerning the role of WT1 in development and disease. DDS is a rare childhood disease characterized by a nephropathy involving mesangial sclerosis, XY pseudohermaphroditism, and͞or Wilms tumor (WT). DDS patients are constitutionally heterozygous for exonic point mutations in WT1, which include mutations predicted to truncate the protein within the Cterminal zinc finger (ZF) region. We report that heterozygosity for a targeted murine Wt1 allele, Wt1 tmT396 , which truncates ZF3 at codon 396, induces mesangial sclerosis characteristic of DDS in adult heterozygous and chimeric mice. Male genital defects also were evident and there was a single case of Wilms tumor in which the transcript of the nontargeted allele showed an exon 9 skipping event, implying a causal link between Wt1 dysfunction and Wilms tumorigenesis in mice. However, the mutant WT1 tmT396 protein accounted for only 5% of WT1 in both heterozygous embryonic stem cells and the WT. This has implications regarding the mechanism by which the mutant allele exerts its effect.WT1 is expressed at high levels in those mesodermally derived tissues that experience mesenchymal-epithelial transition during development, including the genital ridge and developing mesothelium, kidney, and gonads (1, 2). WT1 expression is linked with podocyte differentiation during nephrogenesis, but continues to be expressed in adult podocytes, gonads (Sertoli and granulosa cells), uterus (myometrium), spleen (stromal cells and capsule), and mesothelial cells that line the body cavities and visceral organs. Thus, WT1 has a role in both homeostasis and development. The critical role for WT1 in urogenital development is underlined by the failure of kidney and gonadal development in mice homozygous for a Wt1 null mutation (3). WT1 dysfunction also is implicated in the etiology of certain Wilms tumors (WTs) that derive from the metanephric blastemal cells that should differentiate normally into the epithelial and mesenchymal components of the kidney (4).WT1 encodes a nuclear protein with structural motifs characteristic of transcription factors, including an N-terminal glutamine͞proline-rich transregulatory domain and a Cterminal domain with four Kruppel-type Cys 2 -His 2 zinc fingers (ZFs) (ZF1-4) that bind DNA and RNA and are involved in nuclear localization (5-7). WT1 potentially encodes 16 protein isoforms as a result of RNA editing as well as alternative splicing and translation initiation (8). There are two alternatively spliced exons: ASI and ASII. The first is 17 aa long and constitutes exon 5. ASII results from variation in the splice donor site used at the end of exon 9 and inserts 3 aa (lysine-threonine-s...