Mice lacking the 68-amino-acid, mammal-specific N-terminal extension of WT1 develop normally and are fertile

Abstract

Mutations in the Wilms' tumor 1 gene, WT1, cause pediatric nephroblastoma and the severe genitourinary disorders of Frasier and Denys-Drash syndromes. High levels of WT1 expression are found in the developing kidney, uterus, and testis--consistent with this finding, the WT1 knockout mouse demonstrates that WT1 is essential for normal genitourinary development. The WT1 gene encodes multiple isoforms of a zinc finger-containing protein by a combination of alternative splicing and alternative translation initiation. The use of an upstream, alternative CUG translation initiation codon specific to mammals results in the production of WT1 protein isoforms with a 68-amino-acid N-terminal extension. To determine the function in vivo of mammal-specific WT1 isoforms containing this extension, gene targeting was employed to introduce a subtle mutation into the WT1 gene. Homozygous mutant mice show a specific absence of the CUG-initiated WT1 isoforms yet develop normally to adulthood and are fertile. Detailed histological analysis revealed normal development of the genitourinary system.

FIG. 1.

(A) CLUSTAL W alignment of mouse and human WT1 N-terminal extensions originating from the upstream CUG initiator. Stars indicate conserved amino acids. WT1 proteins initiated by the classical AUG initiator begin with the amino acids MGSD. (B) DOTPLOT comparison of the human WT1 genomic region from −250 bp with those of Fugu, Xenopus, and chick. Sequence conservation begins at the ATG in all comparisons. The gap in all three alignments corresponds to the polyproline tract in WT1 exon 1, which is only found in mammals.

FIG. 2.

Gene targeting strategy for elimination of WT* isoforms. (A) Diagram of WT1 genomic region around exon 1. P, PstI; R, EcoRV; Probe 2, internal ∼500-bp XhoI-EcoRI segment; Probe 1, external ∼800-bp XbaI-PstI segment. (B) Targeting vector. HYGRO-tk, hygromycin-HSVtk fusion gene. (C) Final targeted WT1 allele present in ES cells and mice. (D) DOTPLOT comparison of human and mouse WT1 intron 1 sequences showing the position of the residual loxP site.

FIG. 3.

Creation of mice lacking WT* isoforms. (A) Southern blot of PstI-restricted tail DNA derived from heterozygous intercross showing results for wild-type (+/+), heterozygous mutant (+/−), and homozygous mutant (−/−) littermates. Southern blotting was performed with probe 2. (B) Western blot of fetal kidney nuclear protein separated by SDS-PAGE on a 10% polyacrylamide gel and probed with anti-WT1 monoclonal antibody H2. 66-kDa and 45-kDa size markers are indicated.

FIG. 4.

Hematoxylin- and eosin-stained sections of wild type (+/+) and homozygous mutant (−/−) kidney, testis, ovary, and uterus from ∼3-month-old littermates. Kidney, testis, and ovary, 20× objective; uterus, 10× objective.