The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function. Homozygous mutations of WT1 can be found in approximately 15% of Wilms' tumors. Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males. A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene. Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors. Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide. Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele. In contrast to Denys-Drash syndrome, no mutant protein is produced. The splice mutation leads to an imbalance of WT1 isoforms in vivo , as detected by RT-PCR on streak gonadal tissue. Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform.

We have identified a novel subfamily of mammalian hairy/Enhancer of split (E(spl))-related basic helix-loop-helix (bHLH) genes together with a putative Drosophila homologue. While hairy/E(spl) proteins are characterized by an invariant proline residue in the basic domain and a carboxyterminal groucho-binding WRPW motif, our genes encode a carboxyterminal KPYRPWG sequence and were thus designated as Hey genes (Hairy/E(spl)-related with YRPW motif). Furthermore, they bear a unique C-terminal TE(I/V)GAF motif and the characteristic proline is changed in all Hey family members to glycine. RNA in situ hybridization analysis revealed specific expression of Hey1 during development of the nervous system, the somites, the heart and the craniofacial region. Hey2 is similarly expressed in the somites whereas it shows a complementary expression in the heart, the craniofacial region and the nervous system. The diversity of expression patterns implies unique functions in neurogenesis, somitogenesis and organogenesis.

Vertebrate somitogenesis comprises the generation of a temporal periodicity, the establishment of anteroposterior compartment identity, and the translation of the temporal periodicity into the metameric pattern of somites. Molecular players at each of these steps are beginning to be identified. Especially, members of the Notch signaling cascade appear to be involved in setting up the somitogenesis clock and subsequent events. We had previously demonstrated specific expression of the mHey1 and mHey2 basic helix-loop-helix (bHLH) factors during somitogenesis. Here we show that perturbed Notch signaling in Dll1 and Notch1 knockout mutants affects this expression in the presomitic mesoderm (PSM) and the somites. In the caudal PSM, however, mHey2 expression is maintained and thus is likely to be independent of Notch signaling. Furthermore, we analysed the dynamic expression of the respective chicken c-Hey1 and c-Hey2 genes during somitogenesis. Not only is c-Hey2 rhythmically expressed across the chicken presomitic mesoderm like c-hairy1, but its transcription is similarly independent of de novo protein synthesis. In contrast, the dynamic expression of c-Hey1 is restricted to the anterior segmental plate. Both c-Hey genes are coexpressed with c-hairy1 in the posterior somite half. Further in vitro and in vivo interaction assays demonstrated direct homo- and heterodimerisation between these hairy-related bHLH proteins, suggesting a combinatorial action in both the generation of a temporal periodicity and the anterior-posterior somite compartmentalisation.

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