Åsa Blixt

In the mouse mutant dysgenetic lens (dyl) the lens vesicle fails to separate from the ectoderm, causing a fusion between the lens and the cornea. Lack of a proliferating anterior lens epithelium leads to absence of secondary lens fibers and a dysplastic, cataractic lens. We report the cloning of a gene, FoxE3, encoding a forkhead/winged helix transcription factor, which is expressed in the developing lens from the start of lens placode induction and becomes restricted to the anterior proliferating cells when lens fiber differentiation begins. We show that FoxE3 is colocalized with dyl in the mouse genome, that dyl mice have mutations in the part of FoxE3 encoding the DNA-binding domain, and that these mutations cosegregate with the dyl phenotype. During embryonic development, the primordial lens epithelium is formed in an apparently normal way in dyl mutants. However, instead of the proliferation characteristic of a normal lens epithelium, the posterior of these cells fail to divide and show signs of premature differentiation, whereas the most anterior cells are eliminated by apoptosis. This implies that FoxE3 is essential for closure of the lens vesicle and is a factor that promotes survival and proliferation, while preventing differentiation, in the lens epithelium.

PURPOSE: To evaluate the importance in anterior segment dysgenesis of genetic variation in Foxe3, a gene encoding a forkhead transcription factor specifically expressed in the lens. METHODS: The phenotype of mice heterozygous for a mutation in the DNA-binding domain of Foxe3 was examined from histologic sections, and DNA binding by the encoded protein was investigated by gel-shift assay. FOXE3 from human patients with Peters' anomaly was PCR amplified and sequenced. RESULTS: The dysgenetic lens (dyl) allele of Foxe3 was found to encode a protein unable to bind DNA. Approximately 40% of mice heterozygous for Foxe3(dyl) have corneal and lenticular defects. The phenotype is variable but typically consists of the equivalent of Peters' anomaly in humans, with central corneal opacity, keratolenticular adhesion, and, in some cases, anterior polar cataract. In a small cohort (n = 13) of patients with Peters' anomaly, shown to be normal in the PAX6 locus, one individual was found to be heterozygous for a nonconservative missense mutation in FOXE3. The mutation, which does not occur in 116 chromosomes from a control population, substitutes leucine for arginine 90 at a highly conserved position in the forkhead domain. CONCLUSIONS: Haploinsufficiency of Foxe3 in a mouse model causes anterior segment dysgenesis similar to Peters' anomaly. Although causality could not be shown in the human case, the presence of a rare, nonconservative substitution in FOXE3 of a patient with Peters' anomaly is interesting, in light of the phenotypic similarities with the mutant mice.

Forkhead-related activator 2 (FREAC-2) is a human transcription factor expressed in lung and placenta that binds to cis-elements in several lung-specific genes. We have identified the parts of FREAC-2 responsible for trans-activation and found two functionally redundant activation domains on the C-terminal side of the DNA binding forkhead domain. Activation domain 1 consists of the most C-terminal 23 amino acids of FREAC-2 and contains a sequence motif conserved in an activation domain of another forkhead protein, FREAC-1. Activation domain 2 is built up by three synergistic subdomains in the central part of the FREAC-2 protein. FREAC-2 was shown to interact in vitro with TBP and TFIIB. The target site for FREAC-2 on TBP was localized to the N-terminal repeat in the core domain of TBP. TFIIB binds FREAC-2 close to the cleft between its two globular domains. The part of FREAC-2 that binds TBP was mapped to 21 amino acids in the C-terminal end of the forkhead domain. This sequence is well conserved among forkhead proteins, raising the possibility that interaction with TBP may be a general characteristic of this family of transcription factors. Overexpression of TFIIB potentiates activation by FREAC-2 in a manner dependent on the FREAC-2 activation domains. Nuclear localization of FREAC-2 was found to depend on sequences from both ends of the forkhead domain.

PURPOSE: Mutations in murine and human versions of an ancestrally related gene usually result in similar phenotypes. However, interspecies differences exist, and in the case of two forkhead transcription factor genes (FOXC1 and FOXC2), these differences include corneal or anterior segment phenotypes, respectively. This study was undertaken to determine whether such discrepancies provide an opportunity for identifying novel human-murine ocular phenotypes. METHODS: Four pedigrees with early-onset glaucoma phenotypes secondary to segmental chromosomal duplications or deletions encompassing FOXC1 and 18 individuals from 9 FOXC2 mutation pedigrees underwent detailed ocular phenotyping. Subsequently, mice with mutations in Foxc1 or a related forkhead gene, Foxe3, were assessed for features of the human phenotypes. RESULTS: A significant increase in central corneal thickness was present in affected individuals from the segmental duplication pedigrees compared with their unaffected relatives (mean increase 13%, maximum 35%, P < 0.05). Alterations in corneal thickness were present in mice heterozygous and homozygous for Foxe3 mutations but neither in Foxc1 heterozygotes nor the small human segmental deletion pedigree. Mutations in FOXC2 resulted in ocular anterior segment anomalies. These were more severe and prevalent with mutations involving the forkhead domain. CONCLUSIONS: Normal corneal development is dependent on the precise dose and levels of activity of certain forkhead transcription factors. The altered corneal thickness attributable to increased forkhead gene dosage is particularly important, because it may affect the clinical management of certain glaucoma subtypes and lead to excessive treatment. The FOXC1 and Foxe3 data, taken together with the novel ocular phenotypes of FOXC2 mutations, highlight the remarkable cross-species conservation of function among forkhead genes.