Foxe3 haploinsufficiency in mice: a model for Peters' anomaly.

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.

Novel anterior segment phenotypes resulting from forkhead gene alterations: evidence for cross-species conservation of function.

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.

New, clinically more relevant model for nerve root injury in the rat.

STUDY DESIGN: Exposure to nucleus pulposus and displacement of intraspinal nervous structures with assessment of spontaneous behavioral changes in rats. OBJECTIVE: To develop a controlled, experimental model for nerve root injury. SUMMARY OF BACKGROUND DATA: There are a number of experimental models presented for studies on radiculopathies. One frequently used model is based on exposure to nucleus pulposus and displacement of the dorsal root ganglion (DRG). However, it is clinically more common that the nerve roots are displaced/compressed than the DRG. In this study, we developed a model for displacement of the nerve root by modifying the DRG model. METHODS: After removing the left L3-L4 facet joint, the underlying disc was punctured, and the L4 nerve root was displaced laterally by an injection needle (n = 10). In sham experiments, the same procedure was performed without disc puncture and displacement (n = 10). In 10 rats, the left L4-L5 facet joint was removed. The underlying disc was punctured and the L4 DRG was displaced medially by an injection needle. Assessment of spontaneous behavioral changes was performed on days 1, 3, 7, 14, and 21, postsurgery. RESULTS: There was a clear increase in duration of the behavior "unloading of the paw" after displacement of the DRG that was most pronounced on day 1 and then gradually declined. There was a similar pattern for this behavior induced by nerve root displacement, although the duration was higher than that for the DRG displacement. No apparent trends in behavioral changes were observed for the other behaviors studied. CONCLUSION: Displacement of the nerve root induced more changes in the pain behavior than displacement of the DRG, but only for the behavior unloading of the paw. Because nerve root injury is more common than DRG injury, this model may be more clinically relevant than the DRG model. LEVEL OF EVIDENCE: N/A.

Persistent FoxE3 expression blocks cytoskeletal remodeling and organelle degradation during lens fiber differentiation.

PURPOSE: The anterior hemisphere of the lens is covered by an epithelial monolayer that acts as the stem cell population for lens fiber progenitors. Foxe3, a forkhead transcription factor, is essential for proliferation and survival of the epithelial cells, and cessation of Foxe3 expression at the lens equator coincides with the cell cycle arrest that marks initiation of fiber differentiation. In this study, the consequences of persistent Foxe3 expression during fiber differentiation was investigated. METHODS: The alpha-A-crystallin (Cryaa) promoter was used to drive transgenic expression of Foxe3 in murine differentiating lens fibers. RESULTS: Transgenic mice have a dramatically disturbed lens histology and grave cataracts. Microarray transcript profiling showed an increase of mRNAs normally enriched in epithelial cells, consistent with an epithelialization of the transgenic fibers. Some aspects of fiber differentiation were unaffected, such as the expression of alpha- and beta-crystallins and aquaporins, whereas cytoskeletal remodeling, cell adhesion, organelle degradation, and antimitotic signaling were compromised. CONCLUSIONS: Proper inactivation of FoxE3 expression at the lens equator is important for many aspects of fiber differentiation, and persistent expression leads to a partial epithelialization of fiber cells, with severe consequences for lens function.

Foxe3 is required for morphogenesis and differentiation of the anterior segment of the eye and is sensitive to Pax6 gene dosage.

The dysgenetic lens (dyl) mouse mutant has mutations in Foxe3, which inactivate DNA binding by the encoded forkhead transcription factor. Here we confirm, by targeted inactivation, that Foxe3 mutations are responsible for the dyl phenotype, which include loss of lens epithelium; a small, cataractic lens; and failure of the lens to detach from the surface ectoderm. In contrast to a recent report of targeted Foxe3, we found no phenotypic difference between dyl and Foxe3(-/-) mutants when congenic strains were compared, and thus nothing that argues against Foxe3(dyl) being a null allele. In addition to the lens, most tissues of the anterior segment-iris, cornea, ciliary body and trabecular meshwork-are malformed or show differentiation defects. Many of these abnormalities, such as irido-corneal and irido-lenticular adherences, are present in a less severe form in mice heterozygous for the Foxe3 mutation, in spite of these having an intact lens epithelium. Early Foxe3 expression is highly sensitive to a halved Pax6 gene dosage and there is a striking phenotypic similarity between Pax6 and Foxe3 mutants. We therefore propose that many of the ocular malformations associated with Pax6 haploinsufficiency are consequences of a reduced expression of Foxe3.