Clinical anophthalmia and microphthalmia (absent or small eyes) are significant human birth defects, affecting 0.3 and 1 2 per 10,000 children, respectively (Kallen et al. 1996), and are important causes of congenital blindness. These malformations are thought to arise from an inherent failure in optic vesicle growth or lens induction (DukeElder 1964). In spite of remarkably deep evolutionary conservation among patterning genes and pathways in the optic cup, no molecular mechanism has been found for this disorder. The majority of cases are sporadic, although some pedigrees show simple Mendelian inheritance (Kohn et al. 1988). PAX6 gene mutations have been identified in one case of anophthalmia (Glaser et al. 1594). Teratogenic etiologies, polygenic factors or geneenvironment interactions have also been suggested. However, the small size of affected families, and the possibility of heterogeneity and nongenetic factors make it difficult to perform conventional linkage studies. Likewise, the regression of eyes in numerous fossorial (subteranean) and troglobific (cavedwelling) taxa is a fascinating macroevolutionary problem, which is difficult to approach experimentally. Eye regression in vertebrates may involve relatively few genes and may occur rapidly, even within a single species (Behrens et al. 1997). A mouse model, the eyeless ZRDCT inbred strain, was first described many years ago (Chase and Chase, 19423. It is phenotypically and genetically similar to human clinical anophthalmia. Affected mice lack eyes and optic tracts, have attenuated brain visual centers and primary circadian rhythm disturbances. Although F1 mice are normal, heterozygous embryos are more sensitive to teratogeninduced eye defects than other laboratory mice (Beck, 1963). On the basis of breeding experiments, Chase (1944) proposed that the eyeless trait is oligogenic, involving a synthetic interaction between a single major recessive factor (eyl ) and one or a few modifier loci (ey2). In preliminary studies, we discovered that eyl is a point mutation in the Rx/rax rednal homeobox gene. Rx/rax is conserved among bilateral metazoans; is specifically expressed in the anterior neural fold, hypothalamus, optic cup and neurorefina, beginning at day E7.5 of mouse development; and has the potential to promote neural and pigmented retina formation when ectopically expressed in frog anterior blastomeres (Mathers et al. 1997, Furukawa et al. 1997). However, the molecular role, downstream targets and interacting proteins of this major patterning gene for eye development are unknown. The mutation, a M1OL codon substitution, is hypomorphic in comparison to a null (knockout) Rx/rax allele, which causes severe CNS defects and is lethal (Mathers et al. 1997). The M1OL mutation may thus alter translational initiation or disrupt a previously unknown functional domain at the arninoterminus. Homozygosity for the M1OL allele is necessary but not sufficient to produce the eyeless phenotype The ey2 modifier gene(s) are likely to encode proteins that interact directly with Rx/rax or function in a common pathway for optic cup morphogenesis. Mapping of ey2 is thus conceptually similar to enhancer screens routinely performed in Drosophila. In this proposal, we aim to: (1) characterize the MlOL mutation in detail; (2) find target genes and interacting proteins for Rx/rax using ZR about mice and in vitro assays (3) map, clone and characterize ey2 loci in the mouse genome; (4) compare Rx coding and regulatory sequences from the blind mole rat Spalax erhenbergi to Rx genes of mice, hamsters and other sighted Muridae; (5) collect and screen human anophthalmia cases for Rx mutations; and ( about) test the role of Rx mutahons in two other model organisms, the F33 anophthalmic rat and the eyeless (e/e) axolotl Ambystoma mexicanum. These studies should provide valuable new informahon regarding Rx/rax function, opUc cup formahon and retina1 histogenesis, ontogeny of the hypothalamic circadian clock, the efiology of human clinical anophthalmia and rnicrophthalmia, polygenic developmental disorders and geneenvironrnent interachons, and the evolubon of eyelessness in nature.
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