Anophthalmia, or absence of the eye, is found in 1 in 5,000 to 10,000 individuals and is a devastating birth defect because of the resulting visual impairment. Anopththalmia is closely related to microphthalmia (small eyes) and coloboma (failure of the choroid fissure or the optic fissure to close). We have studied a male patient with severe microphthalmia who had a novel chromosome deletion at chromosome 18q22.1. We have implicated one of the deleted genes at 18q22.1, TXNDC10, in eye development. In situ hybridization showed that Txndc10 was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TXNDC10 in 66 patients with anophthalmia or microphthalmia, and found c.260G>A, predicting a missense substitution, p.R39Q, in an unrelated patient with microphthalmia and retinal coloboma. We used antisense morpholinos targeted against the zebrafish Txndc10 orthologue, Zgc110025, to show that the morphant larvae had microphthalmia and coloboma. In-situ hybridization with Pax2 showed increased choroid fissure staining in morphants compared to controls, suggesting that Pax2 dysregulation was involved in the coloboma formation. The morphant retinas had increased anti-histone H3 staining in the peripheral retina and we observed fewer late-differentiating cell types in the morphant retinas. Co-injection of human wild type TXNDC10 mRNA with a morpholino rescued the morphant phenotype, whereas co-injection of human TXNDC10/(p.R39Q) mutant mRNA with morpholino did not. Our results suggest that haploinsufficiency for TXNDC10 perturbs eye development. We propose to continue our research on TXNDC10 in two directions. We hypothesize that Txndc10 may be part of a known pathway for ventral retinal development, and would like to use zebrafish to determine whether Zgc110025 interacts with other known genes that are involved in choroid fissure closure and the formation of the ventral retina, such as Vax1, Vax2 and Shh (Syu). We will use in-situ hybridization, dual injections of antisense morpholinos and mRNA rescue experiments to accomplish this aim. Our purpose is to try to uncover the mechanism whereby Txndc10 affects eye development. Secondly, we propose to make a mouse model of loss of Txndc10 function to examine the eye phenotype in a genetic null model rather than in an animal model with only partial loss of gene function. We hypothesize that loss of Txndc10 function will produce mice with microphthalmia and coloboma, and we will use the resultant knock-out mice to examine eye morphology in a Txndc10 null animal and to perform expression arrays to evaluate for differences in gene expression between Txndc10 null mice and wildtype litter mates. Our ultimate goal is to improve the existing knowledge about the genes that are required for ventral retina formation and choroid fissure closure so that human patients with anophthalmia and related eye defects can be offered improved genetic counseling and testing. PUBLIC HEALTH RELEVANCE: Birth defects affect an estimated 120,000 (1 in 33) babies born in the United States each year, and are the leading case of mortality in the first year of life. Anophthalmia (absent eye), microphthalmia (small eye) and coloboma (failure of the choroid fissure or the optic fissure to close) can result in a significant burden for affected individuals and their families because of the medical and social effects of reduced vision. This application seeks to improve the existing knowledge pertaining to the genetic causes of these eye defects so that patient counseling, management and treatment can be improved.