The overall goal of the research proposal is to determine whether altered inductive signaling aroung the developing mouse eye leads to abnormalities in the retina and lens. The small eye (Sey) mouse, which has a mutation in the Pax-6 gene, will be used for this study. Sey heterozygotes have a small eye, but are otherwise grossly normal. The proposed experiments are based on the observation that an ectopic population of putative neuronal crest cells is found around the developing eye in Sey heterozygous embryos. The research proposal will test whether a group of mesencephalic neural crest cells that normally migrates to the frontonasal mass and becomes retinoid-producing mesenchyme is arrested at the eye of Sey heterozygous embryos, and whether this ectopic population of cells is able to produce retinoids or other signaling molecules that alter eye development. The experiments are well-designed and should yield important novel findings concerning eye development. However, some preliminary data would increase enthusiasm for these studies. The first specific aim is to determine whether the ecotopic population of putative neural crest cells around the eye of the sey heterozygotes produces retinoic acid. The mesenchyme surrounding the embryonic eye will be dissected and placed on a monolayer of retinoid responsive indicator cells. This experiment should thus be fairly straight-forward to perform and will allow the applicant to determine whether retinoic acid is produced. If the cells do not produce retinoic acid, howeve, it is not chear in which direction the applicant will proceed or which other signaling molecules will be studied. Preliminary data would help to allay this concern. The second specific aim is to assess the effects of this population of cells ongene expression in the eye and in surrounding tissue. The applicant will use in situ hybridization to determine whether specific genes, such as Pax-6, Notch and FGF-2, are expressed preferentially in Sey heterozygotes. Although these experiments will likely yield interesting data, it may be difficult to show that changes in the expression of a specific gene reflect alterations in retinoic acid signaling. In addition, this specific aim is primarily exploratory in nature. Preliminary data would be particularly helpful here. The third specific aim is to determine if the ectopic presence of putative neural crest cells leads to any apoptotic cell death in or around the developing eye in Sey heterozygotes and homozygotes. The applicant proposes to use TUNEL straining, the presence of bc1-2 and induction of tissue transglutaminase to detect thepresence of apoptosis. The applicant is aware that these experiments could result in variable success with some of the methods used and that this would make interpretation difficult. She then proposes to try other markers for apoptoxis such as DNA fragmentation analysis. If apoptosis does not occur, then it is not clear, in the proposal,how the ectopic cell population would interfere with eye development. The fourth specific aim is to determine whether the ectopic population of cells alters the proliferation of mesenchymal cells or retinal cells. This experiment should be straight-forward to perform and will allow the applicant to determine whether cell proliferation is altered. However, it may be difficult to show that changes in proliferation result directly from changes in gene expression such as Pax-6.
