Andrea S. Viczian, PhD - US grants

DESCRIPTION (Verbatim from the applicant's abstract): Sight is critically dependent on the development of appropriate eye morphology and cell types. Although many genes involved in eye development have been isolated, the genetic regulatory pathways that control vertebrate retinal cell differentiation remain undetermined. Moreover, the link between gene activity and cell determination is not well understood. This proposal aims to investigate the cellular and molecular mechanisms of eye formation by (1) decifering the functional relationships among the different genes that control eye formation and then (2) characterizing the role of two retinal genes integral to eye development, Chx10 and Mitf or microphthalmia. By analyzing the expression patterns of the eye transcription factors in wild-type embryos and embryos over-expressing the individual genes, it will be possible to outline a genetic hierarchy. Dominant-negative inhibition and enhancer activation of their function in the developing animal will be used to more precisely define the functional relationships of these genes. In addition, over-expression or inhibition of Chx10 and Mitf at the 2-cell stage and in embryonic optic vesicles (stage 18) may reveal new functions for these genes that previous studies in mice have not.

DESCRIPTION (provided by applicant): Cone photoreceptors are the primarily cell class lost in Age-Related Macular Degeneration. Therapies designed to replace lost cones will require a rich source of these unique cells. Consequently, there is a clear need to identify the molecular mechanisms required to direct more plentiful pluripotent cells types to a cone lineage. We have developed a unique approach to addressing this problem. Our results demonstrate that mouse embryonic stem cells, first converted to primitive ectoderm, can be directed to a cone cell fate in culture. In vitro cone formation requires simultaneous repression of BMP and activation of FGF signaling, respectively. During neural induction, these two signaling pathways regulate SMAD1/5/8 nuclear translocation (BMP) and degradation (FGF), which directs ectodermal cells to a neural fate. In this application, we outline experiments designed to identify how these signaling pathways and their downstream components generate cone photoreceptor fate, by regulating both canonical and non-canonical BMP signaling as well as FGF signaling. Our hypothesis is that modulation of SMAD1/5/8 stability is required for cone formation. Previous studies have clearly demonstrated the developmental stage at which rod progenitors are transplanted to the host retina is critical for successful differentiation and integration of the cells. Our preliminary data demonstrates cone specific proteins and genes of the phototransduction cascade are already expressed in our cells. Characterizing their molecular, morphological and physiological properties will help us to determine the relative age of these in vitro-generated cones. Our research fits well into the National Plan for Eye and Vision Research objectives (http://www.nei.nih.gov/strategicplanning/ np_strab.asp), which include "determine(ing) how stem cells differentiate in the development of the visual system and how they can be used to understand the molecular logic of cell-type- specific identity in the visual system." This project will identify molecular mechanisms that contribute to cone cell generation and form the basis of future studies focusing on the ability of these cells to replace cones lost to retinal damage and degeneration. PUBLIC HEALTH RELEVANCE: Cone photoreceptors account for only 3% of all retinal cells, yet are required for all day vision. Our ability to convert a high proportion of mouse embryonic stem cells to cone photoreceptors provides us with a unique opportunity to study the mechanisms by which these rare cells form. Identifying the molecules drivingcone formation is key to future experiments designed to determine the optimal conditions for cone cell replacement therapies in animal models and for generating human cone cells for further study.

Project Abstract Ocular developmental defects, like Anophthalmia and Microphthalmia, cause 10% of childhood blindness, yet in many cases the cause is unknown. In cases of bilateral anophthalmia and microphthalmia, the underlying cause has been primarily linked to mutations in two anterior neural patterning transcription factors: Sox2 and Otx2. Anterior neural patterning precedes eye field formation, which gives rises to the neural retina and retinal pigment epithelium progenitors, yet how anterior neural plate patterning is molecularly linked to eye field formation is unknown. Our recent work provides insight into this connection. At the start of anterior neural plate formation in Xenopus laevis, we found expression of T-box transcription factor, Tbx3. Our preliminary data shows that Otx2 directly activates Tbx3 at this stage in vivo. Among all the eye field transcription factors that we have investigated, Tbx3 is the only one involved in both early neural induction and eye field determination. In culture Tbx3 is required to generate neuroepithelium from human embryonic stem cells, however the in vivo role of Tbx3 during early mammalian development has not been studied, in part because null mutations are embryonic lethal. Our preliminary data shows that Tbx3 is required for normal eye formation, since conditional ablation in the optic cup results in a thinner optic nerve and fewer dorsal retinal ganglion cells. Our working hypothesis is that mouse Tbx3 is required even earlier, at the onset of neural induction. In Aim 1, we will determine when Tbx3 is first required for mouse eye formation by conditionally deleting Tbx3 prior to neural plate formation, and separately, eye field formation. Using Xenopus laevis we will investigate the regulation of Tbx3 during neural induction and eye field determination. In Aim 2, we will determine how loss of Tbx3 in the optic cup affects the formation of dorsal retinal ganglion, and possibly other retinal cells, in both mouse and frog. Our proposal is in line with NEI?s Programs and Research Priorities on Retinal Disease that states as one of its goals, to ?study the disease pathogenesis and genetic factors that underlie structure, function, and the biology of retinal diseases.? Completion of this R21 will provide an integrated view of the genes linking early neural plate and eye field formation and lead to a better understanding of the signaling pathways that, when altered, result in Anophthalmia and Microphthalmia.