The overall goal of this project is to determine if the genetic background of children with optic nerve hypoplasia (ONH) confers intrinsic defects in retinal ganglion cell (RGC) behavior.
Aim 1 will determine if ONH background confers defective RGC production, survival, or axonogenesis.
Aim 2 will determine if ONH background alters gene expression in immature RGCs. Rationale: ONH has become the leading cause of permanent congenital blindness in Europe and North America. It is usually associated with other neurological and developmental problems, with or without neuroradiographic abnormalities, suggesting that there is an underlying problem with neurogenesis. However, no common genetic mutations have been discovered, and no animal models that mimic the human condition have been developed. This study will compare the development and gene expression of RGCs from nascent retinal tissue derived from children with and without ONH, as a means to determine whether ONH is associated with intrinsic RGC abnormalities. Design: Clones of induced pluripotent stem cells (iPSCs) have been developed from three children with clinically well-characterized bilateral ONH and from three control children. These will be used to create developing retinal tissue in cell culture.
In Aim 1, the number of total and apoptotic RGCs relative to total retinal progenitor cells will be quantified at different stages of retinal development, both by immunostaining of cryosectioned tissue and by flow cytometry. Axon outgrowth from these nascent retinas will be quantified under the influence of known axon growth and guidance factors. This will be further studied in chimeric retinal tissue containing eGFP-labeled ONH iPSC-derived cells and RFP-labeled control iPSC-derived cells in order to compare axon production within the same developing retinal tissue.
In Aim 2, RGCs derived from ONH and control iPSCs will be isolated by fluorescence-activated cell sorting and their RNA analyzed using gene expression microarrays. Data will be analyzed to determine if there are consistent significant differences between the ONH and control RGCs, both in the expression of individual genes and in the expression of groups of genes involved in particular pathways or biological functions. Thus, this study will determine whether the genetic background of ONH patients contributes to intrinsic defects in RGC production, survival, axonogenesis, and gene expression. This represents a first step towards defining the ONH etiology and developing ONH prevention and treatments strategies.

Public Health Relevance

Optic nerve hypoplasia (ONH) is a common congenital cause of childhood blindness characterized by a severe deficit in retinal ganglion cells (RGCs). This project aims to determine if the genetic makeup of children with ONH causes an intrinsic RGC defect by comparing retinal tissue produced from induced pluripotent stem cells (iPSCs) of children afflicted with ONH, to that of normal children. The comparisons will indicate whether children with ONH have a genetic background that causes an intrinsic problem with their retinal neuron development.

National Institute of Health (NIH)
National Eye Institute (NEI)
Exploratory/Developmental Grants (R21)
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Biology of the Visual System Study Section (BVS)
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Chin, Hemin R
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Children's Hospital of Los Angeles
Los Angeles
United States
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