The long-term objective of this application is to gain insights into the molecular events that lead to the differentiation of mammalian retinal ganglion cells. Retinal ganglion cells are essential for normal vision and their loss contributes to major eye diseases in humans. For example, elevated intraocular pressure within the eye can trigger enhanced apoptosis in ganglion cells, which in turn leads to glaucoma. Despite their importance, however, the knowledge base of genes associated with retinal ganglion cell formation and survival is rudimentary. In this application, experiments are proposed that focus on Brn-3b, a POU-domain transcription factor, in retinal ganglion cells. In the mouse, the brn-3b gene is among the first genes activated in postmitotic progenitor cells as ganglion cell differentiation begins. In spite of this early activation, brn-3b is not required for the initial specification of ganglion cells, but it is essential for their normal differentiation and survival. Mice with targeted deletions in brn-3b have defective retinas with a loss of most ganglion cells. Thus, brn-3b is a critical gene that marks the commitment to a ganglion cell fate and is essential for the survival of retinal ganglion cells. The first two aims of this application use the brn-3b locus to probe early events of retinal ganglion cell formation. The final two aims concern the transcriptional properties of Brn-3b.
The Specific Aims will: (1) Test the hypothesis that as retinal ganglion cells form, they inhibit their further production. The proposed experiments will specifically ablate retinal ganglion cells using genetically targeted diphtheria toxin; (2) Identify the cis-regulatory elements within the brn-3b transcriptional control region that activate brn-3b in postmitotic ganglion cell progenitors. The cis-regulatory elements within the brn-3b transcriptional control region will be identified using BAC transgenic analysis; (3) Investigate the functional specificity of Brn-3b in retinal ganglion cell differentiation. The experiments will employ HSV-mediated gene transfer into cultured retinal explants; (4) Determine whether brn-3b can function in committing progenitor cells to a retinal ganglion cell fate. Math5 is a proneural bHLH gene required for retinal ganglion cell formation. Brn-3b will be misexpressed at the math5 locus to determine whether brn-3b is sufficient to promote ganglion cell differentiation in the presence and absence of math5.
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