Through alternative splicing a single gene can generate functionally diverse set of proteins. Indeed, AS is invoked to reconcile the difference between the relatively few (~24,000) protein coding genes in the human genome to its vast proteome. Interestingly, of all the mammal tissues, the central nervous system (CNS) has the highest degree of AS. Thus, it is generally accepted that AS plays a crucial role in neural development, but there is a paucity of information on this issue. Given the complexity of CNS development combined with that of AS, we have chosen to employ the neural retina as our model system. The retina is derived from the CNS, is a relatively simple tissue, has a well defined laminar structure, it has six neural cell types and one glia, birth order of each cell type is known, and importantly it is the most accessible part of the CNS. The overall goal of this research proposal is to investigated the contribution of AS in neural development by studying an AS factor, Sfrs10 in the mouse retina. Previous studies in Drosophila have shown that Sfrs10 is required during sex determination and others have shown that Sfrs10 regulates the AS some neural genes. Based on this and our preliminary work the hypothesis underlying this proposal is that Sfrs10 is essential for neural cell fate determination and differentiation. There are two specific aims and the first specific aim will accomplish the following goals. We will characterize the expression pattern and AS of Sfrs10 during embryonic and postnatal retinal development. In addition, loss (RNAi) and gain of function will be performed by either in vivo DNA electroporation of a postnatal day 0 mouse retina or by in vitro electroporation of embryonic retinal explant cultures. We will employ ultrasound guided delivery technology to deliver viruses with either a gain or a loss of function construct into E10.5 embryo. A significant effort will be dedicated towards finding the targets of Sfrs10. Moreover, a conditional knockout mouse will be generated to validate the aforementioned experiments and provide a tool for the second specific aim. The second specific aim will investigate the AS status of the targets of Sfrs10 at the single cell resolution. The premise here is that tissues consists of several cell types and if each cell type splices an exon differently, then the use of entire tissue for analysis might mask this interesting fact.

Public Health Relevance

Understanding the role of alternative splicing in development is essential to our understanding the underlying mechanism that leads to diseases. There are several diseases such as Alzheimer's disease, myotonic dystrophy, spinal muscular atrophy and autism that are linked to defects in alternative splicing of specific genes or are caused by mutations in genes that regulate the process of alternative splicing.

National Institute of Health (NIH)
National Eye Institute (NEI)
Career Transition Award (K99)
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Special Emphasis Panel (ZEY1-VSN (10))
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Greenwell, Thomas
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Harvard University
Schools of Medicine
United States
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Karunakaran, Devi Krishna Priya; Banday, Abdul Rouf; Wu, Qian et al. (2013) Expression analysis of an evolutionarily conserved alternative splicing factor, Sfrs10, in age-related macular degeneration. PLoS One 8:e75964
Karunakaran, Devi Krishna Priya; Congdon, Sean; Guerrette, Thomas et al. (2013) The expression analysis of Sfrs10 and Celf4 during mouse retinal development. Gene Expr Patterns 13:425-36