The generation of neuronal subtype diversity is controlled by the progression of multi-potent neural progenitors through a series of developmental competence states in which they successively lose and often also acquire the ability to generate different cell types. Recent studies have clearly shown that retinal progenitor cell (RPC) competence is controlled cell autonomously, with competence changes hypothesized to occur by temporally dynamic regulation of transcription factor activity. Despite extensive RNA expression profiling of the developing mouse retina, only a few RPC-expressed transcription factors have been identified whose expression tracks with changes in developmental competence. This suggests that other genes that regulate transcription factor activity may ultimately play a central role in regulating RPC competence. The mouse genome contains over 10,000 long noncoding RNAs (lncRNAs), some of which are known to regulate transcription factor activity. Using RNA-Seq analysis, we have identified over 100 lncRNAs that are differentially expressed between early and late-stage RPCs, and we hypothesize that a subset of these will play a critical role in regulating RPC competence. To test this hypothesis, evolutionarily conserved, lncRNAs that display dynamic expression within progenitors have been identified from mouse retinas. Cellular expression patterns of candidate lncRNAs in developing retina will be examined through in situ hybridization and quantitative RT-PCR experiments. The role of candidate lncRNAs in regulation of RPC competence will then be tested using both overexpression and knockdown in developing retina and genetic approaches. Finally, to determine the molecular mechanism by which lncRNAs regulate RPC competence, we will use protein microarrays and RNA immunoprecipitation to identify proteins that directly interact with candidate lncRNAs, and in turn determine whether overexpression or knockdown of these protein-coding genes regulates RPC competence. These studies will improve our understanding of long noncoding RNA function and provide important insight into mechanisms regulating neural progenitor competence. Furthermore, since approximately one-third of all phenotype-associated polymorphisms identified in genome-wide association studies map to non-protein coding regions of the genome, further characterization of lncRNA function may prove critical in understanding the pathology of multiple genetic diseases.
Although each mammalian genome contains many thousands of long noncoding RNAs, knowledge of how long noncoding RNAs function remains limited, especially within the developing central nervous system. This proposal seeks to investigate the function of lncRNAs in regulating cell fate decisions within mouse retinal progenitors. We anticipate that these studies will provide insight into the mechanisms controlling the generation of specific retinal cell types from retinal progenitor cells and elicit findings that prove pivotalfor the design of stem cell-based therapies for treatment of retinal diseases.
| de Melo, Jimmy; Clark, Brian S; Venkataraman, Anand et al. (2018) Ldb1- and Rnf12-dependent regulation of Lhx2 controls the relative balance between neurogenesis and gliogenesis in the retina. Development 145: |
| Clark, Brian S; Blackshaw, Seth (2017) Understanding the Role of lncRNAs in Nervous System Development. Adv Exp Med Biol 1008:253-282 |
| de Melo, Jimmy; Zibetti, Cristina; Clark, Brian S et al. (2016) Lhx2 Is an Essential Factor for Retinal Gliogenesis and Notch Signaling. J Neurosci 36:2391-405 |
| de Melo, Jimmy; Clark, Brian S; Blackshaw, Seth (2016) Multiple intrinsic factors act in concert with Lhx2 to direct retinal gliogenesis. Sci Rep 6:32757 |
| Thein, Thuzar; de Melo, Jimmy; Zibetti, Cristina et al. (2016) Control of lens development by Lhx2-regulated neuroretinal FGFs. Development 143:3994-4002 |
| Clark, Brian S; Blackshaw, Seth (2014) Long non-coding RNA-dependent transcriptional regulation in neuronal development and disease. Front Genet 5:164 |
