The developing vertebrate eye provides a powerful system for studying the inductive tissue interactions that underlie organogenesis. In the prior grant period, we investigated the function of the Pax6 gene in murine eye development, and identified both upstream and downstream components of the Pax6 genetic pathway. We found that Pax6 regulates a conserved network of subordinate genes, but that in the vertebrate lens it does so imperfectly. Remarkably, however, we also found that this phylogenetically ancient regulatory network has been re-deployed in evolution to function throughout vertebrate organogenesis. In addition, we have also made major inroads into understanding the nature of Pax6 upstream regulation. We identified three distinct Pax6 ocular enhancers that direct expression in different retinal cell types. We then focused on the Pax6 Ectodermal Enhancer or EE, that together with a second SIMO enhancer directs Pax6 expression in developing lens ectoderm. We narrowed the EE to a 107 bp minimal enhancer that remains sufficient to drive robust expression in the developing lens, and we have identified members of the Meis homeoprotein family as key EE regulators. We have now also defined additional cis-regulatory elements that are required for EE activity, and have identified factors that bind them, including members of the Sox and POU families. Work from several groups, including our own, has also revealed a clear role for BMP-signaling in early lens formation. However, little is known about how the BMP-signaling pathway regulates the EE, or if it regulates the transcription factors that regulate the EE. Thus, this renewal application seeks to rectify these gaps in our knowledge by further elucidating how Meis, Sox and POU transcription factors regulate Pax6 expression in the developing lens, and by establishing how the BMP-signaling pathway interacts with them. The underlying hypothesis that informs this grant is that by identifying these genetic and molecular inter- relationships, we can establish distinct regulatory networks for different phases of lens development. To this end, this competing renewal proposes four Specific Aims. First, we will use the power of mouse genetics to establish that the Meis transcription factors and a yet to be identified Meis co-factor that regulate the Pax6 EE behave appropriately from a developmental standpoint. Second, we will use mouse genetics to determine how Sox factors, through cooperative interaction with the co-factor Oct1, regulate the Pax6 EE. Third, we will determine whether the EE and SIMO enhancers are coordinately regulated by Meis, Sox and POU factors, and how their expression properties inter-relate. Lastly, we will employ mouse mutants, experimental embryology and lens epithelial cell lines to test whether the BMP-signaling pathway converges upon transcription factors such as Sox2 that regulate the Pax6 EE. Collectively, these experiments have the potential to significantly expand our knowledge of the upstream regulators and regulatory networks that control Pax6 expression in early lens development. ? ? ? ? ? ?
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| Lin, Haotian; Ouyang, Hong; Zhu, Jie et al. (2016) Lens regeneration using endogenous stem cells with gain of visual function. Nature 531:323-8 |
| Li, Gen; Xu, Fan; Zhu, Jie et al. (2015) Transcription Factor PAX6 (Paired Box 6) Controls Limbal Stem Cell Lineage in Development and Disease. J Biol Chem 290:20448-54 |
| Anchan, Raymond M; Lachke, Salil A; Gerami-Naini, Behzad et al. (2014) Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells. PLoS One 9:e115106 |
| Lachke, Salil A; Higgins, Anne W; Inagaki, Maiko et al. (2012) The cell adhesion gene PVRL3 is associated with congenital ocular defects. Hum Genet 131:235-50 |
| Lachke, Salil A; Ho, Joshua W K; Kryukov, Gregory V et al. (2012) iSyTE: integrated Systems Tool for Eye gene discovery. Invest Ophthalmol Vis Sci 53:1617-27 |
| Lachke, Salil A; Maas, Richard L (2011) RNA Granules and Cataract. Expert Rev Ophthalmol 6:497-500 |
| Lachke, Salil A; Alkuraya, Fowzan S; Kneeland, Stephen C et al. (2011) Mutations in the RNA granule component TDRD7 cause cataract and glaucoma. Science 331:1571-6 |
| Lachke, Salil A; Maas, Richard L (2010) Building the developmental oculome: systems biology in vertebrate eye development and disease. Wiley Interdiscip Rev Syst Biol Med 2:305-323 |
| Rowan, Sheldon; Siggers, Trevor; Lachke, Salil A et al. (2010) Precise temporal control of the eye regulatory gene Pax6 via enhancer-binding site affinity. Genes Dev 24:980-5 |
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