The developing lens constitutes a powerful system for understanding the molecular basis of tissue development, and it is also the target of the leading cause of blindness, cataract. During the prior grant period, we made major progress in elucidating the regulation of a key lens formation gene, Pax6, via signaling pathways (Notch) and transcription factors (Sox, Pou, Meis). We also developed a bioinformatics method, iSyTE, for efficiently using lens expression data to predict functionally important genes in lens development, and we identified three genes, Pvrl3, Sep15 and Tdrd7, that when mutated cause cataracts. Using Tdrd7 as an entry point, we uncovered a new and important mode of post-transcriptional regulation that operates during lens fiber cell differentiation. Tdrd7 encodes an RNA-binding protein, and is mutated in human congenital cataracts, a phenotype also present in Tdrd7 mouse mutants. Tdrd7 is a component of a novel class of RNA granules (Tdrd7-RGs) that are expressed in developing lens fiber cells, and Tdrd7-RGs bind a specific subset of lens mRNAs and, potentially, either sequester them for translation or target them for degradation. In the former class are mRNAs encoding lens fiber cell components mutated in cataracts, while the latter class includes transcripts such as Pax6 that require down- regulation for fiber cell differentiation. These studies provide mechanistic information about the gene regulatory network (GRN) that controls vertebrate lens development. In this renewal, we propose to further expand our understanding of the lens development GRN by focusing on the role of post-transcriptional regulation by Tdrd7 in lens fiber cell differentiation. We hypothesize that the molecular function of Tdrd7 in Tdrd7-RGs is required for normal lens development. By identifying additional genes in the Tdrd7 functional pathway and additional Tdrd7-RG protein components, we propose to define a Tdrd7 regulatory pathway and to evaluate its overall significance.
In Aim 1, we will use conditional and transgenic Tdrd7 mouse alleles, and potentially complementation in mouse and zebrafish to dissect the Tdrd7 function in lens development.
For Aim 2, we have focused on a ligand-receptor pair, Ephrin A5 and its receptor Epha2, that when mutated appears to phenocopy the Tdrd7 null mutant. Epha2 mRNA binds Tdrd7, and hence appears to act directly within the Tdrd7 pathway. We will now establish the nature of the regulatory relationship between these signaling molecules and Tdrd7 continue to identify Tdrd7 phenocopy genes, and test whether they encode Tdrd7 pathway components. Lastly, in Aim 3, we will determine how Tdrd7 executes its post-transcriptional function in lens development by biochemically identifying additional RNA and protein Tdrd7-RG components, and by testing whether these mRNAs are dynamically exchanged between different RG types according to their fate. These studies will increase our understanding of lens development, and provide new information on post-transcriptional regulatory mechanisms in oculogenesis.

Public Health Relevance

This project aims to determine the nature and significance of a specific form of gene regulation, posttranscriptional regulation of mRNAs, in lens development. We will do so by focusing on one particular lens protein, Tdrd7, that when mutated causes cataracts. We will take an interdisciplinary approach involving mouse genetics, developmental biology, genomics and computation, and biochemistry to identify new components of the Tdrd7 pathway and to figure out how it works.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (BVS)
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Araj, Houmam H
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Brigham and Women's Hospital
United States
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Zhang, Ying; Fan, Jieqing; Ho, Joshua W K et al. (2016) Crim1 regulates integrin signaling in murine lens development. Development 143:356-66
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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