State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This abstract is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. The PITX2 transcription factor is essential for normal eye development in vertebrates. Mutations in PITX2 result in Axenfeld-Rieger syndrome (ARS) associated with developmental glaucoma and other systemic defects. Our studies demonstrate conservation of the role of pitx2 in zebrafish ocular development. Using zebrafish model, we identified multiple regulatory regions mediating PITX2/pitx2 expression during eye, brain and craniofacial development. These studies led to the discovery of a novel mechanism of Axenfeld-Rieger syndrome: the deletion of the distant regulatory elements upstream of PITX2. In fact, using a large cohort of ARS patients we showed that virtually all classic ARS cases can be explained by nucleotide or copy number mutations in PITX2 when both coding and regulatory regions are examined. Further exploration of the identified regulatory sequences resulted in tentative identification of upstream regulators of PITX2/pitx2 that are currently being analyzed in terms of their role in normal ocular development and disease. Several downstream genes have also been identified and verified by in vitro and in vivo assays. The genes identified by us and some additional published factors will be further evaluated for their role in PITX2/pitx2 pathway in this application. One of the innovations of this proposal is that we plan to use the resources generated by us during the previous funding period in terms of pitx2 enhancer transgenic lines expressing GFP in periocular mesenchymal (POM) cells and pitx2 knockdown reagents to identify transcripts that distinguish normally differentiating and pitx2-deficient POM cells at different developing stages. This will result in robust in vivo identification of downstream effectors of PITX2/pitx2 as well as aid in general characterization of this migratory population with high significance to normal eye development. Another major innovation is our utilization of zinc finger nucleases- driven genome editing technology to generate pitx2 lines carrying mutations in pitx2 coding region (null allele) and CE4 regulatory element that has been previously shown to be involved in different aspects of pitx2 expression including strong presence in POM cells. Our main hypothesis is that PITX2/pitx2 is a conserved factor that is essential for inducing differentiation of periocular mesenchyme cells upon their migration into the anterior segment of the developing eye. Discovery of the upstream regulators of PITX2 expression and its downstream effectors will provide insight into the mechanisms of vertebrate embryonic eye development and are likely to identify new causes of human glaucoma phenotypes which can be further explored as potential therapeutic targets.

Public Health Relevance

PITX2 is essential for normal eye development. This proposal will investigate how PITX2 is involved in eye development by identifying other genes that regulate PITX2 expression and genes that are controlled by PITX2 activities. The results of this project will provide better understanding of eye development and will likely identify new causes of human ocular conditions like glaucoma, which may lead to new treatment opportunities.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Application #
Study Section
Anterior Eye Disease Study Section (AED)
Program Officer
Chin, Hemin R
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Medical College of Wisconsin
Schools of Medicine
United States
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Happ, Hannah; Weh, Eric; Costakos, Deborah et al. (2016) Case report of homozygous deletion involving the first coding exons of GCNT2 isoforms A and B and part of the upstream region of TFAP2A in congenital cataract. BMC Med Genet 17:64
Deml, Brett; Reis, Linda M; Lemyre, Emmanuelle et al. (2016) Novel mutations in PAX6, OTX2 and NDP in anophthalmia, microphthalmia and coloboma. Eur J Hum Genet 24:535-41
Reis, Linda M; Tyler, Rebecca C; Weh, Eric et al. (2016) Analysis of CYP1B1 in pediatric and adult glaucoma and other ocular phenotypes. Mol Vis 22:1229-1238
Happ, Hannah; Schilter, Kala F; Weh, Eric et al. (2016) 8q21.11 microdeletion in two patients with syndromic peters anomaly. Am J Med Genet A 170:2471-5
Schilter, Kala F; Reis, Linda M; Sorokina, Elena A et al. (2015) Identification of an Alu-repeat-mediated deletion of OPTN upstream region in a patient with a complex ocular phenotype. Mol Genet Genomic Med 3:490-9
Deml, Brett; Kariminejad, Ariana; Borujerdi, Razieh H R et al. (2015) Mutations in MAB21L2 result in ocular Coloboma, microcornea and cataracts. PLoS Genet 11:e1005002
Reis, Linda M; Semina, Elena V (2015) Conserved genetic pathways associated with microphthalmia, anophthalmia, and coloboma. Birth Defects Res C Embryo Today 105:96-113
Deml, Brett; Reis, Linda M; Muheisen, Sanaa et al. (2015) EFTUD2 deficiency in vertebrates: Identification of a novel human mutation and generation of a zebrafish model. Birth Defects Res A Clin Mol Teratol 103:630-40
Reis, Linda M; Tyler, Rebecca C; Zori, Roberto et al. (2015) A Case of 22q11.2 Deletion Syndrome with Peters Anomaly, Congenital Glaucoma, and Heterozygous Mutation in CYP1B1. Ophthalmic Genet 36:92-4
Weh, Eric; Reis, Linda M; Happ, Hannah C et al. (2014) Whole exome sequence analysis of Peters anomaly. Hum Genet 133:1497-511

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