Faculty Mentor: Kristen Kwan (Human Genetics, University of Utah)

The cornea and iris are both important for visual acuity and malformations can lead to impaired vision. Improper formation of these structures is a characteristic of Axenfeld-Rieger syndrome (ARS), a congenital syndrome caused by genetic mutations in pitx2 (Huang et al. 2019; Semina et al., 1996). The cornea and iris are made up of multiple tissue layers that are derived from different embryonic tissues, however the cellular processes that govern tissue assembly during eye development are unknown (Hay 1980; Soules and Link 2005; Zhao et al., 2006). In vertebrates, the cornea arises from ectoderm adjacent to the lens and neural crest-derived mesenchyme, while the iris arises from neural crest-derived mesenchyme and the neural retina (Gage et al. 2005; Johnston et al., 1979; Langenberg et al. 2008). Although these tissues’ origins are known, little is understood about the morphogenesis of the multi-layered cornea and iris. Using the zebrafish (Danio rerio) as a model, we are investigating the cellular mechanisms underlying iris and cornea morphogenesis, including cell proliferation and cell death. By using antibody staining in transgenic zebrafish (bactin2:EGFP-CAAX), and confocal microscopy, we are visualizing and quantifying the cellular basis of anterior segment development in both normal development and in the pitx2 mutant, a model for ARS.

The cornea and iris are both important for visual acuity and malformations can lead to impaired vision. Improper formation of these structures is a characteristic of Axenfeld-Rieger syndrome (ARS), a congenital syndrome caused by genetic mutations in pitx2 (Huang et al. 2019; Semina et al., 1996). The cornea and iris are made up of multiple tissue layers that are derived from different embryonic tissues, however the cellular processes that govern tissue assembly during eye development are unknown (Hay 1980; Soules and Link 2005; Zhao et al., 2006). In vertebrates, the cornea arises from ectoderm adjacent to the lens and neural crest-derived mesenchyme, while the iris arises from neural crest-derived mesenchyme and the neural retina (Gage et al. 2005; Johnston et al., 1979; Langenberg et al. 2008). Although these tissues’ origins are known, little is understood about the morphogenesis of the multi-layered cornea and iris. Using the zebrafish (Danio rerio) as a model, we are investigating the cellular mechanisms underlying iris and cornea morphogenesis, including cell proliferation and cell death. By using antibody staining in transgenic zebrafish (bactin2:EGFP-CAAX), and confocal microscopy, we are visualizing and quantifying the cellular basis of anterior segment development in both normal development and in the pitx2 mutant, a model for ARS.

Generating 3-D renderings from confocal z-stacks of fixed embryos allows for visualization and quantification of cells undergoing apoptosis (AC3-positive cells) or mitosis (PHH3-positive cells), respectively. In the cornea, we found few apoptotic cells throughout cornea development in either wild-type or pitx2 mutants; however, we uncovered temporal differences in proliferation in wild-type and pitx2 mutants in the cornea periphery. Apoptosis was also minimal throughout iris formation and was unaffected by loss of pitx2. In contrast, cell proliferation in the developing iris was much greater than the developing cornea, and proliferation was disrupted in pitx2 mutants compared to wild type. Our results suggest proliferation is disrupted in the pitx2 mutant iris, and that proliferation in the cornea periphery and iris differs temporally when comparing pitx2 mutant and wild type embryos. These disruptions in the pitx2 mutant may contribute to the ARS phenotype.

Bibliography

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Hay E, D., 1980 Development of the vertebrate cornea. International Review of Cytology 63: 263-322.

Huang, L., Meng, Y., & Guo, X. (2019). Novel PITX2 mutations including a mutation causing an unusual ophthalmic phenotype of Axenfeld-Rieger Syndrome. Journal of Ophthalmology, 2019, 1–10. https://doi.org/10.1155/2019/5642126

Ji, Y., Buel, M. S., Amack, J. D. (2016). Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs. Developmental Biology 416: 69-81.

Johnston, M. C., Noden, D. M., Hazelton R. D., Coulombre J. L., Coulombre A. J. (1979). Origins of avian ocular and periocular tissues. Experimental Eye Research 29: 27-43.

Langenberg T., Kahana, A., Wszalek, J. A., Halloran, M. C. (2008). The eye organizes neural crest cell migration. Developmental Dynamics 237: 1645-1652.

Soules, K.A., Link, B.A. (2005). Morphogenesis of the anterior segment in the zebrafish eye. BMC Developmental Biology, 5(12).

Zhao, X. C., Yee, R. W., Norcom, E., Burgess, H., Avanesov, A. S., Barrish, J. P., & Malicki, J. (2006). The Zebrafish Cornea: Structure and Development. Investigative Opthalmology & Visual Science, 47(10): 4341.