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Brenda L. Bohnsack, Donika Gallina, Daniel Kasprick, Daniel Goldman, Alon Kahana; Retinoic Acid Regulation Of Pitx2 Is Essential For Neural Crest Development, Extraocular Muscle Organization And Ocular Morphogenesis In A Zebrafish Model Of Axenfeld-Reiger Syndrome. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2075.
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© ARVO (1962-2015); The Authors (2016-present)
To test the role of retinoic acid (RA) signaling in vivo on development of the eye, extraocular muscles (EOMs), and craniofacial morphology.
Transgenic zebrafish strains expressing GFP in neural crest (sox10::GFP) or muscle (α-actin::GFP) were microinjected with morpholino oligonucleotides (MO; raldh2, pitx2) or mRNA (pitx2a) or treated with all-trans RA or the inhibitor diethylbenzaldehyde (DEAB). Embryos were imaged in vivo and harvested for in situ hybridization and histological analysis.
Craniofacial development, including EOM organization and ocular morphogenesis, is complex and requires coordination of neural crest, mesoderm, and developing eye. We found that RA produced by the developing retina regulated pitx2 expression in periocular mesenchyme. Genetic and pharmacologic inhibition of RA synthesis in zebrafish embryos disrupted jaw cartilage formation (A) and caused mild EOM disorganization. In the eye, raldh2 MO knockdown inhibited retinal patterning and delayed anterior segment development. MO knockdown of pitx2 resulted in jaw (E) and EOM phenotypes that were similar to raldh2 MO knockdown or treatment with DEAB. Expression of dominant negative mutant K50E human pitx2a mRNA, which is associated with Axenfeld-Reiger syndrome, also disrupted neural crest development (N). MO knockdown or overexpression of pitx2 resulted in thinner corneas and loss of corneal and iris stroma. Injection of human pitx2a mRNA (F), but not autosomal recessive T30P mutant pitx2a (G), rescued defects in the pitx2 MO. Furthermore, human pitx2a mRNA partially rescued defects caused by knockdown of raldh2 (B).
RA produced by the developing eye forms a morphogenic gradient that regulates via pitx2 ocular, craniofacial, and EOM development. Mutations in pitx2 are associated with Axenfeld-Reiger syndrome in humans, and using our zebrafish model, we can recapitulate craniofacial and ocular abnormalities to gain a better understanding of mechanisms underlying normal development and human disease.
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