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A. Kahana, F. I. Elsaeidi, D. D. Gallina, B. Bohnsack, D. Kasprick, D. Goldman; Retinoic Acid Regulates a Balance Between PitX2 and PitX3 Expression to Promote Morphogenesis of Extraocular Muscles in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5821.
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Organization of the 4 rectus and 2 oblique extraocular muscles around the eye must be precise to enable binocular vision. This reproducible precision has been termed the Spiral of Tillaux, and dysregulation can lead to strabismus and amblyopia. We aim to understand the process through which the developing EOMs organize and insert onto the globe with such reproducible precision.
We utilize zebrafish to facilitate in vivo experiments while taking advantage of genetic tools available in zebrafish. Morpholino knockdown, RT-PCR, immunohistochemistry, in situ hybridization and in vivo confocal microscopy techniques were used to assess gene expression patterns in the periocular tissues.
Retinoic acid (RA) synthesis by Raldh2 and Raldh3 is required for proper EOM and craniofacial morphogenesis, and pharmacologic inhibition of Raldh leads to EOM disorganization, although muscle differentiation occurs. Raldh inhibition can be rescued with exogenous RA in a dose-dependent fashion, and the craniofacial neural crest is more dose-sensitive than EOMs that are closer to the eye-source of RA. RA disinhibits PitX2 expression, and conversely lack of RA reduces PitX2 expression. RA has the opposite effect on PitX3. PitX2 and PitX3 also influence each other’s expression level and pattern. Confocal microscopy revealed that PitX2 and PitX3 are heavily expressed at the insertion sites of EOMs, as well as within and along the leading edge of the developing muscle.
RA morphogenic gradient is critical for proper orbital development, and regulates the balance between PitX2 and PitX3 expression in the periocular mesenchyme. PitX genes are also important for ocular development, which suggests that ocular and orbital development are interrelated and coregulated. These results also suggest possible targets for inducing EOM tissue regeneration following traumatic injury.
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