Bahaar Chawla

Retinoic Acid and Pitx2 Regulate Early Neural Crest Survival and Migration in Craniofacial and Ocular Development.

Congenital eye and craniofacial anomalies are associated with the dysregulation of retinoic acid (RA) levels during embryogenesis. In the present study, we observed that RA and pitx2a cooperatively regulate early cranial neural crest migration from the rhombencephalon to the pharyngeal arches and from the mesencephalon and prosencephalon to the periocular mesenchyme and frontonasal processes. The cranial neural crest tracked toward areas of high RA activity (i.e., developing eye) and circumvented areas of low RA activity (i.e., mesencephalon). Although previous studies have shown that RA increased pitx2a expression at later stages of cranial neural crest development, in these studies we found that RA inhibited pitx2a expression in the early migrating ventral cranial neural crest. Increased RA or decreased Pitx2a expression decreased cell survival and inhibited ventral neural crest migration. Decreased RA or increased pitx2a expression markedly disrupted both dorsal and ventral neural crest migration. The tight control of RA and subsequent regulation of pitx2 were required for precise cranial neural crest survival and migration. These alterations in the neural crest in the periocular mesenchyme and frontonasal processes may reflect the craniofacial dysmorphism and microphthalmia observed in cases of increased (i.e., as resulting from isoretinoin exposure) or decreased (i.e., as may occur in fetal alcohol syndrome) RA signaling during pregnancy

Retinoic Acid and Pitx2 Regulate Early Neural Crest Survival and Migration in Craniofacial and Ocular Development

Congenital eye and craniofacial anomalies are associated with the dysregulation of retinoic acid (RA) levels during embryogenesis. In the present study, we observed that RA and pitx2a cooperatively regulate early cranial neural crest migration from the rhombencephalon to the pharyngeal arches and from the mesencephalon and prosencephalon to the periocular mesenchyme and frontonasal processes. The cranial neural crest tracked toward areas of high RA activity (i.e., developing eye) and circumvented areas of low RA activity (i.e., mesencephalon). Although previous studies have shown that RA increased pitx2a expression at later stages of cranial neural crest development, in these studies we found that RA inhibited pitx2a expression in the early migrating ventral cranial neural crest. Increased RA or decreased Pitx2a expression decreased cell survival and inhibited ventral neural crest migration. Decreased RA or increased pitx2a expression markedly disrupted both dorsal and ventral neural crest migration. The tight control of RA and subsequent regulation of pitx2 were required for precise cranial neural crest survival and migration. These alterations in the neural crest in the periocular mesenchyme and frontonasal processes may reflect the craniofacial dysmorphism and microphthalmia observed in cases of increased (i.e., as resulting from isoretinoin exposure) or decreased (i.e., as may occur in fetal alcohol syndrome) RA signaling during pregnancy

Cyp1b1 Regulates Ocular Fissure Closure Through a Retinoic Acid–Independent Pathway

Purpose Mutations in the CYP1B1 gene are the most commonly identified genetic causes of primary infantile-onset glaucoma. Despite this disease association, the role of CYP1B1 in eye development and its in vivo substrate remain unknown. In the present study, we used zebrafish to elucidate the mechanism by which cyp1b1 regulates eye development. Methods Zebrafish eye and neural crest development were analyzed using live imaging of transgenic zebrafish embryos, in situ hybridization, immunostaining, TUNEL assay, and methylacrylate sections. Cyp1b1 and retinoic acid (RA) levels were genetically (morpholino oligonucleotide antisense and mRNA) and pharmacologically manipulated to examine gene function. Results Using zebrafish, we observed that cyp1b1 was expressed in a specific spatiotemporal pattern in the ocular fissures of the developing zebrafish retina and regulated fissure patency. Decreased Cyp1b1 resulted in the premature breakdown of laminin in the ventral fissure and altered subsequent neural crest migration into the anterior segment. In contrast, cyp1b1 overexpression inhibited cell survival in the ventral ocular fissure and prevented fissure closure via an RA-independent pathway. Cyp1b1 overexpression also inhibited the ocular expression of vsx2, pax6a, and pax6b and increased the extraocular expression of shha. Importantly, embryos injected with human wild-type but not mutant CYP1B1 mRNA also showed colobomas, demonstrating the evolutionary and functional conservation of gene function between species. Conclusions Cyp1b1 regulation of ocular fissure closure indirectly affects neural crest migration and development through an RA-independent pathway. These studies provide insight into the role of Cyp1b1 in eye development and further elucidate the pathogenesis of primary infantile-onset glaucoma.

Differences in neural crest sensitivity to ethanol account for the infrequency of anterior segment defects in the eye compared with craniofacial anomalies in a zebrafish model of fetal alcohol syndrome

BACKGROUND Ethanol (ETOH) exposure during pregnancy is associated with craniofacial and neurologic abnormalities, but infrequently disrupts the anterior segment of the eye. In these studies, we used zebrafish to investigate differences in the teratogenic effect of ETOH on craniofacial, periocular, and ocular neural crest. METHODS Zebrafish eye and neural crest development was analyzed by means of live imaging, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, immunostaining, detection of reactive oxygen species, and in situ hybridization. RESULTS Our studies demonstrated that foxd3-positive neural crest cells in the periocular mesenchyme and developing eye were less sensitive to ETOH than sox10-positive craniofacial neural crest cells that form the pharyngeal arches and jaw. ETOH increased apoptosis in the retina, but did not affect survival of periocular and ocular neural crest cells. ETOH also did not increase reactive oxygen species within the eye. In contrast, ETOH increased ventral neural crest apoptosis and reactive oxygen species production in the facial mesenchyme. In the eye and craniofacial region, sod2 showed high levels of expression in the anterior segment and in the setting of Sod2 knockdown, low levels of ETOH decreased migration of foxd3-positive neural crest cells into the developing eye. However, ETOH had minimal effect on the periocular and ocular expression of transcription factors (pitx2 and foxc1) that regulate anterior segment development. CONCLUSION Neural crest cells contributing to the anterior segment of the eye exhibit increased ability to withstand ETOH-induced oxidative stress and apoptosis. These studies explain the rarity of anterior segment dysgenesis despite the frequent craniofacial abnormalities in fetal alcohol syndrome. Birth Defects Research 109:1212-1227, 2017.

Retinoic Acid Maintains Function of Neural Crest–Derived Ocular and Craniofacial Structures in Adult Zebrafish

Purpose Retinoic acid (RA) is required for embryonic formation of the anterior segment of the eye and craniofacial structures. The present study further investigated the role of RA in maintaining the function of these neural crest–derived structures in adult zebrafish. Methods Morphology and histology were analyzed by using live imaging, methylacrylate sections, and TUNEL assay. Functional analysis of vision and aqueous humor outflow were assayed with real-time imaging. Results Both decreased and increased RA signaling altered craniofacial and ocular structures in adult zebrafish. Exogenous treatment with all-trans RA for 5 days resulted in a prognathic jaw, while inhibition of endogenous RA synthesis through treatment with 4-diethylaminobenzaldehyde (DEAB) decreased head height. In adult eyes, RA activity was localized to the retinal pigment epithelium, photoreceptors, outer plexiform layer, inner plexiform layer, iris stroma, and ventral canalicular network. Exogenous RA increased apoptosis in the iris stroma and canalicular network in the ventral iridocorneal angle, resulting in the loss of these structures and decreased aqueous outflow. DEAB, which decreased RA activity throughout the eye, induced widespread apoptosis, resulting in corneal edema, cataracts, retinal atrophy, and loss of iridocorneal angle structures. DEAB-treated fish were blind with no optokinetic response and no aqueous outflow from the anterior chamber. Conclusions Tight control of RA levels is required for normal structure and function of the adult anterior segment. These studies demonstrated that RA plays an important role in maintaining ocular and craniofacial structures in adult zebrafish.