Abstract
Purpose :
Tight control of retinoic acid (RA) levels regulates neural crest contributions to the jaw, pharyngeal arches, and anterior segment of the eye. Few downstream targets of RA have been defined. In these studies, we used zebrafish to identify and characterize additional downstream targets of RA in the cranial neural crest.
Methods :
Tg(sox10::EGFP) embryos were treated with 0.1% dimethylsulfoxide, 25 nM all-trans RA or 10 mM diethylaminobenzaldehyde (DEAB) from 24 to 48 hours post fertilization (hpf). Deep sequencing was performed on RNA isolated from EGFP-positive cells obtained from the heads of 48 hpf Tg(sox10::EGFP) embryos (A) using fluorescence-activated cell sorting. In situ hybridization and the injection of morpholino oligonucleotides (MO) were used for further gene analysis.
Results :
Deep sequencing of sox10-positive cranial neural crest cells showed that RA regulated the expression of col2a1, col11a1, col11a2, col9a1a, and col9a3 genes, for which human mutations are associated with Sticklers Syndrome. Treatment with the pan-aldehyde dehydrogenase inhibitor, DEAB, which decreases endogenous RA synthesis, significantly inhibited the expression of these genes (2.48-fold decrease in col2a1a, 2.22-fold decrease in col11a1a, 3.08-fold decrease in col11a2, 2.62-fold decrease in col9a1a, and 2.12-fold decrease in col9a3) in sox10-positive cranial neural crest cells. In situ hybridization demonstrated that these genes are expressed in the optic vesicle and periocular mesenchyme in sox10-positive cells. Knockdown of Col2a1a through MO injections delayed neural crest-derived jaw and pharyngeal arch formation and ocular fissure closure and decreased ocular neural crest migration into the anterior segment. Moreover, knockdown of Col11a1a or Col11a2 inhibited jaw and pharyngeal arch formation. Furthermore, decreased Col11a1a or Col11a2 resulted in abnormal ocular neural crest migration and small eyes.
Conclusions :
Col2a1, col11a1a, col11a2, col9a1a, and col9a3 are downstream targets of RA in the cranial neural crest and are required for craniofacial and eye development. These findings provide insight into the mechanisms underlying Sticklers Syndrome, which in the ocular form, is associated with vitreous degeneration, high myopia, anterior segment dysgenesis and glaucoma.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.