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Elisa Schley, Bahaar Chawla, Antionette L. Williams, Brenda L Bohnsack; Retinoic acid and Pitx2 have reciprocal effects on neural crest migration in a zebrafish model of anterior segment dysgenesis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):227.
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© ARVO (1962-2015); The Authors (2016-present)
Alterations in retinoic acid (RA) levels and mutations in PITX2 are associated with congenital eye diseases such as microphthalmia and anterior segment dysgenesis. We previously showed that dysregulation of RA or pitx2a resulted in zebrafish anterior segment malformations. In these studies, we further elucidated the role of RA and pitx2 specifically during early neural crest migration in craniofacial and ocular development.
Morpholino oligonucleotides (MO) and mRNA injections were used in combination with regulators of RA in transgenic zebrafish [Tg(RARE-gata2::mCherry), Tg(sox10::EGFP)] to analyze RA activity and neural crest development. Embryos were live imaged and harvested for in situ hybridization, RT-PCR and TUNEL assay.
Live imaging of zebrafish embryos demonstrated that RA via RA receptor-a was required prior to and during early neural crest migration. Neural crest migratory pathways tracked to areas of high RA activity (developing eye) via dorsal and ventral waves (C,D,G) and circumvented areas of low RA (posterior to the eye). RA significantly inhibited pitx2a expression in ventral neural crest (H). Treatment with RA (A) or Pitx2a MO knockdown (E) inhibited the ventral neural crest through misdirected dorsal migration and increased apoptosis (L). Inhibition of RA synthesis (B) or pitx2a overexpression (F) led to clustering of neural crest in the hindbrain region, which resulted in decreased dorsal and ventral migration.
RA and pitx2a had reciprocal effects on neural crest development. RA directed dorsal neural crest while pitx2a was required for the ventral wave. Thus, early control of RA and pitx2a expression was essential for neural crest migration and survival. These studies further our understanding of the molecular and cellular basis of congenital eye diseases.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Live-imaging of 24 hours post fertilization Tg(sox10::EGFP) embryos showed that exogenous RA (A) and Pitx2a MO knockdown (E) inhibited ventral neural crest compared to DMSO (C), untreated (D), and control MO (G). Treatment with diethylbenzaldehyde (DEAB) to inhibit RA synthesis (B) or injection of pitx2a mRNA (F) disrupted dorsal and ventral migration. RA decreased pitx2a expression (E) and increased apoptosis (I) compared to compared to DEAB (F,J), control (G,K), and untreated (H,L).
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