Effects of retinoic acid exposure during zebrafish retinogenesis

• Retinoic acid exposure results in persistent microphthalmia mainly due to an increased cell death in the retina.
• Retinal lamination and initial neurogenesis are not affected by retinoic acid exposure.
• Cell differentiation was selectively affected and young axons growing ectopically within the retina.
• Our results provide valuable information about the teratogenic effects of RA in the retinogenesis.

Retinoic acid (RA) is an important morphogen involved in retinal development. Perturbations in its levels cause retinal malformations such as microphthalmia. However, the cellular changes in the retina that lead to this phenotype are little known. We have used the zebrafish to analyse the effects of systemic high RA levels on retinogenesis. For this purpose we exposed zebrafish embryos to 0.1 μM or 1 μM RA from 24 to 48 h post-fertilisation (hpf), the period which corresponds to the time of retinal neurogenesis and initial retinal cell differentiation. We did not find severe alterations in 0.1 μM RA treated animals, but the exposure to 1 μM RA significantly reduced retinal size upon treatment, and this microphthalmia persisted through larval development. We monitored histology and cell death and quantified both the proliferation rate and cell differentiation from 48 hpf onwards, focusing on the retina and optic nerve of normal and 1 μM treated animals. Retinal lamination and initial neurogenesis are not affected by RA exposure, but we found widespread apoptosis after RA treatment that could be the main cause of microphthalmia. Proliferating cells increased their number at 3 days post-fertilisation (dpf) but decreased significantly at 5 dpf maintaining the microphthalmic phenotype. Retinal cell differentiation was affected; some cell markers do not reach normal levels at larval stages and some cell types present an increased number compared to those of control animals. We also found the presence of young axons growing ectopically within the retina. Moreover although the optic axons leave the retina and form the optic chiasm they do not reach the optic tectum. The alterations observed in treated animals become more severe as larvae develop.