Abstract
Purpose: :
Microphthalmia, generally accompanied by vision defects, is a hallmark of fetal alcohol spectrum disorder (FASD) in humans. In zebrafish, embryonic ethanol (EtOH) exposure also results in microphthalmia. This microphthalmia is in part the consequence of reduced retinal cell differentiation, including photoreceptors (Kashyap et al., 2007; Vis Neurosci 24:409-21). Here we pursue two signaling pathways implicated in other aspects of FASD pathogenesis: retinoic acid (RA), and Hedgehog (Hh). EtOH has been shown to reduce RA synthesis, and to reduce Hh activity by inhibiting the synthesis of cholesterol, which is required for proper Hh signaling.
Methods: :
Embryos were treated with 1.5% EtOH during retinal neurogenesis, 24-48 hrs post-fertilization. Rescue experiments were performed either by adding exogenous RA, or by injecting cholesterol into the yolk. Embryos were processed for anatomical measures including eye size, retinal histology and the detection of retina-specific markers. Effects on signal transduction were evaluated through the use of a specific transgenic RA signaling reporter line (RARE-YFP), and measurement of expression of the Hh signaling target genes, ptc1 and ptc2.
Results: :
RA treatment did not rescue EtOH-induced microphthalmia at any concentration tested, using sequential or co-treatment. RA itself caused microphthalmia, though the underlying mechanisms were distinct from those of EtOH. Interestingly, RA treatment appeared to rescue photoreceptor differentiation in a concentration-dependent manner. However, this is likely an independent effect of exogenous RA, as EtOH treatment alone did not affect RA signaling. Cholesterol injection also did not rescue EtOH-induced microphthalmia at any concentration tested. Measurement of ptc expression is in progress.
Conclusions: :
During the time of retinal neurogenesis, the effects of EtOH on eye development are likely independent of the RA and Hh signaling pathways. Moreover, RA exacerbates the microphthalmic effects of EtOH. These studies suggest that FASD intervention strategies based upon augmentation of RA or Hh signaling will not prevent EtOH-induced microphthalmia. Ongoing studies are aimed at discovery of alternative mechanisms through the use of specific strains of zebrafish showing differential susceptibility to embyronic EtOH exposure.
Keywords: retinal development • retinoids/retinoid binding proteins • photoreceptors