Ethanol-Induced Microphthalmia is Not Mediated by Changes in Retinoic Acid or Sonic Hedgehog Signaling During Retinal Neurogenesis

Abstract

Background Microphthalmia (reduced eye size), generally accompanied by vision defects, is a hallmark of fetal alcohol spectrum disorder (FASD) in humans. In zebrafish, embryonic ethanol exposure over the time of retinal neurogenesis also results in microphthalmia. This microphthalmia is in part the consequence of reduced retinal cell differentiation, including photoreceptors. Here we pursue 2 signaling pathways implicated in other aspects of FASD pathogenesis: retinoic acid (RA) and Sonic hedgehog (Shh). Methods We evaluated markers for RA and Shh signaling within the eyes of embryos treated with ethanol during the period of retinal neurogenesis. We also perrormed rescue experiments using administration of exogenous RA and microinjection of cholesterol, which augments Shh signaling. Results Using sequential or co-treatments, RA did not rescue ethanol-induced microphthalmia at any concentration tested. In addition, RA itself caused microphthalmia, although the underlying mechanisms were distinct from those or ethanol. Interestingly, RA treatment appeared to recover photoreceptor differentiation in a concentration-dependent manner. This may be an independent effect or exogenous RA, as ethanol treatment alone did not alter RA signaling in the eye. Cholesterol injection also did not rescue ethanol-induced microphthalmia at any concentration tested, and ethanol treatments did not alter expression of shh, or of ptc-2, which is normally regulated by Shh signaling. Conclusions Together these findings indicate that, during the time of retinal neurogenesis, effects of ethanol on eye development are likely independent of the RA and Shh signaling pathways. These studies suggest that FASD intervention strategies based upon augmentation or RA or Shh signaling may not prevent ethanol-induced microphthalmia.

Zebrafish retinal defects induced by ethanol exposure are rescued by retinoic acid and folic acid supplement

“…Previous zebrafish-FASD model studies demonstrated loss of visual function at higher concentrations of ethanol (1–2% v/v) using electroretinography, due to reduced rod photoreceptor function, leading to scotopic vision loss (Bilotta et al, 2002; Matsui et al, 2006). Other studies showed that persistent microphthalmia resulted from ethanol exposure when ethanol was administered during retinal neurogenesis (24–48 hpf), showing slight rescue of photoreceptor differentiation by RA treatment (Kashyap et al, 2007; Kashyap, Frey, & Stenkamp, 2011). However, these studies did not differentiate particular retinal cell specification and differentiation events.…”

Molecular and morphological changes in zebrafish following transient ethanol exposure during defined developmental stages

“…While the zebrafish has been gaining traction as an animal model for FAS and FASD, most developmental zebrafish studies have used chronic ethanol exposure times (Bilotta et al, 2004; Arenzana et al, 2006; Dlugos and Rabin, 2007; Kashyap et al, 2007; Loucks et al, 2007; Loucks and Ahlgren, 2009; Kashyap et al, 2011; McCarthy et al, 2013) that likely suffer from not truly representing the behavior of a pregnant woman drinking alcohol during pregnancy. Importantly, the majority of zebrafish embryos exposed to ethanol for these chronic exposures do not survive past the larval stage, suggesting that these chronic exposure models are not representative of human FASD.…”

Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis

“…76 However, retinoic acid supplementation fails to rescue microphthalmia in ethanol-exposed zebrafish. 77 It is likely that the involvement of retinoic acid in ethanol teratogenicity is cell-type specific, as Zhang et al also found retinoic acid failed to rescue eye development but did restore mid-hindbrain development. 56 The precise mechanism by which retinoic acid may be involved in ethanol teratogenesis is unknown but it may relate to cross talk with another signaling pathway, Sonic hedgehog (Shh), 56 which is an indirect target of retinoic acid signaling.…”