Ethanol-induced microphthalmia is not mediated by changes in retinoic acid or sonic hedgehog signaling during retinal neurogenesis.

ETHANOL CONSUMPTION DURING pregnancy results in a spectrum of irreversible disorders in the fetus ranging from minor birth defects to a severe syndrome involving the brain, eyes, heart, and limbs referred to as fetal alcohol spectrum disorder (FASD; Stratton et al., 1996) Ophthalmic involvement is very common in FASD. Eye defects range from microphthalmia (reduced eye size), optic nerve hypoplasia, and coloboma of the iris and retina, as well as more subtle effects including strabismus and epicanthus (Stromland and Pinazo-Duran, 2002). Microphthalmia is a diagnostic criterion for FASD (Rosett, 1980) and ocular manifestations are seen in 90% of the children with FASD (Stromland and Pinazo-Duran, 2002). The mechanism(s) through which embryonic and fetal ethanol exposure results in visual system defects are not known. The zebrafish has recently achieved credibility as an animal model appropriate for the pursuit of the understanding of the mechanisms underlying FASD in general, and the effects of ethanol on the developing visual system in particular. The vertebrate retina is highly conserved, such that anatomical, physiological, and developmental characteristics of the zebrafish retina resemble those of mammals. During the second and third day of embryonic development, the zebrafish retina undergoes a proliferative phase that overlaps with the generation of ganglion cells, then the cells of the inner nuclear layer, and finally the photoreceptor cells of the outer nuclear layer (Hu and Easter, 1999; Schmitt and Dowling, 1999). The zebrafish embryo develops externally, and therefore can be easily manipulated pharmacologically. A host of molecular and genetic tools are available to evaluate effects on the developing visual system (Easter and Malicki, 2002; Kashyap et al., 2007; Stenkamp, 2007). The zebrafish embryo shows developmental abnormalities at doses of ethanol predicted to match those estimated to occur in human embryos as a consequence of maternal social drinking (Li et al., 2007). These developmental abnormalities resemble the phenotype seen in human FASD, including specific defects in the eye. These defects depend upon the timing of ethanol administration and include cyclopia (Blader and Strahle, 1998; Loucks and Ahlgren, 2009; Loucks et al., 2007), microphthalmia (Bilotta et al., 2002; Kashyap et al., 2007; Matsui et al., 2006), reduced retinal lamination (Arenzana et al., 2006), reduced photoreceptor differentiation (Dlugos and Rabin, 2007; Kashyap et al., 2007; Matsui et al., 2006), and optic nerve hypoplasia (Matsui et al., 2006). Our earlier study (Kashyap et al., 2007) revealed that exposure to ethanol over the period of retinal proliferation and neurogenesis (24 to 48 hours postfertilization [hpf]) results in significant microphthalmia, and that reduced eye size is a consequence of overall developmental delay, increased cell death in the lens, and reduced retinal cell differentiation. The molecular mechanism(s) underlying FASD are not completely known. Various mechanisms have been proposed, including effects via reduced retinoic acid (RA) synthesis (Duester, 1991 ; Marrs et al., 2010; Yelin et al., 2005), and impairment of Sonic hedgehog (Shh) signaling (Ahlgren et al., 2002; Blader and Strahle, 1998; Li et al., 2007). RA, a Vitamin A metabolite, is known to be essential in the normal development of the central nervous system, eyes, limbs, and heart (Zachman and Grummer, 1998). The systems involved in FASD are similar to those seen in Hypovitaminosis A (decreased vitamin A), supporting the hypothesis that the spectrum of manifestations seen in FASD is due to decreased RA signaling (Deltour et al., 1996; Duester, 1991). Ethanol is thought to act by inhibiting the enzymes catalyzing the synthesis of retinaldehyde and RA (Duester, 1998; Duester et al., 1997; Kot-Leibovich and Fainsod, 2009; Fig. 1A). In support of this mechanism, studies in Xenopus (Yelin et al., 2005, 2007), chick (Satiroglu-Tufan and Tufan, 2004), mouse (Johnson et al., 2007), quail (Twal and Zile, 1997), and zebrafish embryos (Marrs et al., 2010) have shown reduced RA levels due to ethanol exposure and/ or were able to rescue, or partially rescue, phenotypes caused by ethanol exposure by treating with RA. Of particular relevance to our current studies, RA plays a vital role in retinal development. An excess of RA during eye morphogenesis results in a double retina phenotype (Hyatt et al., 1996a), whereas inhibition of RA synthesis results in the lack of a ventral retina (Marsh-Armstrong et al., 1994). Later in development, RA promotes the expression of some photoreceptor-specific genes and inhibits the expression of others (Hyatt et al., 1996b; Prabhudesai et al., 2005). However, roles for RA in mediating the effects of ethanol on retinal development have not been tested. Fig. 1 Rationale and experimental design. (A) Flow chart depicting a mechanism through which ethanol may result in developmental defects by inhibition of enzymes involved in retinoic acid (RA) synthesis. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ... Shh is another important morphogen involved in the development of the vertebrate eye. Shh signaling is essential for eye morphogenesis (Chiang et al., 1996; Ekker et al., 1995; Macdonald et al., 1995; Roessler et al., 1996; Stenkamp and Frey, 2003), retinal lamination (Wang et al., 2002), and the development of ganglion cells (Neumann and Nuesslein-Volhard, 2000; Zhang and Yang, 2001) and photoreceptors (Shkumatava et al., 2004; Stenkamp et al., 2000, 2002). Shh has been implicated in pathogenesis of FASD because phenotypic features such as holoprosencephaly and cyclopia are commonly seen as a consequence of defective Shh signaling (Blader and Strahle, 1998; Loucks et al., 2007; Maity et al., 2005; Sulik and Johnston, 1982). In support of this mechanism, studies in mouse (Yamada et al., 2005), chick (Ahlgren et al., 2002), and zebrafish (Li et al., 2007; Loucks and Ahlgren, 2009; Loucks et al., 2007) embryos have shown reduction in Shh signaling after ethanol treatment, and were able to rescue specific phenotypes associated with FASD. The proposed mechanisms for the reduced Shh signaling are reduction in Shh transcripts (Loucks and Ahlgren, 2009; Loucks et al., 2007), or the disruption of cholesterol homeostasis and/ or synthesis (Aguilera et al., 2004; Guizzetti and Costa, 2007; Li et al., 2007), resulting in impaired post translational modification of Shh (Fig. 1B). As with RA, roles for Shh in mediating the effects of ethanol specifically on retinal development have not been pursued. In this study, we test the hypothesis that the microphthalmic effects of embryonic ethanol exposure are mediated by effects on the RA or Shh signaling systems. We use rescue strategies involving supplementation with exogenous RA or cholesterol to ethanol-treated zebrafish embryos. Surprisingly, we find no evidence of rescue of microphthalmia using either RA or cholesterol, suggesting that alternative mechanisms mediate the effects of ethanol on eye development. In support of this interpretation, molecular indicators of RA and Shh signaling within the eye were not affected by ethanol treatments. These studies also revealed that RA itself can cause microphthalmia by reducing retinal cell proliferation. Finally, we report that RA can recover photoreceptor differentiation in ethanol-treated embryos, but likely due to an independent mechanism.