Zebrafish Models of Fetal Alcohol Spectrum Disorders

Abstract

Fetal Alcohol Spectrum Disorders (FASD) describes a wide range of structural deficits and cognitive impairments. FASD impacts up to 5% of children born in the United States each year, making ethanol one of the most common teratogens. Due to limitations and ethical concerns, studies in humans are limited in their ability to study FASD. Animal models have proven critical in identifying and characterizing the mechanisms underlying FASD. In this review, we will focus on the attributes of zebrafish that make it a strong model in which to study ethanol-induced developmental defects. Zebrafish have several attributes that make it an ideal model in which to study FASD. Zebrafish produced large numbers of externally fertilized, translucent embryos. With a high degree of genetic amenability, zebrafish are at the forefront of identifying and characterizing the gene-ethanol interactions that underlie FASD. Work from multiple labs has shown that embryonic ethanol exposures result in defects in craniofacial, cardiac, ocular and neural development. In addition to structural defects, ethanol-induced cognitive and behavioral impairments have been studied in zebrafish. Building upon these studies, work has identified ethanol-sensitive loci that underlie the developmental defects. However, analyses show there is still much to be learned of these gene-ethanol interactions. The zebrafish is ideally suited to expand our understanding of gene-ethanol interactions and their impact on FASD. Because of the conservation of gene function between zebrafish and humans, these studies will directly translate to studies of candidate genes in human populations and allow for better diagnosis and treatment of FASD.

Birth defects: the strength of animal models

Birth defects are complex, multifactorial conditions that impact up to 3% of newborns in the US every year, accounting for approximately 20% of all infant deaths (Hoyert, 2006; Yoon, 1997). Yet, while rates of infant mortality have decreased of the last 40 years (Christianson et al., 2006; Matthews et al., 2015), birth defects still cause lifelong physiological and psychological issues requiring extensive, expensive medical care that can cost upwards of $1 billion dollars annually (CDC 2007). Thus, birth defects continue to be a major public health issue. Unfortunately, since the underlying etiologies remain poorly understood, we lack strategies to prevent and/or treat the causes of birth defects (Durham et al., 2017; Harris et al., 2017; Khokha et al., 2017).

Birth defects develop from complex interactions between genetic and environmental factors and the overall number of these factors can be inconceivably large. Just understanding how environmental factors influence birth defects is daunting given that annually, at least 80,000 synthetic chemicals are produced (Landrigan et al., 2006) with a greater magnitude more being produced naturally. Our understanding of the etiology of birth defects is further complicated by the fact that other factors such as the onset, duration and dosage of the ethanol exposure, as well as genetics can modulate the effects environmental factors have on the developing fetus (C. Lovely et al., 2017). Genetic factors can be equally confounding. Genetic variation can take many forms, from single nucleotide polymorphisms to insertion/deletions and from splicing anomalies to large chromosomal rearrangements. These changes underlie the variation in phenotypes we see in the human population (Corsello & Giuffrè, 2012; Webber et al., 2015). In addition, many causal genetic changes can be cryptic in nature, only expressing phenotypes in combination with environmental and/or other genetic factors (Durham et al., 2017; Harris et al., 2017; Khokha et al., 2017). Determining how genetic and environmental factors cause birth defects in clinical populations can be difficult because sample sizes, dependency on self-reports and ethics are major hurdles (reviewed in Khokha et al., 2017).

Animal models can overcome hurdles affecting research in the clinical population. Animal models provide a genetically tractable platform that allows consistently reproducible analyses and afford a degree of genetic and environmental control that would be impossible in human studies (Barkley-Levenson & Crabbe, 2012). For example, using animal models we can precisely examine how environmental inputs, such as ethanol interact with genes, which in turn expands our understanding of the mechanisms underlying gene-environment interactions and birth defects (reviewed extensively elsewhere Eberhart & Parnell, 2016; Khokha et al., 2017; Lovely, 2020; Van Otterloo et al., 2016). The nature of gene-environment interactions underlying birth defects has been studied in multiple model systems. Multiple vertebrate and non-vertebrate models of FASD have been reviewed elsewhere (Comeau et al., 2015; Kelly et al., 2009; Kiecker, 2016; Patten et al., 2014; Sulik, 2005) and are beyond the scope of our review. While no single animal model fully recapitulates all aspects of human FASD, each model has strengths that make them ideal for determining the etiology of ethanol-induced birth defects. In this review, we will focus on the use of zebrafish which have been widely used to understand both the genetic and the environmental factors driving birth defects (Raterman et al., 2020); specifically we will focus on zebrafish studies that examine how the most common environmental cause of birth defects, ethanol interacts with genes.

Ethanol (alcohol) causes many human health problems

Historically, ethanol has been known to have an impact on a developing fetus (Calhoun & Warren, 2007; Dangardt & Chikritzhs, 2020). In 1968, Lemoine and colleagues first described a set of birth defects identified in children exposed prenatally to ethanol (originally published in French in 1968, translated and published in English in 2003; Lemoine et al., 2003). Shortly after, in 1973, Jones and Smith coined the term Fetal Alcohol Syndrome (FAS) to described the constellation of birth deficits associated with prenatal ethanol exposure, which included growth retardation, craniofacial malformations and central nervous system (CNS) abnormalities (KennethL. Jones et al., 1973; KennethL. Jones & Smith, 1973). Since 1973, the spectrum of birth defects associated with prenatal ethanol exposure has grown to include a wider range of structural and organ malformations as well as cognitive impairments; currently these deficits are collectively known as Fetal Alcohol Spectrum Disorders (FASD) (Denny et al., 2017; Popova et al., 2016; Wilhoit et al., 2017; Wozniak et al., 2019).

While FAS is the most severe outcome of prenatal ethanol exposure it is not the most common outcome. FAS has a worldwide prevalence of 0.15% of live births (Popova et al., 2017) while the overall rates of FASD incidence is approximately 0.77% of the general population (Lange et al., 2018; Popova et al., 2019). Furthermore, FASD subpopulations can yet be much higher, ranging from 2–5% in the US and nearly 30% of individuals in some parts of the world with higher incidences of binge drinking (May et al., 2009, 2014, 2018; Popova et al., 2019). The wide variation in prevalence estimates are due to differing sampling techniques using passive (existing medical records), direct, clinic-based (direct from pregnant patients during clinic visits) or active (recruitment for research studies) methods (May et al., 2014; Wilhoit et al., 2017). Differences in diagnostic criteria may also impact these estimates (May et al., 2014). Complicating our understanding of the rates of FASD is the fact that in the US, over 50% of women consumed at least one drink in the previous 30 days (CDC 2009) and nearly half of all pregnancies are unplanned (Finer & Zolna, 2016) with anywhere from 10–30% of pregnant women reporting alcohol consumption (Ethen et al., 2009; Popova et al., 2017, 2018). Furthermore, many pediatricians either fail to recognize FASD (Rojmahamongkol et al., 2015) or are reluctant to enter a diagnosis of FAS into a patient’s medical record (Sampson et al., 1997). Thus, the proposed rates of FASD are likely underestimated (Ethen et al., 2009; Wilhoit et al., 2017) and it is strongly suggested that prenatal ethanol exposure is likely a leading cause of birth defects worldwide.

Interestingly, not every prenatal ethanol exposure leads to birth defects. Only 1 in 67 fetuses exposed to ethanol develop FAS (Popova et al., 2017), while 1 in 13 will develop FASD (Popova et al., 2019). Therefore, it is very likely that multiple factors contribute to the high degree of variability in ethanol induced birth defects. Several clinical studies show that genetics plays a role in FASD. For example, in monozygotic twins there is 100% concordance for FAS phenotypes, while only 64% concordance in dizygotic twins (Streissguth & Dehaene, 1993). Moreover, alleles of ALCOHOL DEHYDROGENASE (ADH) correlate to different sensitivities of FASD (Chevrier et al., 2005; Dodge et al., 2014; Viljoen et al., 2001; Warren & Li, 2005); while both SMC2 and MLLT3 strongly associate with ethanol-induced facial clefting (Beaty et al., 2011). These studies strongly suggest that genetic factors underlie and modulate FASD pathology.

Even though animal models do not fully replicate the complexity of human development, their role in examining the impact of ethanol on human development cannot be understated. For instance, animal models have proven crucial in showing that ethanol is the teratogenic source of FASD, developing our understanding of FASD and the gene-ethanol interactions that underlie it (Ali et al., 2011; Kiecker, 2016; Lovely et al., 2016; Lovely, 2020; Riley et al., 2011; Smith et al., 2014; Stockard, 1910; Sulik, 2005). To fully recapitulate the impact of prenatal ethanol exposure on human development, many different species have been used, including nonhuman primates, rodents, birds, frogs and fish (Comeau et al., 2015; Fainsod & Kot-Leibovich, 2018; Fujisawa et al., 2019; Hong & Zha, 2019; Kelly et al., 2009; Kiecker, 2016; Kot-Leibovich & Fainsod, 2009, p.; Pai & Adams, 2019; Patten et al., 2014; Sulik, 2005; Wang et al., 2006; Wentzel & Eriksson, 2008). By combing the strengths of all these model systems, we have made great inroads in understanding the mechanistic underpinnings of the gene-ethanol interactions driving FASD. This has been extensively reviewed elsewhere (Eberhart & Parnell, 2016; Fernandes et al., 2018; C. B. Lovely, 2020) and will not be covered in this review. However, despite the early success of fish being used to show that ethanol disrupts development of several organ systems, including the brain, face and eyes (Stockard, 1910), fish models of FASD have been historically scarce. Only recently have zebrafish models of FASD gained traction. The goal of this review is to give an overview of the tools available in zebrafish and review how these tool make the zebrafish an ideal model system to identify and understand gene-ethanol interactions.

Zebrafish FASD models are the new kids on the block

The zebrafish was established as a model system in the early 1980s when a small group of researchers, led by George Streisinger and Charles Kimmel helped develop them into a model system for early vertebrate development (Grunwald & Eisen, 2002). The formation of multiple organ systems, including the face, brain, sensory organs, and heart are highly conserved from fish to human; accordingly work in zebrafish has successfully modeled developmental disorders and disease states in these organ systems (Lieschke & Currie, 2007; Santoriello & Zon, 2012). Since more than 70% of genes are homologues between humans and zebrafish (Howe et al., 2013) genetics lie at the heart of this conservation. Interestingly, the percentage of homologous genes between humans and zebrafish increases to 82% when the genes are known to cause human disease (Howe et al., 2013). The combination of zebrafish tools and the conservation rate with humans have caused the use of these fish to grow exponentially in the study of human disease.

The zebrafish possess a constellation of experimental, biological, and behavioral characteristics that make it a well-suited model for studying the impact of embryonic ethanol on development. Zebrafish are highly fecund, with a single mating pair capable of producing hundreds of embryos. These embryos are externally fertilized, which allows for repeated use of the same breeding pairs over multiple experiments as the mothers are not sacrificed to examine the embryos. This controls for variation due to maternal physiology and rearing; thus, enabling high-throughput analyses not capable in mammalian models. In addition, zebrafish embryos allow for control of the onset, duration and dosage of the ethanol exposure. Simply, to generate FASD phenotypes, zebrafish embryos are allowed to develop to a desired embryonic stage. The embryos are then placed in an ethanol solution with a known concentration for a pre-specified exposure duration. To end the ethanol exposure, the embryos are simply removed from the ethanol solution and placed in untreated embryo media. Thus, using zebrafish easily allows for the study of acute, discreet, and/or chronic ethanol exposures in a wide range of doses in hundreds of embryos simultaneously. Moreover, zebrafish embryos are translucent, providing a powerful tool for in vivo, imaging analyses. This allows researchers to connect early developmental impact to later structural/behavioral outcomes. Most importantly, zebrafish have a wealth of tools available for a wide range of genetic manipulation, ranging from transgenesis to the most recent advances in CRISPR/Cas mutagenesis. The advent of CRISPR/Cas mutagenesis approaches have been key in generating targeted knockout and knock-in alleles which are used for genetic/epigenetic editing, genome imaging and lineage tracing (Liu et al., 2019), which in turn have been critical in generating mutant models of rare genetic diseases (Adamson et al., 2018). In addition, many mutant zebrafish models for monogenic human genetic diseases have been created through forward-genetic and transgenic approaches (Lieschke & Currie, 2007).

High fecundity and external fertilization are not unique to zebrafish. Both Xenopus and other fish species produce large numbers of external embryos for ethanol-treatment paradigms and live imaging analyses. Chickens also allow for live imaging and explant analyses albeit with lower experimental numbers. However, Xenopus, chickens nor other fish species have the genetic capabilities of zebrafish. Mice are a powerful genetic model, though the direct manipulation of embryos and subsequent imaging can be difficult and often leads to sacrificing of the mother and embryo preventing longitudinal embryonic analyses. Additionally, compared to rodent models the onset, duration and dosage of the ethanol exposure in generating the FASD phenotypes in zebrafish is simple and precise. While no single research attribute is unique to zebrafish, nor was it the first to begin to characterize the complex etiology of FASD, the zebrafish has quickly shown strong conservation of the impact ethanol has on development, which is similar to other models systems. Ultimately, the unique contribution of zebrafish to FASD research is the combination of the tools available in zebrafish that make it a powerful model to study the etiology of FASD. Next we will focus on how the zebrafish has contributed to our understanding of FASD and how through the use of less-biased, high-throughput genetic screens, it is strongly positioned to drive future analyses of the complex etiology of FASD.

Ethanol exposure paradigms: How much do zebrafish “drink”?

The key to establishing zebrafish as a model of FASD was to determine the physiologically relevant dosages of ethanol. As mentioned above, creating the FASD phenotype in zebrafish is simple and readily controlled, however knowing the ethanol concentration inside the embryo relative to the media concentration and how these zebrafish embryonic concentrations relate to human exposures were not well understood. Based on this lack of understanding, previous zebrafish studies used a wide variety of ethanol doses, ranging from 0.12% to as high as 10% ethanol volume by volume (v/v) in the media (Fernandes et al., 2018). Furthermore, in studies that quantified tissue levels of ethanol the dose also varied widely, ranging from .42% to 70% v/v (Lovely & Eberhart, 2014).

Several possible reasons exist to explain these tissue level differences, including onset, duration and dosage of the ethanol exposure, yet, the cause for these differences is most likely embryonic processing (C. B. Lovely & Eberhart, 2014). More recent work has come to a consensus. Using differing enzymatic assays and Headspace Gas Chromatography, multiple groups have reported the tissue levels of ethanol to be approximately 30% of the concentration of ethanol in the media (Flentke et al., 2014; C. B. Lovely et al., 2014; Reimers et al., 2004; C. Zhang et al., 2013). Critically, regardless of media concentration, the ratio of tissue to media ethanol concentration remains consistently around 30%, (Flentke et al., 2014; C. Zhang et al., 2014); from this, researchers can determine tissue concentrations based on media concentrations. In addition, from the tissue concentrations we can calculate equivalent blood alcohol concentration (BAC) g/dL (BAC). For example, 1% v/v ethanol results in tissue concentrations of 0.3% v/v (C. B. Lovely et al., 2014). Based on embryonic volume (C. B. Lovely et al., 2014), a 0.3% v/v ethanol tissue concentration is approximately equivalent to 0.23 g/dL (BAC). While a 0.23 BAC dose is high, it is still physiologically relevant, since humans are capable of achieving much higher alcohol levels (Canfield et al., 2019; A. W. Jones, 2008; Whaley et al., 2019). Work in zebrafish and other fish species has also shown media doses lower than 1% v/v impacts embryonic development (Buckley et al., 2019; Dlugos et al., 2011; Dlugos & Rabin, 2003; Flentke et al., 2014; Gerlai et al., 2008; Li et al., 2007; E. Loucks & Carvan, 2004; Reimers et al., 2004; Oxendine et al., 2006; Wang et al., 2006). Taken together, the data suggest that zebrafish researchers can generate target ethanol tissue concentrations that replicate human ethanol levels.

While embryonic processing appears to greatly impact tissue quantification, the chorion or genetic background do not appear to have a significant influence on ethanol tissue concentration. The chorion is an acellular structure that envelops zebrafish eggs (Bonsignorio et al., 1996) and does not appear to be a major barrier to ethanol, though it does alter total ethanol levels due to the increase in overall volume of the embryo (Flentke et al., 2014; C. B. Lovely et al., 2014). Genetic background also does not appear to have a substantial effect on tissue levels of ethanol (C. B. Lovely et al., 2014). However, while tissue concentration levels are consistent, the genetic background of developing zebrafish embryos (E. Loucks & Carvan, 2004) and adults (Arenzana et al., 2006; Dlugos & Rabin, 2003; Gerlai et al., 2008, 2009; Pan et al., 2011) can show varying ethanol sensitivities. This phenomena of genetic background modulating ethanol sensitivity is not specific to zebrafish as work in rodent and chick also show genetic background modulates ethanol sensitivity (Cavieres & Smith, 2000; Y. Chen et al., 2011; de Licona et al., 2009; Debelak & Smith, 2000; Dou et al., 2013; Downing et al., 2009; Garic et al., 2014; Gilliam, 2014; Su et al., 2001; Wentzel & Eriksson, 2008). Given that researchers can generate ethanol tissue concentrations in zebrafish capable of recapitulating human ethanol levels; the conserved pathways between zebrafish and other models systems and the vast number of genetics tools available in zebrafish make them a strong model for the study of the genetic mechanisms of FASD.

Zebrafish insights into ethanol teratogenicity

Ethanol is incredibly pleiotropic suggesting that FASD is not driven by a single mechanism. Given the wide range of phenotypes, extensive research has gone into understanding the many abnormalities in ethanol teratogenesis. Studies in multiple model systems have shown that the most sensitive developmental window to ethanol is gastrulation (Becker et al., 1996; Kiecker, 2016; Ponnappa, 2000; Stockard, 1910; Sulik, 2005). Gastrulation is when the germ layers that will eventually give rise to the Central Nervous System (CNS), heart and face, among others, are being generated. Therefore, ethanol exposure during these early windows can disrupt the formation of these structures (Boschen et al., 2021; Dunty et al., 2001; Godin et al., 2010; Higashiyama et al., 2007; Lipinski et al., 2012), ultimately generating the sentinel features of FAS (Stratton et al., 1996). The combination of the tools in zebrafish, described above, place the model system at the forefront of these early exposure analyses and the genetic contexts in which these defects arise.

We will begin by reviewing how zebrafish have been used to understand the structural defects of FASD, then follow up by examining how they have also been used to study the behavioral and cognitive defects associated with FASD. We will end the review by examining how the genetic tools of zebrafish provide the framework to greatly expand our understanding of the gene-ethanol interactions underlying FASD.

Early FASD studies focused on the similarities of holoprosencephaly and some children with FASD (Su et al., 2001; Sulik, 2005; Yelin et al., 2005, 2007). The precision of ethanol exposure paradigms in zebrafish have further clarified the impact of ethanol exposure during early gastrulation. Work from Blader and Strähle (Blader & Strähle, 1998) showed that ethanol-treatment from dome to 30% epiboly (roughly 4.3–4.7 hours post-fertilization, hpf – late blastula stages), resulted in cyclopic phenotypes. A similar ethanol exposure window also resulted in split body axis which is associated with altered gastrulation cell movements (Y. Zhang et al., 2010). Additional work showed that genetic background modulates ethanol-induced cyclopia with the zebrafish strain EK being most sensitive, followed by AB and GL strains (Arenzana et al., 2006). Combined, these results are highly similar to phenotypes observed in the PCP mutant wnt11 (Heisenberg et al., 2000; Ulrich, 2003). Work in Xenopus and zebrafish show that the Wnt/PCP pathway plays a critical role in regulating early gastrulation cell movements, suggesting that ethanol is disrupting cell signaling during these cell movements (Hardy et al., 2008; Heisenberg et al., 2000; Ohkawara, 2003; Roszko et al., 2009; Ulrich, 2003). Recent work from zebrafish has shown that Wnt/PCP members vangl2 and gcp4 interact with ethanol during gastrulation disrupting convergence extension leading to cyclopia, an extreme form of holoprosencephaly (Sidik et al., 2021; Swartz et al., 2014).

Beyond the early cell movements driving gastrulation, these early ethanol exposure windows lead to many other ethanol-induced phenotypes. One of the main phonotypes observed in FASD is growth retardation (Wozniak et al., 2019). Multiple animals models show that ethanol exposure during gastrulation results in significant reductions to the overall embryo size (Peng et al., 2005; Sulik, 2005). Zebrafish have also shown a dose dependent decrease in embryo size with higher doses resulting in greater size reductions (Ali et al., 2011; Bilotta et al., 2004; E. J. Loucks & Ahlgren, 2009). Interestingly, this may be in part due to disruption to Shh signaling (E. J. Loucks & Ahlgren, 2009). In addition to general growth retardation, brain, cardiac and eye defects are extremely common (Bilotta et al., 2002, 2004; Cavieres & Smith, 2000; Dlugos & Rabin, 2010; Ponnappa, 2000; Wilhoit et al., 2017; Wozniak et al., 2019). Ethanol exposure in zebrafish results in significant increases in cardiac edema, reduction in heart volume and reduced ventricular thickness at (Dlugos & Rabin, 2010). Using the live imaging capabilities in zebrafish Sarmah et al. (Sarmah et al., 2016; Sarmah & Marrs, 2013) report that ethanol exposure during gastrulation disrupts precursor migration, heart looping and gene expression in cardiac development. This work showed that ethanol alters both Bmp and Notch signaling, tying these signaling pathways to ethanol-induced cardiac defects (Sarmah et al., 2016). In addition to heart development, ethanol exposure also disrupts eye and brain development and function (Bilotta et al., 2002, 2004; Dlugos & Rabin, 2007; Muralidharan et al., 2018; C. Zhang et al., 2013, 2014). Zhang et al (C. Zhang et al., 2011, 2013, 2014) showed ethanol treatment at similar time windows resulted in defects in eye development as well as midbrain-hindbrain formation and these defects may be Shh- and Fgf-dependent (C. Zhang et al., 2011). Wnt-ethanol interactions may also play a role in eye development as well protecting retinal cell differentiation (Muralidharan et al., 2018). Furthermore, other sensory systems are ethanol sensitive as well. Ethanol exposure results in the reduction of cell numbers in both the inner ear and lateral line (Uribe et al., 2013; Zamora & Lu, 2013). One possible explanation for this cell number reduction is increased cell death, which is observed in the sensory hair cells of the inner ear (Uribe et al., 2013).

Ethanol-induced cell death is also observed in the cranial neural crest in chicken, mouse and zebrafish (Debelak & Smith, 2000; Dunty et al., 2001; Flentke et al., 2014; McCarthy et al., 2013; Smith et al., 2014). Cranial neural crest cells give rise to the majority of the facial skeleton as well as several other cell types in the developing head (Mork & Crump, 2015). Multiple studies in various model systems have found that ethanol alters facial shape and structure which can be modulated by genetic background (Ali et al., 2011; Carvan et al., 2004a; Downing et al., 2009; Flentke et al., 2014; Lipinski et al., 2012; Marrs et al., 2010; McCarthy et al., 2013; Su et al., 2001; Swartz et al., 2014, 2020). Several of the studies in zebrafish show that genetic background modulates the increase in cell death in the cranial neural crest (Flentke et al., 2014; McCarthy et al., 2013; Swartz et al., 2014). Importantly, the live imaging capabilities of zebrafish show that beyond cell death, cell migration of the cranial neural crest is also disrupted by ethanol exposure (Boric et al., 2013). Along with more recent work in frog showing ethanol-induced defects in neural crest migration (Shi et al., 2014), this suggest that ethanol-induced migration defects may underlie the subtle craniofacial phenotypes observed in humans.

Beyond the face and organs, data from animal models (Schneider et al., 2011) including zebrafish (Fernandes et al., 2015a, 2015b) show that the brain is similarly sensitive to embryonic ethanol exposure. For example, ethanol exposure from gastrulation to pharyngeal stages (6–24 hours post fertilization (hpf)) disrupts formation of the mid-hindbrain boundary (C. Zhang et al., 2013, 2015) and reduces the number of elavl3-positive neural progenitors (Joya et al., 2014). Using transgenic lines that label unique CNS neurons, Buckley et al. (2019) found migration of facial branchial motor neurons to be ethanol sensitive. Additionally, the branching of secondary motor neurons increased and the diameter of Mautner axons decreased due to ethanol exposure (Shan et al., 2015). Ethanol also disrupts migration of hypocretin/orexin neurons (Collier et al., 2019). Furthermore, embryonic ethanol exposure in zebrafish, in a very narrow window, 24 to 26 hpf, has been shown to reduce the expression of brain‐derived neurotrophic factor (BDNF), neuronal cell adhesion molecule (NCAM) and synaptophysin which are important for brain development and plasticity (Mahabir et al., 2018). Embryonic ethanol exposure during this developmental window has also been shown to reduce L-, T- and N- type Ca++ and the SCN1A Na+ voltage-gated cation channels in the pallium and cerebellum of zebrafish (Chatterjee et al., 2021b) and glial cells and cell membrane components in the hindbrain (Chatterjee et al., 2021a).

Furthermore, structural changes in brain formation has been linked to impaired behavior and cognition (Nuñez & Sowell, 2011; Riley & McGee, 2005). Individuals with FASD demonstrate impaired social functioning (Rockhold et al., 2021). Data from animal models also strongly suggest that embryonic ethanol exposure alters social behavior (Kelly et al., 2009). Unlike any other animal model system, zebrafish form social groups called shoals (Norton & Bally-Cuif, 2010; Pitcher, 1983), thus they are ideal model organism in which to study the impact embryonic ethanol exposure has on social behavior (Fernandes et al., 2018). Research has shown that exposure to 1% ethanol from 24 to 26 hpf or exposure to 0.12% v/v ethanol from (48 to 216 hpf - 9 days) impairs the shoaling response to a virtual shoal or a live zebrafish (Buske & Gerlai, 2011; Fernandes et al., 2015a, 2015b; Fernandes & Gerlai, 2009; Parker et al., 2014a). Additionally individuals with FASD also show deficits in learning and memory (Kodituwakku, 2007), which can be recapitulated in zebrafish (Bailey, Oliveri, & Levin, 2015; Bailey, Oliveri, Zhang, et al., 2015; Carvan et al., 2004b; Fernandes et al., 2014).

The effects of prenatal alcohol exposure persist through child and adulthood (Streissguth, Ann P et al., 1991). More recently the effects of embryonic ethanol exposure on young fish called larva have begun to be examined. Researchers have found hypoactivity in 5-day old fish following embryonic ethanol exposure (Ali et al., 2011) as well as impaired locomotor activity in 2-day old fish (Shan et al., 2015). Additionally, exposure to 0.25% v/v and 0.50% v/v ethanol from 24 to 26 hpf has been shown to increase anxiety in 10-day old larvae (Pinheiro-da-Silva et al., 2020). Altogether, this demonstrates that the zebrafish is ideally suited to study the link between brain structure and function in FASD.

Gene-ethanol interactions and zebrafish

Ample evidence shows that genetics play an important role in ethanol teratogenesis. Studies in multiple model systems show that different strains have different sensitivities to ethanol-induced defects (Cavieres & Smith, 2000; S. Chen et al., 2000; Dlugos & Rabin, 2003; E. Loucks & Carvan, 2004; Su et al., 2001). While we know genetics play a role in FASD, to date we have identified relatively few ethanol-sensitive loci. Zebrafish possess a number of genetic tools available which have proven extremely powerful in our understanding of ethanol-sensitive loci. Morpholinos, while controversial (Kok et al., 2015), have been used to rapidly knock down gene expression and screen potential ethanol sensitive genetic loci (C. Zhang et al., 2011, 2013, 2015). More large-scale, high throughput genetic approaches, tilling projects, such as the Zebrafish Mutation Project (ZMP), transgenic and forward genetic screens have created mutations in more than 60% of the zebrafish protein coding genes. In addition, the newer gene editing techniques, CRISPR/Cas9 approaches, have made it possible to directly target nearly any gene of interest and have been highly successful in zebrafish. More recent work has shown the utility of CRISPR/Cas9 technologies in large scale genetic screens in animals (Parvez et al., 2021). Most importantly, work from multiple labs have shown that these zebrafish genetic tools are readily applicable to FASD studies and have already begun to inform our understanding of FASD.

Some of the early evidence for gene-ethanol interactions in zebrafish came from studies of the candidate genes. Ethanol degradation is a multi-step process where each step can be a target of ethanol sensitivity. In zebrafish, Retinoic Acid (RA) is critical for many developmental processes (Samarut et al., 2015). Retinaldehyde dehydrogenases (RALDHs) convert retinal to retinoic acid but also degrade ethanol. Thus, one hypothesis is that ethanol is a competitive inhibitor of RA synthesis (Duester, 1991; Pullarkat, 1991). Recent work in frog has shown that the ethanol metabolite, acetaldehyde, acts as the competitive inhibitor of both Xenpous RALDH2 and Human RALDH2 disrupting RA synthesis, which is rescued with exogenous RA (Shabtai et al., 2018). Multiple studies have shown that RA supplementation plays a protective role in multiple tissues exposed to ethanol (Burton et al., 2017; Ferdous et al., 2017; Marrs et al., 2010; Rah et al., 2019; C. Zhang et al., 2015). However, RA supplementation fails to rescue microphthalmia in ethanol-exposed zebrafish, suggesting that the role of RA may be tissue or gene specific (Kashyap et al., 2011; C. Zhang et al., 2015). A promising role for RA signaling is regulating Sonic hedgehog (Shh) signaling (Petrelli et al., 2019; C. Zhang et al., 2015).

Loss of Shh signaling results in neural tube defects, loss of midline craniofacial structures and neural crest-specific cell death (Chiang et al., 1996; Eberhart et al., 2006; Jeong, 2004; Pan et al., 2011; Roessler & Muenke, 2010; Wada et al., 2005). These structural defects in FASD are strikingly similar and these phenotypes present in children exposed prenatally to ethanol (Johnson & Rasmussen, 2010; K. L. Jones, 2011). This suggests that Shh signaling may be ethanol sensitive leading to several of the structural defects of FASD. Work in mice, chicken and zebrafish show that ethanol exposure reduces Shh signaling leading to increased CNCC death, as well as disrupted midline and eye development (Ahlgren et al., 2002; Aoto et al., 2008; Higashiyama et al., 2007; Kashyap et al., 2011; Li et al., 2007; Parker et al., 2014b) has shown that these phenotypes can be rescued by injection of shh mRNA (E. J. Loucks & Ahlgren, 2009). One potential mechanism attenuation of Shh signaling by ethanol is disruption of cholesterol modification (Li et al., 2007) and supplemental cholesterol protects against these ethanol induced defects (Ehrlich et al., 2012). Work in zebrafish provides more evidence that gene-ethanol interactions in the Shh pathway drive FASD phenotypes. Morpholino knockdown of shha disrupts GABAergic and glutamatergic neural development (C. Zhang et al., 2013), while ethanol interacts with agrin, a mediator of Shh signaling during eye development (C. Zhang et al., 2011).

The studies in gene-ethanol interactions described above are informative but are biased towards FASD phenotypes, which limits their scope. One of the biggest strengths of zebrafish is the ability to perform large scale, genetic screens. Zebrafish have been extensively and successfully used in these large scale genetic screens to expand our understanding of developmental biology (Haffter & Nüsslein-Volhard, 1996; Huang et al., 2012; Lawson & Wolfe, 2011; Piotrowski et al., 1996; Schilling et al., 1996). Recently, these genetic screens have been modified to identify ethanol-sensitive genetic loci. The first was a pilot screen for gene-ethanol interactions in five craniofacial mutant lines, smoothened (a Shh pathway member), cyp26b1 (RA catabolizing enzyme), smad5 (Bone Morphogenetic Protein (BMP) pathway member), gata3 (transcription factor) and pdgfra (growth factor signaling receptor). Interestingly, neither smoothened nor cyp26b1, members of the ethanol sensitive Shh and RA pathway, respectively, interacted with ethanol. However, from this screen, pdgfra (platelet-derived growth factor receptor alpha), was found to be highly ethanol sensitive (McCarthy et al., 2013) which suggests that growth factor signaling is a target of ethanol in FASD.

Work in zebrafish, mice and human have shown that loss of pdgfra leads to cleft palate (Eberhart et al., 2008; Hoch & Soriano, 2003; Rattanasopha et al., 2012; Soriano, 1997). When exposed to ethanol, zebrafish embryos lacking pdgfra showed increases in neural crest cell death, leading to defects in the craniofacial skeleton, particularly the palate (McCarthy et al., 2013). Critically, single nucleotide polymorphisms (SNPs) in both PDGFRA and PDGFRB in humans are significantly associated ethanol-induced facial shape changes (McCarthy et al., 2013). McCarthy et al., went on to show that ethanol acts downstream of Pdgfra, at the level of mTOR. Pdgfra signaling through PI3K/mTOR regulating cell survival, proliferation and growth (Dibble & Cantley, 2015; Klinghoffer & Hamilton, 2002). As a result, the ethanol-induced facial defects can be rescued by upregulation of the mTOR pathway through either knockdown of the PI3K inhibitor, pten or supplementation of L-leucine (McCarthy et al., 2013). This demonstrated that zebrafish genetic screens could be used to identify novel, ethanol-sensitive genes that predict ethanol-sensitive genes in human FASD but can also identify therapeutic interventions for those gene-ethanol interactions.

This initial screen was expanded to screen 20 mutants available from the Zebrafish International Resource Center (ZIRC). From this group of 20 mutants, five ethanol-sensitive mutant lines were identified (Swartz et al., 2014). Surprisingly these novel ethanol-sensitive genes that spanned a wide range of cellular functions with no obvious link between them. The ethanol-sensitive loci identified included transcription factor foxi1, mars, a methionine-tRNA synthetase required for protein translation, cell cycle components hinfp and plk1 and the planar cell polarity pathway member, vangl2. The observed phenotypes displayed a range of structural defects, including general growth retardation, defects to the craniofacial skeleton and eyes defects, reduced axonal projections and increased in cell death.

For four of genes, foxi1, mars, hinfp and plk1, ethanol exacerbates the existing phenotypes (Swartz et al., 2014). Embryos lacking foxi1 exhibit the posterior craniofacial defects, in particular to specific craniofacial cartilage elements called the hyosymplectic, and ceratobranchials (Solomon et al., 2003). When treated with ethanol these cartilage elements are lost. In addition, axon projections are mislocalized and ear size is reduced. Embryos lacking mars also exacerbates existing ventral craniofacial cartilage elements and eye defects. Loss of the cell cycle components, hinfp and plk1, leads to microcephaly (small head) and microphthalmia (small eyes) in both mutants, while plk1 mutants exhibit cell apoptosis and mislocalized axonal projections. However, ethanol exposure results in different phenotypic outcomes. In embryos lacking hinfp, ethanol exposure exacerbated the microcephaly and microphthalmia and led to loss of the lower jaw. Loss of plk1 led to complete loss of the craniofacial skeleton, a large expansion of cell apoptosis and near complete loss of axon projections. The planar cell polarity pathway member, vangl2, interacted with ethanol the strongest. Required for convergent extension, loss of vangl2 results in a low incidence of cyclopia (Marlow et al., 1998). Ethanol exposure results in full penetrance of cyclopia with severe defects to the palate and ventral craniofacial cartilage elements and disrupted cranial nerve projections. More recent work from the Eberhart lab shows that ethanol exposure in vangl2 mutants disrupts the number and polarity of cell protrusions. Strikingly, this ethanol exposure does not impact expression of PCP members, but acts indirectly through localization of shha expression (Sidik et al., 2021). This work demonstrates ethanol can interact with and disrupt multiple pathways underlying FASD, though human vangl2 has yet to be implicated in FASD.

Both of these initial zebrafish screens were shelf-screens, i.e. screens of existing mutant lines. While incredibly informative, these less biased approaches, these screens are still limited to previously curated mutant lines. To eliminate this, the most recent zebrafish ethanol screen utilized a forward genetic approach (Swartz et al., 2020). This approach eliminates bias towards known mutants by randomly mutagenizing the genome through ENU treatment (Lawson & Wolfe, 2011) and screening for unique ethanol-induced phenotypes to the craniofacial skeleton, then identifying the causal mutation. Using this approach, Swartz et al (Swartz et al., 2020), identified five novel ethanol-sensitive mutants from a screen of 126 inbred F2 families, au15, au27, au28, au29 and au32. A 6^th spontaneous mutant, au26, was identified screening for background ethanol sensitivity in wild type zebrafish. Of the six mutants identified, five were recessive in nature and only one, au29, exhibited phenotypes in the absence of ethanol, reduced palate and reduced and malformed ventral craniofacial cartilage elements. The ethanol-induced phenotypes spanned of a range of facial defects. Ethanol-treated au26 embryos displayed cleft palate, while au15 showed jaw loss and au28 had a very narrow hypoplasia to the hyosymplectic facial cartilages. Bone loss, but not cartilage defects were observed in au27 and au32 mutants had reductions in embryonic size and defects to both the palate and jaw. One gene, au29 was mapped to a previously uncharacterized gene, si:dkey-88l16.3. This suggests that the rest of the lines will map to previously uncharacterized mutations and provide new novel gene-ethanol interactions to interrogate. Overall, this demonstrates that the importance of zebrafish genetic screens in expanding our understanding of ethanol teratogenesis.

Conclusions

FASD continues to be a significant problem in our society. Estimating to impact up to 5% of children in the US, though this is likely an underestimate (Ethen et al., 2009; May et al., 2009, 2014, 2018). Combined with the fact that more than 50% of childbearing women drink and nearly 50% of pregnancies are unplanned (Finer & Zolna, 2016), FASD continues to impact the lives of millions of people. Building a complete understanding of FASD is critical to human health and animal models have been key in the process (C. B. Lovely, 2020).

The etiology of FASD is exceedingly complex with multiple factors driving its pathology. The zebrafish is ideally suited to study the complex interplay between these factors. In this review we discussed how the features of the zebrafish has contributed to our understanding of FASD. The ultimate strength of zebrafish is in combining these various tools to address fundamental questions in FASD etiology. Recent work in zebrafish has shown that the combined high throughput genetic analyses and imaging capabilities can successfully identify novel gene-ethanol interactions and give insight to their mechanistic underpinnings (McCarthy et al., 2013; Swartz et al., 2014). Due to the high degree of conservation of gene function between zebrafish and human, studies in zebrafish will directly inform human studies. To support this, McCarthy et al (McCarthy et al., 2013) showed that the ethanol-sensitive locus pdgfra associates with facial dysmorphology in human FASD patients. This demonstrates that zebrafish studies are vital to our understanding of FASD and to our ability to identify therapeutic approaches to overcome its catastrophic effects. Zebrafish and its combined strengths, is becoming one of the prominent model systems in FASD research.