Zebrafish raptor mutation inhibits the activity of mTORC1, inducing craniofacial defects due to autophagy-induced neural crest cell death.

The mechanistic target of rapamycin (mTOR) coordinates metabolism and cell growth with environmental inputs. mTOR forms two functional complexes: mTORC1 and mTORC2. Proper development requires both complexes but mTORC1 has unique roles in numerous cellular processes, including cell growth, survival and autophagy. Here, we investigate the function of mTORC1 in craniofacial development. We created a zebrafish raptor mutant via CRISPR/Cas9, to specifically disrupt mTORC1. The entire craniofacial skeleton and eyes were reduced in size in mutants; however, overall body length and developmental timing were not affected. The craniofacial phenotype associates with decreased chondrocyte size and increased neural crest cell death. We found that autophagy is elevated in raptor mutants. Chemical inhibition of autophagy reduced cell death and improved craniofacial phenotypes in raptor mutants. Genetic inhibition of autophagy, via mutation of the autophagy gene atg7, improved facial phenotypes in atg7;raptor double mutants, relative to raptor single mutants. We conclude that finely regulated levels of autophagy, via mTORC1, are crucial for craniofacial development.

Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling.

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant's phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.

Predicting Modifiers of Genotype-Phenotype Correlations in Craniofacial Development.

Most human birth defects are phenotypically variable even when they share a common genetic basis. Our understanding of the mechanisms of this variation is limited, but they are thought to be due to complex gene-environment interactions. Loss of the transcription factor Gata3 associates with the highly variable human birth defects HDR syndrome and microsomia, and can lead to disruption of the neural crest-derived facial skeleton. We have demonstrated that zebrafish gata3 mutants model the variability seen in humans, with genetic background and candidate pathways modifying the resulting phenotype. In this study, we sought to use an unbiased bioinformatic approach to identify environmental modifiers of gata3 mutant craniofacial phenotypes. The LINCs L1000 dataset identifies chemicals that generate differential gene expression that either positively or negatively correlates with an input gene list. These chemicals are predicted to worsen or lessen the mutant phenotype, respectively. We performed RNA-seq on neural crest cells isolated from zebrafish across control, Gata3 loss-of-function, and Gata3 rescue groups. Differential expression analyses revealed 551 potential targets of gata3. We queried the LINCs database with the 100 most upregulated and 100 most downregulated genes. We tested the top eight available chemicals predicted to worsen the mutant phenotype and the top eight predicted to lessen the phenotype. Of these, we found that vinblastine, a microtubule inhibitor, and clofibric acid, a PPAR-alpha agonist, did indeed worsen the gata3 phenotype. The Topoisomerase II and RNA-pol II inhibitors daunorubicin and triptolide, respectively, lessened the phenotype. GO analysis identified Wnt signaling and RNA polymerase function as being enriched in our RNA-seq data, consistent with the mechanism of action of some of the chemicals. Our study illustrates multiple potential pathways for Gata3 function, and demonstrates a systematic, unbiased process to identify modifiers of genotype-phenotype correlations.

Exposure to ethanol leads to midfacial hypoplasia in a zebrafish model of FASD via indirect interactions with the Shh pathway.

BACKGROUND: Gene-environment interactions are likely to underlie most human birth defects. The most common known environmental contributor to birth defects is prenatal alcohol exposure. Fetal alcohol spectrum disorders (FASD) describe the full range of defects that result from prenatal alcohol exposure. Gene-ethanol interactions underlie susceptibility to FASD, but we lack a mechanistic understanding of these interactions. Here, we leverage the genetic tractability of zebrafish to address this problem. RESULTS: We first show that vangl2, a member of the Wnt/planar cell polarity (Wnt/PCP) pathway that mediates convergent extension movements, strongly interacts with ethanol during late blastula and early gastrula stages. Embryos mutant or heterozygous for vangl2 are sensitized to ethanol-induced midfacial hypoplasia. We performed single-embryo RNA-seq during early embryonic stages to assess individual variation in the transcriptional response to ethanol and determine the mechanism of the vangl2-ethanol interaction. To identify the pathway(s) that are disrupted by ethanol, we used these global changes in gene expression to identify small molecules that mimic the effects of ethanol via the Library of Integrated Network-based Cellular Signatures (LINCS L1000) dataset. Surprisingly, this dataset predicted that the Sonic Hedgehog (Shh) pathway inhibitor, cyclopamine, would mimic the effects of ethanol, despite ethanol not altering the expression levels of direct targets of Shh signaling. Indeed, we found that ethanol and cyclopamine strongly, but indirectly, interact to disrupt midfacial development. Ethanol also interacts with another Wnt/PCP pathway member, gpc4, and a chemical inhibitor of the Wnt/PCP pathway, blebbistatin, phenocopies the effect of ethanol. By characterizing membrane protrusions, we demonstrate that ethanol synergistically interacts with the loss of vangl2 to disrupt cell polarity required for convergent extension movements. CONCLUSIONS: Our results show that the midfacial defects in ethanol-exposed vangl2 mutants are likely due to an indirect interaction between ethanol and the Shh pathway. Vangl2 functions as part of a signaling pathway that regulates coordinated cell movements during midfacial development. Ethanol exposure alters the position of a critical source of Shh signaling that separates the developing eye field into bilateral eyes, allowing the expansion of the midface. Collectively, our results shed light on the mechanism by which the most common teratogen can disrupt development.

Loss of tumor protein 53 protects against alcohol-induced facial malformations in mice and zebrafish.

BACKGROUND: Alcohol exposure during the gastrulation stage of development causes the craniofacial and brain malformations that define fetal alcohol syndrome. These malformations, such as a deficient philtrum, are exemplified by a loss of midline tissue and correspond, at least in part, to regionally selective cell death in the embryo. The tumor suppressor protein Tp53 is an important mechanism for cell death, but the role of Tp53 in the consequences of alcohol exposure during the gastrulation stage has yet to be examined. The current studies used mice and zebrafish to test whether genetic loss of Tp53 is a conserved mechanism to protect against the effects of early developmental stage alcohol exposure. METHODS: Female mice, heterozygous for a mutation in the Tp53 gene, were mated with Tp53 heterozygous males, and the resulting embryos were exposed during gastrulation on gestational day 7 (GD 7) to alcohol (two maternal injections of 2.9 g/kg, i.p., 4 h apart) or a vehicle control. Zebrafish mutants or heterozygotes for the tp53zdf1  M214K mutation and their wild-type controls were exposed to alcohol (1.5% or 2%) beginning 6 h postfertilization (hpf), the onset of gastrulation. RESULTS: Examination of GD 17 mice revealed that eye defects were the most common phenotype among alcohol-exposed fetuses, occurring in nearly 75% of the alcohol-exposed wild-type fetuses. Tp53 gene deletion reduced the incidence of eye defects in both the heterozygous and mutant fetuses (to about 35% and 20% of fetuses, respectively) and completely protected against alcohol-induced facial malformations. Zebrafish (4 days postfertilization) also demonstrated alcohol-induced reductions of eye size and trabeculae length that were less common and less severe in tp53 mutants, indicating a protective effect of tp53 deletion. CONCLUSIONS: These results identify an evolutionarily conserved role of Tp53 as a pathogenic mechanism for alcohol-induced teratogenesis.

Visible Light Mediated Bidirectional Control over Carbonic Anhydrase Activity in Cells and in Vivo Using Azobenzenesulfonamides.

Two azobenzenesulfonamide molecules with thermally stable cis configurations resulting from fluorination of positions ortho to the azo group are reported that can differentially regulate the activity of carbonic anhydrase in the trans and cis configurations. These fluorinated probes each use two distinct visible wavelengths (520 and 410 or 460 nm) for isomerization with high photoconversion efficiency. Correspondingly, the cis isomer of these systems is highly stable and persistent (as evidenced by structural studies in solid and solution state), permitting regulation of metalloenzyme activity without continuous irradiation. Herein, we use these probes to demonstrate the visible light mediated bidirectional control over the activity of zinc-dependent carbonic anhydrase in solution as an isolated protein, in intact live cells and in vivo in zebrafish during embryo development.

Multifactorial Genetic and Environmental Hedgehog Pathway Disruption Sensitizes Embryos to Alcohol-Induced Craniofacial Defects.

BACKGROUND: Prenatal alcohol exposure (PAE) is perhaps the most common environmental cause of human birth defects. These exposures cause a range of structural and neurological defects, including facial dysmorphologies, collectively known as fetal alcohol spectrum disorders (FASD). While PAE causes FASD, phenotypic outcomes vary widely. It is thought that multifactorial genetic and environmental interactions modify the effects of PAE. However, little is known of the nature of these modifiers. Disruption of the Hedgehog (Hh) signaling pathway has been suggested as a modifier of ethanol teratogenicity. In addition to regulating the morphogenesis of craniofacial tissues commonly disrupted in FASD, a core member of the Hh pathway, Smoothened, is susceptible to modulation by structurally diverse chemicals. These include environmentally prevalent teratogens like piperonyl butoxide (PBO), a synergist found in thousands of pesticide formulations. METHODS: Here, we characterize multifactorial genetic and environmental interactions using a zebrafish model of craniofacial development. RESULTS: We show that loss of a single allele of shha sensitized embryos to both alcohol- and PBO-induced facial defects. Co-exposure of PBO and alcohol synergized to cause more frequent and severe defects. The effects of this co-exposure were even more profound in the genetically susceptible shha heterozygotes. CONCLUSIONS: Together, these findings shed light on the multifactorial basis of alcohol-induced craniofacial defects. In addition to further implicating genetic disruption of the Hh pathway in alcohol teratogenicity, our findings suggest that co-exposure to environmental chemicals that perturb Hh signaling may be important variables in FASD and related craniofacial disorders.

Novel Ethanol-Sensitive Mutants Identified in an F3 Forward Genetic Screen.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) collectively refer to all deleterious outcomes due to prenatal alcohol exposures. Alterations to the face are common phenotypes in FASD. While alcohol exposure is the underlying cause of FASD, many variables modify the outcomes of such exposures. Genetic risk is one such variable, yet we still have a limited understanding of the nature of the genetic loci mediating susceptibility to FASD. METHODS: We employed ENU-based random mutagenesis in zebrafish to identify mutations that enhanced the teratogenicity of ethanol (EtOH). F3 embryos obtained from 126 inbred F2 families were exposed to 1% EtOH in the medium (approximately 41 mM tissue levels). Zebrafish stained with Alcian Blue and Alizarin Red were screened for qualitative alterations to the craniofacial skeleton between 4 and 7 days postfertilization (dpf). RESULTS: In all, we recovered 6 EtOH-sensitive mutants, 5 from the genetic screen itself and one as a background mutation in one of our wild-type lines. Each mutant has a unique EtOH-induced phenotype relative to the other mutant lines. All but 1 mutation appears to be recessive in nature, and only 1 mutant, au29, has apparent craniofacial defects in the absence of EtOH. To validate the genetic screen, we genetically mapped au29 and found that it carries a mutation in a previously uncharacterized gene, si:dkey-88l16.3. CONCLUSIONS: The phenotypes of these EtOH-sensitive mutants differ from those in previous characterizations of gene-EtOH interactions. Thus, each mutant is likely to provide novel insights into EtOH teratogenesis. Given that most of these mutants only have craniofacial defects in the presence of EtOH and our mapping of au29, it is also likely that many of the mutants will be previously uncharacterized. Collectively, our findings point to the importance of unbiased genetic screens in the identification, and eventual characterization, of risk alleles for FASD.

In vivo zebrafish morphogenesis shows Cyp26b1 promotes tendon condensation and musculoskeletal patterning in the embryonic jaw.

Integrated development of diverse tissues gives rise to a functional, mobile vertebrate musculoskeletal system. However, the genetics and cellular interactions that drive the integration of muscle, tendon, and skeleton are poorly understood. In the vertebrate head, neural crest cells, from which cranial tendons derive, pattern developing muscles just as tendons have been shown to in limb and trunk tissue, yet the mechanisms of this patterning are unknown. From a forward genetic screen, we determined that cyp26b1 is critical for musculoskeletal integration in the ventral pharyngeal arches, particularly in the mandibulohyoid junction where first and second arch muscles interconnect. Using time-lapse confocal analyses, we detail musculoskeletal integration in wild-type and cyp26b1 mutant zebrafish. In wild-type fish, tenoblasts are present in apposition to elongating muscles and condense in discrete muscle attachment sites. In the absence of cyp26b1, tenoblasts are generated in normal numbers but fail to condense into nascent tendons within the ventral arches and, subsequently, muscles project into ectopic locales. These ectopic muscle fibers eventually associate with ectopic tendon marker expression. Genetic mosaic analysis demonstrates that neural crest cells require Cyp26b1 function for proper musculoskeletal development. Using an inhibitor, we find that Cyp26 function is required in a short time window that overlaps the dynamic window of tenoblast condensation. However, cyp26b1 expression is largely restricted to regions between tenoblast condensations during this time. Our results suggest that degradation of RA by this previously undescribed population of neural crest cells is critical to promote condensation of adjacent scxa-expressing tenoblasts and that these condensations are subsequently required for proper musculoskeletal integration.

Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish.

The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face.

Social deficits following embryonic ethanol exposure arise in post-larval zebrafish.

Prenatal alcohol exposure is the leading cause of birth defects, collectively termed fetal alcohol spectrum disorders (FASD). In the United States and Canada, 1 in 100 children will be born with FASD. Some of the most commonly debilitating defects of FASD are in social behavior. Zebrafish are highly social animals, and embryonic ethanol exposure from 24 to 26 hours post-fertilization disrupts this social (shoaling) response in adult zebrafish. Recent findings have suggested that social behaviors are present in zebrafish larvae as young as 3 weeks, but how they relate to adult shoaling is unclear. We tested the same ethanol-exposed zebrafish for social impairments at 3 weeks then again at 16 weeks. At both ages, live conspecifics were used to elicit a social response. We did not find alcohol-induced differences in behavior in 3-week-old fish when they were able to see conspecifics. We do find evidence that control zebrafish are able to use nonvisual stimuli to detect conspecifics, and this behavior is disrupted in the alcohol-exposed fish. As adults, these fish displayed a significant decrease in social behavior when conspecifics are visible. This surprising finding demonstrates that the adult and larval social behaviors are, at least partly, separable. Future work will investigate the nature of these nonvisual cues and how the neurocircuitry differs between the larval and adult social behaviors.

Diving into the world of alcohol teratogenesis: a review of zebrafish models of fetal alcohol spectrum disorder.

The term fetal alcohol spectrum disorder (FASD) refers to the entire suite of deleterious outcomes resulting from embryonic exposure to alcohol. Along with other reviews in this special issue, we provide insight into how animal models, specifically the zebrafish, have informed our understanding of FASD. We first provide a brief introduction to FASD. We discuss the zebrafish as a model organism and its strengths for alcohol research. We detail how zebrafish has been used to model some of the major defects present in FASD. These include behavioral defects, such as social behavior as well as learning and memory, and structural defects, disrupting organs such as the brain, sensory organs, heart, and craniofacial skeleton. We provide insights into how zebrafish research has aided in our understanding of the mechanisms of ethanol teratogenesis. We end by providing some relatively recent advances that zebrafish has provided in characterizing gene-ethanol interactions that may underlie FASD.

An Fgf-Shh signaling hierarchy regulates early specification of the zebrafish skull.

The neurocranium generates most of the craniofacial skeleton and consists of prechordal and postchordal regions. Although development of the prechordal is well studied, little is known of the postchordal region. Here we characterize a signaling hierarchy necessary for postchordal neurocranial development involving Fibroblast growth factor (Fgf) signaling for early specification of mesodermally-derived progenitor cells. The expression of hyaluron synthetase 2 (has2) in the cephalic mesoderm requires Fgf signaling and Has2 function, in turn, is required for postchordal neurocranial development. While Hedgehog (Hh)-deficient embryos also lack a postchordal neurocranium, this appears primarily due to a later defect in chondrocyte differentiation. Inhibitor studies demonstrate that postchordal neurocranial development requires early Fgf and later Hh signaling. Collectively, our results provide a mechanistic understanding of early postchordal neurocranial development and demonstrate a hierarchy of signaling between Fgf and Hh in the development of this structure.

Pdgfra and Pdgfrb genetically interact during craniofacial development.

BACKGROUND: One of the most prevalent congenital birth defects is cleft palate. The palatal skeleton is derived from the cranial neural crest and platelet-derived growth factors (Pdgf) are critical in palatogenesis. Of the two Pdgf receptors, pdgfra is required for neural crest migration and palatogenesis. However, the role pdgfrb plays in the neural crest, or whether pdgfra and pdgfrb interact during palatogenesis is unclear. RESULTS: We find that pdgfrb is dispensable for craniofacial development in zebrafish. However, the palatal defect in pdgfra;pdgfrb double mutants is significantly more severe than in pdgfra single mutants. Data in mouse suggest this interaction is conserved and that neural crest requires both genes. In zebrafish, pdgfra and pdgfrb are both expressed by neural crest within the pharyngeal arches, and pharmacological analyses demonstrate Pdgf signaling is required at these times. While neither proliferation nor cell death appears affected, time-lapsed confocal analysis of pdgfra;pdgfrb mutants shows a failure of proper neural crest condensation during palatogenesis. CONCLUSIONS: We provide data showing that pdgfra and pdgfrb interact during palatogenesis in both zebrafish and mouse. In zebrafish, this interaction affects proper condensation of maxillary neural crest cells, revealing a previously unknown interaction between Pdgfra and Pdgfrb during palate formation. Developmental Dynamics 245:641-652, 2016. © 2016 Wiley Periodicals, Inc.

Pdgfra protects against ethanol-induced craniofacial defects in a zebrafish model of FASD.

Human birth defects are highly variable and this phenotypic variability can be influenced by both the environment and genetics. However, the synergistic interactions between these two variables are not well understood. Fetal alcohol spectrum disorders (FASD) is the umbrella term used to describe the wide range of deleterious outcomes following prenatal alcohol exposure. Although FASD are caused by prenatal ethanol exposure, FASD are thought to be genetically modulated, although the genes regulating sensitivity to ethanol teratogenesis are largely unknown. To identify potential ethanol-sensitive genes, we tested five known craniofacial mutants for ethanol sensitivity: cyp26b1, gata3, pdgfra, smad5 and smoothened. We found that only platelet-derived growth factor receptor alpha (pdgfra) interacted with ethanol during zebrafish craniofacial development. Analysis of the PDGF family in a human FASD genome-wide dataset links PDGFRA to craniofacial phenotypes in FASD, prompting a mechanistic understanding of this interaction. In zebrafish, untreated pdgfra mutants have cleft palate due to defective neural crest cell migration, whereas pdgfra heterozygotes develop normally. Ethanol-exposed pdgfra mutants have profound craniofacial defects that include the loss of the palatal skeleton and hypoplasia of the pharyngeal skeleton. Furthermore, ethanol treatment revealed latent haploinsufficiency, causing palatal defects in ∼62% of pdgfra heterozygotes. Neural crest apoptosis partially underlies these ethanol-induced defects in pdgfra mutants, demonstrating a protective role for Pdgfra. This protective role is mediated by the PI3K/mTOR pathway. Collectively, our results suggest a model where combined genetic and environmental inhibition of PI3K/mTOR signaling leads to variability within FASD.

The Genetics of Fetal Alcohol Spectrum Disorders.

The term "fetal alcohol spectrum disorders" (FASD) defines the full range of ethanol (EtOH)-induced birth defects. Numerous variables influence the phenotypic outcomes of embryonic EtOH exposure. Among these variables, genetics appears to play an important role, yet our understanding of the genetic predisposition to FASD is still in its infancy. We review the current literature that relates to the genetics of FASD susceptibility and gene-EtOH interactions. Where possible, we comment on potential mechanisms of reported gene-EtOH interactions. Early indications of genetic sensitivity to FASD came from human and animal studies using twins or inbred strains, respectively. These analyses prompted searches for susceptibility loci involved in EtOH metabolism and analyses of candidate loci, based on phenotypes observed in FASD. More recently, genetic screens in animal models have provided an additional insight into the genetics of FASD. Understanding FASD requires that we understand the many factors influencing phenotypic outcome following embryonic EtOH exposure. We are gaining ground on understanding some of the genetics behind FASD, yet much work remains to be carried out. Coordinated analyses using human patients and animal models are likely to be highly fruitful in uncovering the genetics behind FASD.

Gene-ethanol interactions underlying fetal alcohol spectrum disorders.

Fetal alcohol spectrum disorders (FASD) is an umbrella term that describes a diverse set of ethanol-induced defects. The phenotypic variation is generated by numerous factors, including timing and dosage of ethanol exposure as well as genetic background. We are beginning to learn about how the concentration, duration, and timing of ethanol exposure mediate variability within ethanol teratogenesis. However, little is known about the genetic susceptibilities in FASD. Studies of FASD animal models are beginning to implicate a number of susceptibility genes that are involved in various pathways. Here we review the current literature that focuses on the genetic predispositions in FASD.

Commentary: catching a conserved mechanism of ethanol teratogenicity.

BACKGROUND: Due to its profound impact on human development, ethanol (EtOH) teratogenicity is a field of intense study. The complexity of variables that influence the outcomes of embryonic or prenatal EtOH exposure compels the use of animal models in which these variables can be isolated. METHODS: Numerous model systems have been used in these studies. The zebrafish is a powerful model system, which has seen a recent increase in usage for EtOH studies. RESULTS: Those using zebrafish for alcohol studies often face 2 questions: (i) How physiologically relevant are the doses of EtOH administered to zebrafish embryos? and (ii) Will the mechanisms of EtOH teratogenesis be conserved to other model systems and human? CONCLUSIONS: The current article by Flentke and colleagues () helps to shed important light on these questions and clearly demonstrates that the zebrafish will be a valuable model system with which to understand EtOH teratogenicity.

A screen of zebrafish mutants identifies ethanol-sensitive genetic loci.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a highly variable set of phenotypes caused by fetal alcohol exposure. Numerous factors influence FASD phenotypes, including genetics. The zebrafish is a powerful vertebrate model system with which to identify these genetic factors. Many zebrafish mutants are housed at the Zebrafish International Resource Center (ZIRC). These mutants are readily accessible and an excellent source to screen for ethanol (EtOH)-sensitive developmental structural mutants. METHODS: We screened mutants obtained from ZIRC for sensitivity to EtOH teratogenesis. Embryos were treated with 1% EtOH (41 mM tissue levels) from 6 hours postfertilization onward. Levels of apoptosis were evaluated at 24 hours postfertilization. At 4 days postfertilization, the craniofacial skeleton, peripheral axon projections, and sensory neurons of neuromasts were examined. Fish were genotyped to determine whether there were phenotype/genotype correlations. RESULTS: Five of 20 loci interacted with EtOH. Notable among these was that vangl2, involved in convergent extension movements of the embryonic axis, interacted strongly with EtOH. Untreated vangl2 mutants had normal craniofacial morphology, while severe midfacial defects including synophthalmia and narrowing of the palatal skeleton were found in all EtOH-treated mutants and a low percentage of heterozygotes. The cell cycle gene, plk1, also interacted strongly with EtOH. Untreated mutants have slightly elevated levels of apoptosis and loss of ventral craniofacial elements. Exposure to EtOH results in extensive apoptosis along with loss of neural tissue and the entire craniofacial skeleton. Phenotypes of hinfp, mars, and foxi1 mutants were also exacerbated by EtOH. CONCLUSIONS: Our results provide insight into the gene-EtOH interactions that may underlie EtOH teratogenesis. They support previous findings that EtOH disrupts elongation of the embryonic axis. Importantly, these results show that the zebrafish is an efficient model with which to test for gene-EtOH interactions. Understanding these interactions will be crucial to understanding of the FASD variation.