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.

Loss of Nicotinamide nucleotide transhydrogenase sensitizes embryos to ethanol-induced neural crest and neural apoptosis via generation of reactive oxygen species.

Fetal alcohol spectrum disorders (FASD) are a continuum of birth defects caused by prenatal alcohol exposure. FASD are the most common environmentally induced birth defect and are highly variable. The genetics of an individual influence the severity of their FASD phenotype. However, the genes that sensitize an individual to ethanol-induced birth defects are largely unknown. The ethanol-sensitive mouse substrain, C57/B6J, carries several known mutations including one in Nicotinamide nucleotide transhydrogenase (Nnt). Nnt is a mitochondrial transhydrogenase thought to have an important role in detoxifying reactive oxygen species (ROS) and ROS has been implicated in ethanol teratogenesis. To directly test the role of Nnt in ethanol teratogenesis, we generated zebrafish nnt mutants via CRISPR/Cas9. Zebrafish embryos were dosed with varying concentrations of ethanol across different timepoints and assessed for craniofacial malformations. We utilized a ROS assay to determine if this could be a contributing factor of these malformations. We found that exposed and unexposed mutants had higher levels of ROS compared to their wildtype counterparts. When treated with ethanol, nnt mutants experienced elevated apoptosis in the brain and neural crest, a defect that was rescued by administration of the antioxidant, N-acetyl cysteine (NAC). NAC treatment also rescued most craniofacial malformations. Altogether this research demonstrates that ethanol-induced oxidative stress leads to craniofacial and neural defects due to apoptosis in nnt mutants. This research further supports the growing body of evidence implicating oxidative stress in ethanol teratogenesis. These findings suggest that antioxidants can be used as a potential therapeutic in the treatment of FASD.

Embryonic ethanol exposure disrupts craniofacial neuromuscular integration in zebrafish larvae.

Forming a vertebrate head involves the meticulous integration of multiple tissue types during development. Prenatal alcohol exposure is known to cause a variety of birth defects, especially to tissues in the vertebrate head. However, a systematic analysis of coordinated defects across tissues in the head is lacking. Here, we delineate the effects of ethanol on individual tissue types and their integration during craniofacial development. We found that exposure to 1% ethanol induced ectopic cranial muscle and nerve defects with only slight effects on skeletal pattern. Ectopic muscles were, however, unaccompanied by ectopic tendons and could be partially rescued by anesthetizing the larvae before muscle fibers appeared. This finding suggests that the ectopic muscles result from fiber detachment and are not due to an underlying muscle patterning defect. Interestingly, immobilization did not rescue the nerve defects, thus ethanol has an independent effect on each tissue even though they are linked in developmental time and space. Time-course experiments demonstrated an increase in nerve defects with ethanol exposure between 48hpf-4dpf. Time-lapse imaging confirmed the absence of nerve pathfinding or misrouting defects until 48hpf. These results indicate that ethanol-induced nerve defects occur at the time of muscle innervation and after musculoskeletal patterning. Further, we investigated the effect of ethanol on the neuromuscular junctions of the craniofacial muscles and found a reduced number of postsynaptic receptors with no significant effect on the presynaptic terminals. Our study shows that craniofacial soft tissues are particularly susceptible to ethanol-induced damage and that these defects appear independent from one another. Thus, the effects of ethanol on the vertebrate head appear highly pleiotropic.

Gene-alcohol interactions in birth defects.

Most human birth defects are thought to result from complex interactions between combinations of genetic and environmental factors. This is true even for conditions that, at face value, may appear simple and straightforward, like fetal alcohol spectrum disorders (FASD). FASD describe the full range of structural and neurological disruptions that result from prenatal alcohol exposure. While FASD require alcohol exposure, evidence from human and animal model studies demonstrate that additional genetic and/or environmental factors can influence the embryo's susceptibility to alcohol. Only a limited number of alcohol interactions in birth defects have been identified, with many sensitizing genetic and environmental factors likely yet to be identified. Because of this, while unsatisfying, there is no definitively "safe" dose of alcohol for all pregnancies. Determining these other factors, as well as mechanistically characterizing known interactions, is critical for better understanding and preventing FASD and requires combined scrutiny of human and model organism studies.

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.

High-throughput detection of craniofacial defects in fluorescent zebrafish.

Losses and malformations of cranial neural crest cell (cNCC) derivatives are a hallmark of several common brain and face malformations. Nevertheless, the etiology of these cNCC defects remains unknown for many cases, suggesting a complex basis involving interactions between genetic and/or environmental factors. However, the sheer number of possible factors (thousands of genes and hundreds of thousands of toxicants) has hindered identification of specific interactions. Here, we develop a high-throughput analysis that will enable faster identification of multifactorial interactions in the genesis of craniofacial defects. Zebrafish embryos expressing a fluorescent marker of cNCCs (fli1:EGFP) were exposed to a pathway inhibitor standard or environmental toxicant, and resulting changes in fluorescence were measured in high-throughput using a fluorescent microplate reader to approximate cNCC losses. Embryos exposed to the environmental Hedgehog pathway inhibitor piperonyl butoxide (PBO), a Hedgehog pathway inhibitor standard, or alcohol (ethanol) exhibited reduced fli1:EGFP fluorescence at one day post fertilization, which corresponded with craniofacial defects at five days post fertilization. Combining PBO and alcohol in a co-exposure paradigm synergistically reduced fluorescence, demonstrating a multifactorial interaction. Using pathway reporter transgenics, we show that the plate reader assay is sensitive at detecting alterations in Hedgehog signaling, a critical regulator of craniofacial development. We go on to demonstrate that this technique readily detects defects in other important cell types, namely neurons. Together, these findings demonstrate this novel in vivo platform can predict developmental abnormalities and multifactorial interactions in high-throughput.

Embryonic Nicotine Exposure Disrupts Adult Social Behavior and Craniofacial Development in Zebrafish.

Cigarette smoking remains the leading cause of preventable death and morbidity worldwide. Smoking during pregnancy is associated with numerous adverse birth outcomes, including craniofacial and behavioral abnormalities. Although tobacco smoke contains more than 4000 toxic substances, nicotine is addictive and is likely the most teratogenic substance in cigarette smoke. However, much remains to be determined about the effects of embryonic nicotine exposure on behavior and craniofacial development. Therefore, this study evaluated adult social behavior in zebrafish, craniofacial defects, and nicotine metabolism in embryos after embryonic nicotine exposure. Zebrafish embryos were exposed to different doses of nicotine beginning at 6 h post fertilization. To evaluate craniofacial defects, the embryos were collected at 4 days post fertilization and stained with Alizarin Red and Alcian Blue. For behavioral testing, embryos were reared to adulthood. To evaluate nicotine metabolism, cotinine levels were analyzed at various time points. Our findings demonstrate that embryonic exposure to nicotine modifies social behavior in adulthood, causes craniofacial defects with reduced size of craniofacial cartilages, and that zebrafish metabolize nicotine to cotinine, as in humans. Together, our data suggest that zebrafish are useful as a model for studying nicotine-related diseases.

Divergent cis-regulatory evolution underlies the convergent loss of sodium channel expression in electric fish.

South American and African weakly electric fish independently evolved electric organs from muscle. In both groups, a voltage-gated sodium channel gene independently lost expression from muscle and gained it in the electric organ, allowing the channel to become specialized for generating electric signals. It is unknown how this voltage-gated sodium channel gene is targeted to muscle in any vertebrate. We describe an enhancer that selectively targets sodium channel expression to muscle. Next, we demonstrate how the loss of this enhancer, but not trans-activating factors, drove the loss of sodium channel gene expression from muscle in South American electric fish. While this enhancer is also altered in African electric fish, key transcription factor binding sites and enhancer activity are retained, suggesting that the convergent loss of sodium channel expression from muscle in these two electric fish lineages occurred via different processes.

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.

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.

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.

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.

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.

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.

Differentially sensitive neuronal subpopulations in the central nervous system and the formation of hindbrain heterotopias in ethanol-exposed zebrafish.

BACKGROUND: A cardinal feature of prenatal ethanol exposure is CNS damage, resulting in a continuum of neurological and behavioral impairments that are described by the term fetal alcohol spectrum disorders (FASD). FASDs are variable and depend on several factors, including the amount, timing, and duration of prenatal ethanol exposure. To enhance interventions for CNS dysfunction, it is necessary to identify ethanol-sensitive neuronal populations and expand the understanding of factors that modify ethanol teratogenesis. METHODS: To investigate the susceptibility of different neuronal subtypes, we exposed transgenic zebrafish (Danio rerio) to several ethanol concentrations (0.25, 0.5, 1.0, 1.5, or 2.0%), at different hours post fertilization (hpf; 0, 6, or 24 hpf), for various durations (0-24, 0-48, 4-24, 6-24, 6-48,or 24-48 hpf). Following exposure, embryo survival rates were determined, and CNS neurogenesis, differentiation, and patterning were assessed. RESULTS: Embryo survival rates decrease as ethanol concentrations increase and drastically decline when exposed from 0-24 hpf compared to 4-24 hpf. Abnormal tangential migration of facial motor neurons is observed in isl1:gfp embryos exposed to ethanol concentrations as low as 0.25%, and the formation of IVth ventricle heterotopias are revealed by embryos exposed to ≥1.0% ethanol. Whereas, expression of olig2:dsred and ptf1a:gfp in the cerebellum and spinal cord are largely unaffected. While levels of etv4 mRNA are overtly resistant to ethanol, we observe significant reductions in ptch2 mRNA levels. CONCLUSIONS: These data show differentially sensitive CNS neuron subpopulations with susceptibility to low levels of ethanol. In addition, these data reveal the formation of ethanol-induced hindbrain heterotopias.

Social behavioral phenotyping of the zebrafish casper mutant following embryonic alcohol exposure.

The term Fetal Alcohol Spectrum Disorder (FASD) describes all the deleterious consequences of prenatal alcohol exposure. Impaired social behavior is a common symptom of FASD. The zebrafish has emerged as a powerful model organism with which to examine the effects of embryonic alcohol exposure on social behavior due to an innate strong behavior, called shoaling. The relative transparency of the embryo also makes zebrafish powerful for cellular analyses, such as characterizing neural circuitry. However, as zebrafish develop, pigmentation begins to obscure the brain and other tissues. Due to mutations disrupting pigmentation, the casper zebrafish strain remains relatively transparent throughout adulthood, potentially permitting researchers to image neural circuits in vivo, via epifluorescence, confocal and light sheet microscopy. Currently, however the behavioral profile of casper zebrafish post embryonic alcohol exposure has not been completed. We report that exposure to 1% alcohol from either 6 to 24, or 24 to 26 h postfertilization reduces the social behavior of adult casper zebrafish. Our findings set the stage for the use of this important zebrafish resource in studies of FASD.

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.

PDGFRA gene, maternal binge drinking and obstructive heart defects.

Obstructive heart defects (OHDs) are a major health concern worldwide. The platelet-derived growth factor (PDGF) genes are known to have regulatory functions that are essential for proper heart development. In a zebrafish model, Pdgfra was further demonstrated to interact with ethanol during craniofacial development. In this article, we investigated interactions between variants in PDGF genes and periconceptional alcohol exposure on the risk of OHDs by applying log-linear models to 806 OHD case and 995 control families enrolled in the National Birth Defects Prevention Study. The interactions between four variants in PDGFA and maternal binge drinking reached a nominal significance level. The maternal T allele of rs869978 was estimated to increase OHD risk among women who binge drink, while infant genotypes of rs2291591, rs2228230, rs1547904, and rs869978 may reduce the risk. Although none of these associations remain statistically significant after multiple testing adjustment and the estimated maternal effect may be influenced by unknown confounding factors, such as maternal smoking, these findings are consistent with previous animal studies supporting potential interactions between the PDGFRA gene and maternal alcohol exposure. Replication studies with larger sample sizes are needed to further elucidate this potential interplay and its influence on OHD risks.

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.

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.