Like me, when you were a kid many of you probably puckered your lips and placed your hands on the side of you neck to make believe that you were a fish. At the time, you were probably just being silly and didn’t realize how similar you actually are to a fish. The zebrafish is a common model organism used in research because it shares a remarkable amount of genetic information with humans. Because of this similarity, the zebrafish model system has been useful for identifying and characterizing the genetics behind many different human birth defects, including anomalies to the craniofacial skeleton. It is increasingly clear that the environment plays an important role in the genesis of birth defects. Because zebrafish are fertilized externally, this tiny fish is a useful model for understanding these gene/environment interactions.
Gene/environment interactions may be beneficial, such as when a mother eats a healthy diet, or detrimental, like when an expectant mother is using drugs or alcohol. It is not new knowledge that alcohol can have detrimental impacts on the growth of the fetus. The first manuscript identifying Fetal Alcohol Syndrome (FAS) was published 40 years ago and the phenotypes associated with FAS were noted even earlier in children of alcoholic mothers. However, the effect of alcohol on a developing embryo can be incredibly variable, and the broader term Fetal Alcohol Spectrum Disorders (FASD) covers these variable, ethanol-induced phenotypes. FASD patients can suffer from both neural and craniofacial defects, and although timing and volume of exposure can influence the variability of this disease, genetic predisposition most likely adds to this variability.
In our study (McCarthy et al, 2013), we sought to identify genes that protected against ethanol-induced defects and, thus, might contribute to this variability. When these protective genes are mutated, embryos should be more susceptible to the deleterious effects of ethanol. Therefore, we exposed embryos to a level of ethanol that did not disrupt development in normal wild-type embryos. We examined several of the zebrafish mutant lines that we house in our fish facility for susceptibility to ethanol-induced developmental defects. We found that one of these fish lines, a hypomorphic pdgfra allele, was strikingly susceptible to ethanol. In fact, most of the heterozygous embryos, which still have one good copy of pdgfra, also had craniofacial defects; under normal conditions these heterozygotes do not have defects. We were particularly struck with how different the ethanol-treated heterozygotes and mutants looked from the untreated mutants. The difference in phenotypes suggested that ethanol interacted synergistically with pdgfra.
While untreated pdgfra mutants have defects in migration of skeletal precursors, ethanol treatment causes an increase in apoptosis of craniofacial precursor cells in ethanol-treated pdgfra mutants and heterozygotes. This provided support that pdgfra and ethanol did indeed synergistically interact. Mechanistically, we show that mTOR signaling, downstream of pdgfra, was reduced in ethanol-treated pdgfra mutants. mTOR is important in cellular growth and survival, and has been implicated to interact with ethanol in other studies. L-leucine is a common dietary supplement that can elevate mTOR signaling. Elevating mTOR signaling with L-leucine in ethanol-treated mutant zebrafish partially rescues the ethanol-induced phenotype. But the question remained does this information, obtained from a fish, help us understand FASD in humans?
Luckily for us, we were able to collaborate with the Foroud lab to get at this question. In a human sample, they found concordance between facial phenotypes and PDGFRA genotype, or more specifically single nucleotide polymorphisms, SNPs, associated with the PDGFRA gene. This sample included children who either were or were not exposed to alcohol during pregnancy and who underwent a series of craniofacial measurements. Dr Foroud and her colleagues found a significant association between a Pdgfra SNP and differences in craniofacial measurements in ethanol-exposed children, compared to their unexposed counterparts. Importantly, the SNP had no effect on facial phenotypes by itself. It was only when the SNP was present and there had been an alcohol exposure that the phenotype was altered. Thus, what began as an interesting phenotype caused by exposing a little fish to ethanol has lead to a human SNP that might help us understand FASD.
Whether other genes, such as other growth-factor genes, could also interact with ethanol is of ongoing interest in the lab. Working with the Foroud lab, we hope to sustain the collaboration we have formed and build a better understanding of the genetic predispositions to FASD. By using a small fish we can provide the mechanistic details of these gene-ethanol interactions. So while we may not look much like fish, they can certainly tell us a lot about ourselves.
McCarthy, N., Wetherill, L., Lovely, C.B., Swartz, M.E., Foroud, T.M., and Eberhart, J.K. Pdgfra protects against ethanol-induced craniofacial defects in a Zebrafish model of FASD. Development 2013 Aug; 140(15): 3254-65.