Application of fish models for studying mechanisms of human fetal alcohol spectrum disorders (FASDs)
Date: 29 August 2023
Thesis or dissertation
University of Exeter
PhD in Biological science
In this project, embryos from two small fish species, zebrafish (freshwater) and Arabian killifish (AKF) (seawater and brackish water), have been used as suitable models to study the mechanism of human fetal alcohol spectrum disorders (FASDs). A. dispar was used as a novel model species for ethanol (EtOH) toxicity research. FASDs are ...
In this project, embryos from two small fish species, zebrafish (freshwater) and Arabian killifish (AKF) (seawater and brackish water), have been used as suitable models to study the mechanism of human fetal alcohol spectrum disorders (FASDs). A. dispar was used as a novel model species for ethanol (EtOH) toxicity research. FASDs are caused by an elevated alcohol level in the pregnant mother’s body. The symptoms of FASDs include microcephaly, holoprosencephaly, craniofacial abnormalities, and cardiac defects with a birth defect in severe cases and milder cases, leading to developmental and learning disabilities. The action of alcohol on embryo development is highly complicated, having many target tissues and cellular events, including gene expression, cell migration, metabolism, cell cycle regulation, DNA replication, cell differentiation and others. Therefore, the actual mechanism of alcohol effect (direct and indirect) on each tissue development is not fully understood. Here, we hypothesised that early cell movement and gene expression change at the gastrula stage might cause direct and indirect consequences to induce later-stage embryonic abnormalities, including microcephaly, eye deformity, short body axis and many other defects. To test this hypothesis, we aim to investigate the mechanism of the impact of alcohol on cell movement and gene expression during the gastrulation stage. Firstly, we overview the studies on FASDs (general introduction in Chapter 1 and In Chapters 3 and 5, we discuss with a specific focus the mechanisms by which alcohol alters cell migration during early embryogenesis, including blastula, gastrula and organogenesis stages, which later cause morphological defects in the brain and other tissues. In Chapter 4, we describe the AKF embryonic staging in detail that was needed to use the species as a novel model for embryonic toxicological studies. Developmental stages of embryos were named with the hour post fertilisation at 28˚C. We found that A. dispar can also tolerate a wide range of temperatures (Chapter 4). Toxicities of EtOH were investigated using zebrafish (Chapter 3) and AKF (Chapter 5). The early embryogenesis data with EtOH exposure were collected from WT and TG strains: WT zebrafish (Danio rerio), TG (H2A:GFP), TG (Gsc:GFP) and Zebrabow. WT Arabian killifish A. dispar, TG (EF1α:Kaede) and TG (beta-actin:Dsred). In the zebrafish, the effects of alcohol were observed in many places during embryo development, from cell lineage-specific gene expression at the blastula/gastrula stage, gastrulation cell movement, morphogenesis of the central nervous system and neuronal development. ACQUIFER multiwall live Imaging Machine microscope was used to image the EtOH treated embryos. With a large data set, quantitative and qualitative analyses were accomplished. The data revealed that EtOH suppresses convergence-extension and epiboly cell movement at the gastrula stage and causes the failure of normal neural plate formation, brain development, eye development and body extension. In addition to the malformation of the body, reduction of the cell pigment (melanophore) was observed with a higher dose of EtOH treated embryos (Chapter 3). The transparent zebrafish embryo offers an ideal model system to investigate the genetic, cellular and organismal response to alcohol. However, there is a limitation in using zebrafish as a model because the short time for independent feeding and irregular shape of the blastoderm cause a compromise of the precise measurement of the epiboly cell movement in the ACQUIFER multiwell imaging with random orientation. Therefore, in this thesis, besides zebrafish, AKF embryos were also used for examining the effect of EtOH (Chapter 5). Finally, a new transgenic line of A. dispar fish was developed using the EF1α:Kaede transgene. We intend to employ it for single-cell tracing by UV photo conversion in embryos with control and EtOH treated. Our data of Kaede fish with Zeiss LSM suggest we can trace and analyse single-cell migration at the gastrula stage. It would further clarify our understanding of the effect of EtOH on different cell movements, including involution, convergence-extension and epiboly cell movement. Overall, these results present that gastrulation cell movement will become a very sensitive, quantitative, and specific biomarker for chemical toxicities. In addition, it has been proved that our novel TG (EF1α:Kaede) AKF would contribute to the study of alcohol toxicity’s effect on cell migration during embryo development. It demonstrates the potential advantages of using this species in future studies of chemical toxicity research.
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