Date Published: May 14, 2019
Publisher: Public Library of Science
Author(s): Sundeep S. Dhillon, Frida Torell, Magdalena Donten, Katrin Lundstedt-Enkel, Kate Bennett, Stefan Rännar, Johan Trygg, Torbjörn Lundstedt, Daniel Monleon.
The zebrafish embryo is a popular model for drug screening, disease modelling and molecular genetics. In this study, samples were obtained from zebrafish at different developmental stages. The stages that were chosen were 3/4, 4/5, 24, 48, 72 and 96 hours post fertilization (hpf). Each sample included fifty embryos. The samples were analysed using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Principle component analysis (PCA) was applied to get an overview of the data and orthogonal projection to latent structure discriminant analysis (OPLS-DA) was utilised to discriminate between the developmental stages. In this way, changes in metabolite profiles during vertebrate development could be identified. Using a GC-TOF-MS metabolomics approach it was found that nucleotides and metabolic fuel (glucose) were elevated at early stages of embryogenesis, whereas at later stages amino acids and intermediates in the Krebs cycle were abundant. This agrees with zebrafish developmental biology, as organs such as the liver and pancreas develop at later stages. Thus, metabolomics of zebrafish embryos offers a unique opportunity to investigate large scale changes in metabolic processes during important developmental stages in vertebrate development. In terms of stability of the metabolic profile and viability of the embryos, it was concluded at 72 hpf was a suitable time point for the use of zebrafish as a model system in numerous scientific applications.
The zebrafish (Danio rerio) has emerged as a successful scientific platform for studies of metabolism and metabolic diseases . The embryonic developmental stages of zebrafish have been characterized by Kimmel et al. , while Parichy et al.  described the normal table of post-embryonic zebrafish development. These two excellent studies make it possible to assign individual zebrafish to particular developmental stages, and describe the developmental changes in a variety of anatomical traits and how these traits vary over the zebrafish life cycle.
The results obtained in this study showed high similarity to those obtained in previous studies, where zebrafish metabolomic profiling at different developmental stages was also performed [7, 8, 10, 21]. Clear dynamic changes in metabolites were observed, which were found to be stage-specific and showed consistency such as a constant increase, constant decrease or no change during development, see boxplots available as supplementary information. The final PCA plot without the outlier showed clear clustering of stages with a directed shift from high levels of DNA building blocks at early stages to increased amino acid levels at intermediate stages and at later stages a shift towards elevated levels of metabolic waste products.
The metabolic stability and the viability of the embryos suggest that 72 hpf is the most suitable time point for use of zebrafish as a model system in applications including for example vertebra development, toxicology and genotype studies. One metabolite differed significantly in concentration between dechorinated embryos and those still in their chorion. This was spermidine which was reported to be important for viability, proliferation and differentiation of cells. The time period between the earliest developmental stage and 24 hpf was characterized by significant metabolic changes where there was a marked increase in building blocks, i.e. amino acids and nucleic acids as well as a significant increase in energy metabolites i.e. sugars and TCA cycle intermediates. The same pattern was observable between 24 hpf and 48 hpf, as well as 48 hpf and 72 hpf, however with fewer and fewer significant differences. The smallest significant differences were observed between 72 and 96 hpf. The time point between 72 and 96 hpf was characterized by an increased DNA synthesis, reduced energy demand and a peak in neurotransmitter precursors around 72 hpf. Interestingly, the levels of free fatty acids and sterols remained stable between the investigated developmental stages while fatty alcohols decreased over the studied time period. We propose further studies to investigate the role of fatty acids and alcohols in zebrafish development.