Microarray based transcriptomics and the search for biomarker genes in zebrafish

In the past, zebrafish genes were mapped to human or mouse orthologs in order to perform Gene Ontology or pathway analyses. Therefore, genes without orthologs were removed and zebrafish-specific pathways were not taken into account. After the zebrafish genome has been sequenced almost completely, a growing number of biological databases for zebrafish have been made available. The increasing availability of gene function descriptions and specific pathways improves the applicability of zebrafish for transcriptomics studies. To make full use of the enhanced capabilities, however, new methods need to be developed. In this thesis, I describe results of two different transcriptional studies. In the first one, I analyzed gene expression data of zebrafish embryos treated with 10 different compounds at 24-48 hpf. I employed multivariate statistical methods to identify compounds that lead to similar expression pattern changes. Furthermore, I tried to identify similarities by comparing co-regulated genes. A gene function analysis of the significantly differentially expressed genes was performed in order to gain a better understanding of the modes of action of the compounds. The findings were validated using literature data. In order to identify biomarker genes, I grouped the compounds based on the identified modes of action and searched for genes that were only de-regulated after treatment with compounds with the same mode of action. I defined sets of biomarker genes for the following modes of action: disruption of mitochondrial potential, Acetylcholinesterase inhibition, Glutathione metabolism, and induction of apoptosis. During the studies of the 10 compounds, it became obvious that commercially available zebrafish microarrays lack several important genes. To overcome this problem, I designed a new array that covers almost the whole zebrafish genome. I could show that the newly designed whole genome array clearly improves microarray experiments. Additionally, we aimed at gaining deeper insights into the transcriptional regulation during zebrafish development. For this reason, I designed a new microarray consisting only of transcription factors. This array was employed to study six different developmental stages, covering the complete development from egg till larva. We were also interested in variations of transcription factor expression in certain tissues like muscle and brain. The microarray data was analyzed with a newly developed approach using two color arrays to detect expressed transcription factors. Using the new method, I could detect groups of transcription factors that exhibited a similar expression pattern over time. With the help of Gene Ontology, I was able to identify different gene function mechanisms associated with specific developmental stages. Transcription factors with highest expression before gastrulation were mostly involved in protein metabolism, and factors expressed at similar levels during the whole development period were likely to be involved in organ development. Transcription factors with expression peaking at the end of the development seemed to be mostly involved in development of the nervous system and biosynthesis. Additionally, I defined biomarker genes specific for the 6 developmental stages and the tissue samples used in this study

Transcriptional profiling reveals barcode-like toxicogenomic responses in the zebrafish embryo

Microarray profiling of zebrafish embryos exposed to a range of environmental toxicants revealed distinct expression profiles for each of the toxicants tested

Evaluation of the effects of acetylcholinesterase inhibitors in the zebrafish touch-evoked response: quantitative vs. qualitative assessment

Background: The difficulty of finding new treatments for neurological diseases with great impact in our society like Alzheimer's disease can be ascribed in part to the complexity of the nervous system and the lack of quick and costeffective screening tools. Such tools could not only help to identify potential novel treatments, but could also be used to test environmental contaminants for their potential to cause neurotoxicity. It has been estimated that 5-10% of the anthropogenic chemicals are developmental neurotoxic (DNT) and exposure to DNT compounds has been linked to several neurological diseases. Within this study we were testing the applicability of a quick and cost-effective behavioural test using zebrafish embryos: the touch-evoked response assay, in this case, an assay evaluating the swimming response to a tap in the tail. Two acetylcholinesterase (AChE) inhibitors positive controls (paraoxon and huprine Y), as well as 10 huprine-derivative compounds were tested and the results were evaluated using 2 different methods, a quantitative and a qualitative one. Results: We could show that the methodology presented is able to detect behavioural effects of AChE inhibitors. A good correlation between the results obtained with the quantitative and the qualitative method was obtained (R2 = 0.84). Conclusions: Our proposed method enables combination of screening for new drugs with toxicity screening in a whole embryo model alternative to animal experimentation, thereby merging 2 drug development steps into one

Lmx1b is required for the glutamatergic fates of a subset of spinal cord neurons

Background: Alterations in neurotransmitter phenotypes of specific neurons can cause imbalances in excitation and inhibition in the central nervous system (CNS), leading to diseases. Therefore, the correct specification and maintenance of neurotransmitter phenotypes is vital. As with other neuronal properties, neurotransmitter phenotypes are often specified and maintained by particular transcription factors. However, the specific molecular mechanisms and transcription factors that regulate neurotransmitter phenotypes remain largely unknown. Methods: In this paper we use single mutant, double mutant and transgenic zebrafish embryos to elucidate the functions of Lmx1ba and Lmx1bb in the regulation of spinal cord interneuron neurotransmitter phenotypes. Results: We demonstrate that lmx1ba and lmx1bb are both expressed in zebrafish spinal cord and that lmx1bb is expressed by both V0v cells and dI5 cells. Our functional analyses demonstrate that these transcription factors are not required for neurotransmitter fate specification at early stages of development, but that in embryos with at least two lmx1ba and/or lmx1bb mutant alleles there is a reduced number of excitatory (glutamatergic) spinal interneurons at later stages of development. In contrast, there is no change in the numbers of V0v or dI5 cells. These data suggest that lmx1b-expressing spinal neurons still form normally, but at least a subset of them lose, or do not form, their normal excitatory fates. As the reduction in glutamatergic cells is only seen at later stages of development, Lmx1b is probably required either for the maintenance of glutamatergic fates or to specify glutamatergic phenotypes of a subset of later forming neurons. Using double labeling experiments, we also show that at least some of the cells that lose their normal glutamatergic phenotype are V0v cells. Finally, we also establish that Evx1 and Evx2, two transcription factors that are required for V0v cells to acquire their excitatory neurotransmitter phenotype, are also required for lmx1ba and lmx1bb expression in these cells, suggesting that Lmx1ba and Lmx1bb act downstream of Evx1 and Evx2 in V0v cells. Conclusions: Lmx1ba and Lmx1bb function at least partially redundantly in the spinal cord and three functional lmx1b alleles are required in zebrafish for correct numbers of excitatory spinal interneurons at later developmental stages. Taken together, our data significantly enhance our understanding of how spinal cord neurotransmitter fates are regulated

Evaluation of LC-MS and LC×LC-MS in analysis of zebrafish embryo samples for comprehensive lipid profiling

In this study, both conventional one-dimensional liquid chromatography (1DLC) and comprehensive two-dimensional liquid chromatography (2DLC) coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF MS) were used for full-scale lipid characterization of lipid extracts from zebrafish embryos. We investigated the influence on annotated lipids and different separation mechanisms (HILIC, C18, and PFP), and their different orders arranged in the first and the second dimensions. As a result, the number of lipid species annotated by conventional one-dimensional LC-MS was between 212 and 448. In contrast, the number of individual lipids species annotated by C18×HILIC, HILIC×C18, and HILIC×PFP were 1784, 1059, and 1123, respectively. Therefore, it was evident that the performance of comprehensive 2DLC, especially the C18×HILIC method, considerably exceeded 1DLC. Interestingly, a comparison of the HILIC×C18 and C18×HILIC approaches showed, under the optimized conditions, similar orthogonality, but the effective separation power of the C18×HILIC was much higher. A comparison of the HILIC×C18 and the HILIC×PFP methods demonstrated that the HILIC×PFP separation had superior orthogonality with a small increase on its effective peak capacity, indicating that the HILIC×PFP combination maybe a promising platform for untargeted lipidomics in complex samples. Finally, from the comprehensive lipid profiling respective, the C18×HILIC was selected for further studies

Using comprehensive lipid profiling to study effects of PFHxS during different stages of early zebrafish development

PFHxS (Perfluorohexane sulfonic acid) is one of the short-chain perfluoroalkyl substances (PFASs) which are widely used in many industrial and consumer applications. However, limited information is available on the molecular mechanism of PFHxS toxicity (e.g. lipid metabolism). This study provides in-depth information on the lipid regulation of zebrafish embryos with and without PFHxS exposure. Lipid changes throughout zebrafish development (4 to 120 h post fertilization (hpf)) were closely associated with lipid species and lipid composition (fatty acyl chains). A comprehensive lipid analysis of four different PFHxS exposures (0, 0.3, 1, 3, and 10 μM) at different zebrafish developmental stages (24, 48, 72, and 120 hpf) was performed. Data on exposure concentration, lipids, and developmental stage showed that all PFHxS concentrations dysregulated the lipid metabolism and these were developmental-dependent. The pattern of significantly changed lipids revealed that PFHxS caused effects related to oxidative stress, inflammation, and impaired fatty acid β-oxidation. Oxidative stress and inflammation caused the remodeling of glycerophospholipid (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)), with increased incorporation of omega-3 PUFA and a decreased incorporation of omega-6 PUFA