Graduate Thesis Or Dissertation
 

Vitamin E Protects Developmental Gene Expression and Metabolic Networks in Zebrafish Embryos

Public Deposited

Downloadable Content

Download PDF
Request Accessible Version
https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/0k225j811

Descriptions

Attribute NameValues
Creator
Abstract
  • Vitamin E (VitE) is necessary for vertebrate embryonic development. VitE prevents lipid peroxidation (LPO), which requires detoxification by cellular antioxidant systems subsequently involving reducing power derived from energy metabolism. Thus, VitE protects metabolic networks in the developing embryo and the integrated gene expression networks compensating for and impaired by LPO-induced errors. In this thesis, I present four experimental chapters regarding VitE’s vital role in the time-dependent protection of embryo developmental gene expression and metabolic networks using the zebrafish model. I begin by evaluating gastrulation, neural plate formation and neural crest cell migration defined by expression of key transcription factors goosecoid, sox10, and pax2a. VitE-deficient (E–) zebrafish embryos appear normal during neuroectoderm formation at 6 hours post-fertilization (hpf), however, by 12 hpf E– embryos are developmentally abnormal with mis-localized expression of pax2a and sox10 in the early midbrain-hindbrain boundary and neural crest cells. At 24 hpf E– further have reduced expression of these latter transcription factors distributed throughout the spinal neurons and in trunk neural crest cells. Patterning defects precede the obvious morphological impairments in E– embryos as well as histologic evidence that all brain regions develop erroneously by 24 hpf. In addition, the -tocopherol transfer protein (TTPA) gene is expressed at the leading edges of the brain ventricle border; the brains of E– embryos were often over- or under inflated by 24 hpf. I then evaluated the whole transcriptome profile of VitE-sufficient (E+) and E– embryos at 12-, 18- and 24 hpf using RNAseq. By gene annotation, gene set enrichment, and integrated metabolomic analyses I found that E– embryos experience significant disruption to expression of genes associated with energy metabolism. Specifically, E– relative to E+ embryos have increased expression of genes involved in glycolysis and the pentose phosphate pathway, while they have decreased expression of genes involved in anabolic pathways and gene transcription. More importantly, when both gene expression and the metabolome in embryos at 24 hpf were analyzed together, the mechanistic Target of Rapamycin (mTOR) signaling pathway was found diminished in E– embryos. VitE protected gene expression associated with energy metabolism, as integrated by the cell cycle survival and growth protein complex mTOR. Gene expression disruption may precede the errors in embryonic patterning visualized with sox10 and pax2a. To ascertain the metabolic impacts of a VitE deficiency, we then developed sensitive UPLC-MS/MS methods to identify and quantify amino acids and thiol-related antioxidants in the E+ and E– embryos. Betaine, the oxidation product of choline was increased in E– embryos at 12-, 24- and 48 hpf. At 24 hpf, E– embryos contained less choline and by 48 hpf glutathione (GSH) was also decreased. It is likely that choline, an integral component of the membrane lipid, phosphatidyl choline, is used to generate betaine, which is a substrate for the methionine cycle and needed to produce methyl donors. These methyl donors, including S-adenosyl methionine (SAM) were also decreased in E– embryos. SAM is used to generate homocysteine for synthesis of cysteine, the rate-limiting amino acid in glutathione (GSH) synthesis. Ultimately, our analyses indicate that thiols become depleted in E– embryos because LPO generates products that requires compensation using limited amino acids and methyl donors that are also developmentally relevant. The first three experimental chapters demonstrate the interconnected reality of VitE-deficiency induced LPO that disrupts antioxidant balance; thiol and energy metabolic status; and gene expression patterns leading to increased morbidity and mortality outcomes. Finally, we aimed to generate a transgenic model to track VitE-dependent cells in vivo with a fluorescently tagged TTPA protein. Using CRISPR-Cas technology, pigment-less Casper zebrafish embryos were injected with cas9 complex proteins and a plasmid containing the mScarlet coding sequence targeted 5’ of the first exon of ttpa. Homozygote mutant fish, named the RedEfish, were found to express the mScarlet protein and produce red fluorescence in the olfactory pits, digestive tract including the liver, and posterior tail fin by 7 days post-fertilization (dpf). At 14 dpf the RedEfish also expressed mScarlet in the caudal vertebrae edges identified as likely dorsal root ganglia and the caudal vein plexus. The TTPA protein function was not impaired by the addition of the mScarlet genomic sequence, however, the VitE status of the RedEfish was 30% lower relative to Casper zebrafish of the same age, suggesting some TTPA dysfunction. This model provides a valuable new means of testing and visualizing VitE necessity during vertebrate development. This thesis provides ample evidence supporting VitE as a necessary dietary factor for embryonic survival. It is thus critical to continue evaluating the physiologically relevant nature of a simple dietary deficiency in a developmental window defined by neurogenesis and specific gene expression and metabolic needs.
Contributor
License
Resource Type
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Committee Member
Rights Statement
Funding Statement (additional comments about funding)
  • BAH was supported in part by the Provost's Graduate Fellowship, Mark Sponenburgh-Linus Pauling Institute Endowed Fellowship and the Marion T. Tsefelas-Linus Pauling Institute Endowed Fellowship.
Publisher
Peer Reviewed
Language
Accessibility Feature

Relationships

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details HeadBrianA2021.pdf 2021-03-12 Public Download