Vitamin E is necessary for zebrafish nervous system development

Abstract

Vitamin E (VitE) deficiency results in embryonic lethality. Knockdown of the gene ttpa encoding for the VitE regulatory protein [α-tocopherol transfer protein (α-TTP)] in zebrafish embryos causes death within 24 h post-fertilization (hpf). To test the hypothesis that VitE, not just α-TTP, is necessary for nervous system development, adult 5D strain zebrafish, fed either VitE sufficient (E+) or deficient (E−) diets, were spawned to obtain E+ and E− embryos, which were subjected to RNA in situ hybridization and RT-qPCR. Ttpa was expressed ubiquitously in embryos up to 12 hpf. Early gastrulation (6 hpf) assessed by goosecoid expression was unaffected by VitE status. By 24 hpf, embryos expressed ttpa in brain ventricle borders, which showed abnormal closure in E− embryos. They also displayed disrupted patterns of paired box 2a (pax2a) and SRY-box transcription factor 10 (sox10) expression in the midbrain-hindbrain boundary, spinal cord and dorsal root ganglia. In E− embryos, the collagen sheath notochord markers (col2a1a and col9a2) appeared bent. Severe developmental errors in E− embryos were characterized by improper nervous system patterning of the usually carefully programmed transcriptional signals. Histological analysis also showed developmental defects in the formation of the fore-, mid- and hindbrain and somites of E− embryos at 24 hpf. Ttpa expression profile was not altered by the VitE status demonstrating that VitE itself, and not ttpa, is required for development of the brain and peripheral nervous system in this vertebrate embryo model.

Vitamin E Deficiency Disrupts Gene Expression Networks during Zebrafish Development

“…Morphological deformities including yolk sac and pericardial edemas, bent axes, and apparent developmental delays generally occur after 24 hpf in E– embryos as previously reported [ 12 ]. To evaluate gene expression differences based on VitE status alone and not phenotypic changes, only embryos identified as morphologically normal were used.…”

“…The outcomes of this study show that VitE deficiency disrupts numerous gene expression networks, including energy metabolism, oxidoreductase activity, intra- and intercellular signaling, and developmental transcriptional regulation, during critical developmental windows in zebrafish embryos. We previously identified that VitE deficiency prevents proper neural crest cell migration and impairs midbrain–hindbrain development [ 12 ].…”

“…This period coincides with segmentation of the mesoderm into somites, notochord vacuolation, and primary and secondary neurulation resulting in brain regionalization and neural tube expansion. We showed both by (1) using morpholinos to block the embryonic translation of the mRNA for the α-tocopherol transfer protein (α-TTP) [ 9 ] and by (2) evaluating neuronal structures in VitE-deficient embryos (E–) [ 10 , 11 , 12 ] that VitE is essential during the early stages of zebrafish neurogenesis. Specifically, blocking TTP mRNA translation with an oligonucleotide in zebrafish was 100% lethal by 24 hpf with noticeable impairment of brain and eye development beginning at 12 hpf [ 9 ].…”

“…We hypothesize that VitE is necessary to protect zebrafish transcriptional networks associated with metabolism, cell signaling and embryo energy status. In addition, we sought to identify transcription factors necessary for zebrafish embryo nervous system development that are VitE-dependent and produce the morphological defects previously observed [ 12 , 19 ].…”

Vitamin E deficiency dysregulates thiols, amino acids and related molecules during zebrafish embryogenesis

“…These latter studies suggested that choline, betaine and the methionine cycle were dysregulated by inadequate VitE protection during embryogenesis. The dysregulation of these pathways in E− embryos causes abnormal nervous system formation, especially the midbrain-hindbrain boundary, spinal cord and dorsal root ganglia [ 14 ]. Thus, we raise the question “why is a water-soluble methyl donor dysregulated during VitE deficiency?” We hypothesize that an increased requirement for GSH during the increased lipid peroxidation observed in E− embryos drives a complex mechanism of overlapping biochemical pathways needed to maintain thiol homeostasis that is dependent on betaine and methyl group donation.…”