Functional analyses of vertebrate tissue-globins in zebrafish
General Genetics and Functional Genome Biology
Animal Physiology and Biochemistry
Final Report Abstract
(Hemo-)globins are among the best-investigated proteins in biological sciences but still offer surprises. Previous studies led to the discovery of six novel globins in gnathostome (jawed) vertebrates, mostly with still poorly defined functions. In this project we proposed to study the functions of cytoglobin (Cygb1 and Cygb2), neuroglobin (Ngb), and globin X (GbX) in the zebrafish Danio rerio. We employed the CRISPR/Cas9 method to generate knockouts of the globin genes. The method has been established in the lab and we generated a Cygb1, Cygb2 and Ngb knockout zebrafish line. The globin knockouts developed without any obvious (d)effects. We proceeded to test for (hidden) developmental defects via RNAseq analysis and also tested the hypoxia tolerance and response of larvae and adult Knockouts. We further also compared the globin specific protection from reactive oxygen species (ROS) in embryos. Further, with the comparative RNAseq approach (knockout vs. wildtype) we could identify the corresponding metabolic pathways in which the globins are involved thus allowing us to exclude potential functions that are discussed in the literature. According to our results, Ngb is mainly involved in O2 supply and ROS decomposition within the Zebrafish brain. In this application, we specifically focused on Cygb1 and Cygb2. In contrast to other vertebrates, teleosts harbor two distinct Cygb genes. We hypothesized that they diverged by sub-functionalization after gene duplication. The duplicated genes may play a role in the supply of O2 for collagen synthesis, protection from ROS, or in the NO metabolism. Cygb1 deficiency led to the upregulation of immune regulatory and cell cycle regulatory transcripts (e.g. tp53) in the cygb1 knockout liver. Additionally, the expression of transcripts involved in lipid metabolism and transport, the antioxidative defence and iron homeostasis was affected in the cygb1 knockout. Thus, Cygb1 may function as an anti-inflammatory and cytoprotective factor in zebrafish liver, and is involved in lipid-, iron-, and ROS-dependent signalling.
Publications
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The knockout of cytoglobin 1 in zebrafish (Danio rerio) alters lipid metabolism, iron homeostasis and oxidative stress response. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1870(8), 119558.
Schlosser, Annette; Helfenrath, Kathrin; Wisniewsky, Michelle; Hinrichs, Kira; Burmester, Thorsten & Fabrizius, Andrej
