Functional analyses of vertebrate tissue-globins in zebrafish

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(Hemo-)globins are among the best-investigated proteins in biological sciences but still offer surprises. Our studies led to the discovery of six novel globins in gnathostome (jawed) vertebrates, mostly with still poorly defined functions. Here we propose to study the functions of cytoglobin (Cygb1 and Cygb2), neuroglobin (Ngb), and globin X (GbX) in the zebrafish Danio rerio. We will employ the CRISPR/Cas9 method to generate knockouts of the globin genes. The method has been established in the lab by generating a Cygb1 knockout zebrafish line. The globin knockouts will be tested for developmental defects, changes in O2 consumption, hypoxia tolerance and response, protection from reactive oxygen species (ROS) and nitric oxide, and protection of membrane lipids. Further, the comparative RNA-Seq approach (knockout vs. wildtype) will help to identify the corresponding metabolic pathways in which the globins are involved. According to our hypothesis, Ngb may be involved in O2 supply or ROS decomposition, while GbX possibly protects the cell membrane. In this application, we will specifically focus on Cygb1 and Cygb2. In contrast to other vertebrates, teleosts harbor two distinct Cygb genes. We hypothesize that they diverged by subfunctionalization 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. The results are expected to reveal the physiological roles of Ngb, Cygb1, Cygb2, and GbX in zebrafish in particular, and help to better understand the functional diversity of these globins in vertebrates in general.