Survival of Antarctic notothenioid fishes in the context of global climate change will depend upon the impact of rising oceanic temperatures on their embryonic development, yet little is known regarding the molecular mechanisms underlying this complex suite of processes. Many notothenioids are characterized by secondary pelagicism, which enables them to exploit food sources in the water column and is supported in part by skeletal pedomorphism. Here the PI proposes to examine the hypothesis that reactive oxygen species (ROS) regulate notothenioid skeletal pedomorphism. The PI proposes to examine the hypothesis that reactive oxygen species (ROS) regulate notothenioid skeletal pedomorphism. The research objectives are : 1) To quantify and localize ROS production and identify the point(s) of origin of ROS production in embryonic Antarctic fishes that differ in skeletal phenotypes 2) To determine whether the time course of embryogenesis and the extent of osteological development in embryonic Antarctic fishes can be altered by changing the oxidative status of the animal during embryogenesis 3) To evaluate whether transgenic alteration of oxidative status can induce skeletal pedomorphism in a fish model. Broader Impacts will include teaching undergraduate lectures, recruiting undergraduate students to help with lab analyses (and possibly field work), lectures and demonstrations to high school students, and allowing secondary educators access to personal photos and videos of research animals for curriculum development.
Antarctic fishes lack a swim bladder, the gas-filled organ used in most fishes to control the animal's position in the water column. Additionally, the ancestor of the Antarctic fishes lived on the ocean bottom and was likely heavily-ossified. We now know that fully developed adult Antarctic fishes live throughout the water column and have a range of skeletal characteristics from negatively buoyant and heavily ossified (mostly bone) to neutrally buoyant and lightly ossified (mostly cartilage). Since vertebrates (including humans) typically develop lightly ossified skeletons early and transition into relatively heavily ossified skeletons later in development, we utlized embryos of these Antarctic fish species to investigate the cellular and molecular mechanisms involved in the process of endochondral ossifcation (bone development in the presence of cartilage). Specifically we were interested in the generation of reactive oxygen species (ROS) and processes which balance the production of these molecules. To this end, we first had to devise and optimize procedures for conducting in vitro fertilizations of Antarctic fishes in order to obtain embryos early in development. We were successful in fertilizing three Antarctic fish species (C. aceratus, N. coriiceps, and C. gunnari). Unfortunately, however, we were only able to maintain populations of N. coriiceps embryos through the duration of our experiment. Given the basic lack of information regarding the reproductive ecology of the other species, our trial and error in maintaining our experimental embryos of these species will be invaluable in future attempts to study these species at early developmental stages. We were able to quantify the amounts of ROS being produced within in-tact N. coriiceps embryos over developmental time using a fluorescent dye and imaging software. Additionally we could quantify the activity of antioxidant enzymes (e.g., superoxide dismutase - SOD and catalase - CAT) responsible for neutralizing these molecules. We hypothesized that an imbalance in the processes which generate and neutralize ROS may partially explain changing skeletal characteristics. As expected, there is a change in the activity of the antioxidant enzymes relative to the amount of ROS being produced. These results indicate that more defenses are active as development of these embryos progresses, possibly altering the oxidative status of the organism. We will continue to explore how and/or if this changing oxidative status correlates with the process of endochondral ossification.
