CENTRAL QUESTION. How are marine-derived fluorescent proteins used to investigate fundamental questions in biology?
OBSERVATIONAL FIELD STUDIES
Background. Primarily a marine phenomenon, bioluminescence is cold light produced by biochemical reactions that occur within biological organisms. In the deep ocean, the largest habitable volume of Earth, bioluminescence is the predominant source of light. Throughout evolution, bioluminescence has appeared in many phylogenetically distinct groups of marine organisms, where it serves an important role in predation, defense, and courtship/mating.
Different groups of organisms utilize distinct biochemical strategies to produce light. In the Crystal jellyfish (Aequorea victoria), a cylindrical fluorescent protein called green fluorescent protein (GFP) is responsible for converting blue light emitted by another protein (aequorin) into intense green light. GFP is among a group of closely related proteins that possess similar properties. Interestingly, GFP-like proteins appear in many non-bioluminescent organisms, including invertebrate species found in the Channel Islands National Park and Marine Sanctuary. Although the biological function of these proteins is poorly understood, several interesting (and controversial) hypotheses have been proposed for their presence in non-luminous organisms.
Field Work. In preparation for field studies, students will explore the phenomenon of marine bioluminescence, its biological roles, the chemical bases of light production in bacteria, dinoflagellates, and cnidarians, and the spectral properties of GFP and some of its color variants. In the field, students will conduct underwater observational studies within the kelp forests surrounding Anacapa or Santa Cruz Islands. Equipped with specially designed ultraviolet lights and mask filters, certified student divers will visualize GFP-like proteins produced by non-bioluminescent invertebrates that reside on the kelp forest floor, including brown cup corals (Astrangia lajollaensis), club-tipped corallimorphs (Corynactis californica), aggregating anemones (Anthopleura elegantissima), and many others.
EXPERIMENTAL LABORATORY STUDIES
Background. Together with advances in fluorescence microscopy and molecular genetics, GFP and other marine-derived fluorescent proteins have revolutionized our ability to explore fundamental questions in biology, including central nervous system (CNS) hard-wiring. During CNS development, nerve cells (neurons) extend wire-like projections or axons that often travel long distances before finding and establishing connections with target nerve cells in the same biological circuit. The exquisite precision with which axons locate their targets is under the control of guidance molecules that provide navigational instructions to nerve cells. The leading tips of axons, the growth cones, bear receptors or sensors that read the instructions provided by guidance molecules and respond by steering growing axons along very specific pathways toward their targets.
To understand how navigational molecules control the trillions of connections that form during brain development, neurobiologists study relatively simpler systems in model organisms, including the spinal cord of chick, rodents and zebrafish, and its invertebrate counterpart, the ventral nerve cord, in fruit flies (Drosophila melanogaster). By genetically manipulating neurons within these systems to produce GFP, scientists can visualize their axons and thereby assess the role of molecular cues in guiding them to distant targets and establishing the appropriate synaptic connections.
Laboratory work. In this component of the NeuroLab program, students will explore our current understanding of axon guidance and participate in cutting-edge scientific research aimed at understanding the role of axon guidance receptors in spinal cord axon pathfinding. In the laboratory, students will use recombinant DNA technology to create genetic constructs that drive the production of fusion proteins containing GFP linked to modified axon guidance receptors in certain subsets of spinal cord neurons and axons. Based on previous studies, expression of these fusion proteins in spinal cord axons is expected to disrupt their normal axon pathfinding behavior. During a critical window of chick embryonic development, students will apply in ovo electroporation, a rapid, reliable, and well-established method for gene transfer, to introduce GFP fusion protein constructs into spinal cord neurons. The effects of these genetic manipulations on axon pathfinding will then be visualized and quantified using epifluorescence microscopy.
During each session, students will also examine axon pathfinding in the ventral nerve cord of genetically manipulated Drosophila embryos. After using the GAL4/UAS transactivation system to drive GFP expression in ventral nerve cord axons, students will apply immunohistochemistry and epifluorescence microscopy to visualize and characterize the trajectories of CNS axons in different genetic backgrounds.