Michael Bastiani - US grants

We have identified and begun the molecular and functional characterization of two new cell surface molecules involved in neurogenesis. Lazarillo and Conulin were found by making mAbs specific to the developing nervous system and then using those mAbs as biochemical reagents to purify and characterize each antigen. We have cloned the gene encoding Lazarillo and we are proposing to clone the gene encoding Conulin in this application. Lazarillo represents a new class of molecule, a lipocalin, involved in neuronal path findings and suggests a new molecular signaling pathway. We are proposing experiments to characterize how Lazarillo regulates growth cone and filopodial behavior to guide neurons along a specific pathway. This will entail developing a "molecular knockout" anti-sense technology to specifically inhibit the expression of Lazarillo in identified neurons. We will test the hypothesis that Lazarillo mediates its regulation of growth cone behavior via a signal transduction pathway that includes a small lipophilic ligand and a protein ligand. Structural and functional experiments are proposed to identify both the small lipophilic ligand that binds in the lipocalin hydrophobic pocket and the protein ligand that binds to the putative protein protein interaction domain suggested by our modeling studies. We predict that homologues of Lazarillo exist in other animals, including vertebrates, and function in a similar way during the development of their nervous systems. We have proposed a straightforward PCR based approach to identify these homologues and will use sequence information and in situ hybridization to verify the homology. Conulin's unique spacial and temporal localization to a subset of neuronal growth cones makes it a good candidate for a novel molecular function. Experiments to identify Conulin include immunoaffinity and biochemical purification, and expression cloning. We hypothesize that Conulin functions in neuronal path finding as growth cones switch from one axonal fascicle to another in the central ganglionic neuropil. We will test this hypothesis by observing the behavior of identified growth cones that have had conulin removed from their surfaces. It is clear that many of the basic molecular mechanisms in developmental processes have been highly conserved during the evaluation of nervous systems as diverse as nematode, fly and mouse. We propose that Lazarillo and Conulin will lead us to new molecular mechanisms used by all developing nervous systems.

The goals of this proposal are to determine (1) what cellular interactions are essential for pioneering the initial scaffold of axon pathways in the embryonic grasshopper central nervous system, (2) how later neurons are guided in their cell specific choices among this scaffold of axon pathways, and (3) what molecules are involved in guiding growth cones in their initial and later pathway choices. The grasshopper embryonic nervous system will be used to answer these questions. This model system is uniquely suited for these experiments because of the in vivo cellular resolution and accessibility it offers. Single identified growth cones in an identified cellular environment can be observed and manipulated in the grasshopper embryo. Specific neuron-epithelial, neuron-glial, and neuron-neuron interactions will be characterized at light and EM level and then experimental manipulations will be performed to determine the role of each interaction in growth cone guidance and pathway choice. Monoclonal antibodies will generated against this same set of epithelial, glial, and neuronal cells to find molecular correlates of the specific interactions required for growth cone guidance.

This proposal requests funds toward the purchase of a JEOL model JEM-1210 Transmission Electron Microscope (TEM) equipped with a high resolution video camera, a digital image archiving system, and image processing software. Equipment that will directly support the operation of this TEM is also requested. This instrument will replace an aging Philips 201 microscope currently housed in the Biology Microscopy Facility at the University of Utah. Although the Philips 201 TEM was a capable microscope in its day, it is now 16 years old and suffers from continual mechanical and electrical breakdowns. Components are no longer manufactured to support this instrument making it a challenge to keep the microscope operational. It is likely that the Philips 201 microscope will fail permanently in the near future. The requested TEM will have a dramatic impact on the research programs of many faculty at the University of Utah. A minimum of thirteen faculty in the Department of Biology alone are pursuing research programs that focus on structure/function relationships at the cellular or subcellular level. For example, the microscope will be used by researchers in the Biology neuroscience group to 1) analyze connectivity changes in C. elegans mutants defective in neurite outgrowth, synaptic specificity or synapse formation, and 2) to study the cellular contacts among developing neurons in grasshoppers and flies. Other research groups in Biology will use the microscope in immunogold-labeling studies to examine the structure and localization of molecules involved in cell adhesion, neuronal differentiation, neurotransmission, cytoskeletal function, cell division, development of cell polarity and bacterial flagellar motility. In addition, a number of investigators use a genetic approach to study the early development of multicellular plants and animals. These investigators need a transmission electron microscope to perform morphological analyses on mut ants arrested in specific developmental stages. Many of the projects listed above will require specialized features that are only available on a state-of-the-art transmission electron microscope like the JEOL JEM-1210. The research needs of our existing faculty, staff, and students simply cannot be met by the antique Philips 201 microscope currently available in the Biology Microscope Facility. The University of Utah has committed 50% in matching funds toward the purchase of a transmission electron microscope. This commitment indicates the high degree of institutional priority for our request. In addition, the Biology Department has guaranteed 1) full salary support for the facility manager, Dr. Edward King, 2) 50% support for a full time microscopy technician for two years, and 3) funds for a four year service contract. Together, these contributions represent an institutional commitment of greater than 50%. The JEOL JEM-1210 will be part of a core facility used by faculty, staff, graduate and undergraduate students in the Biology Department and by researchers from other departments and colleges on the University of Utah campus. The Biology Microscopy Facility already maintains a heavily used laser scanning confocal microscope and a scanning electron microscope. The addition of a modern transmission electron microscope to the facility will enhance campus wide research efforts addressing a variety of basic biological questions.

We want to characterize DD and UD growth cone behavior during embryonic and post embryonic (LI) development of the nematode nervous system. We plan to use multiphoton confocal microscopy and 41) software to observe GFP expressing UD and DD neurons in wild-type and mutant nematodes to better understand the role played by specific molecules in growth cone pathfinding and target recognition. Initial studies will be performed using confocal microscopy. However, it appears likely we will need the multiphoton scope to maintain viability during long term fiequent collection of data sets.

fluorescent dye /probe

The Summer Molecular Research Experience for Students Underrepresented in Science will provide an intensive 10-week research experience at the University of Utah in Salt Lake City. A total of 10 students will be selected to participate. Students who come from underrepresented backgrounds and from institutions with limited research opportunities are especially well served in this program. Students will receive a stipend of $3000 for 10 weeks of full time research, as well as room and board at the newly constructed student housing which was used as the Olympic Village during the 2002 Winter Olympics. Travel expenses to the University of Utah will be covered. Students accepted to the Program will have the opportunity to choose from a wide variety of projects that employ molecular techniques. A few potential projects are:

Paternity tests for birds. Techniques include DNA extraction and microsatellite-based paternity analysis.
Phylogenetics of chemical defenses in tropical trees. Techniques include High Pressure Liquid Chromatography and testing the toxicity of compounds in bioassays with insects.
Co-speciation of doves, lice and microbes. Techniques include DNA extraction, PCR, cloning, and DNA sequencing.
Regulation of cytoskeletal arrays in algae and plants. Techniques include transcript identification by RT-PCR and protein levels by Western blot.
Detoxification capacities of mammalian herbivores. Techniques include DNA microarray analysis and microsomal assays.
Systematics of Solanaceae. Techniques include PCR and sequence analysis.
Molecular machineries that regulate division and fusion of mitochondria in budding yeast.
Development of the macronucleus from the germline micronucleus of the ciliate, Oxytricha. Use PCR to infer the nature of the rearrangements and sequence modifications that are employed in generating the macronucleus.
Promotion of homologous recombination by the generation of linear
DNA in the nematode, C. elegans.
The role of chemoreceptor clustering in chemotactic signaling by
Escherichia coli.

Upon arrival at the University of Utah students will spend two days becoming acquainted with the campus, their research groups and the surrounding area. To maintain the cohesiveness of the group, 4 weekly meetings will be scheduled for the duration of the program. A GRE preparation class and classes to assist students with their graduate school applications will be offered. At the conclusion of the program students will write a research report and present their research in an oral Bioscience Symposium. Students will be encouraged to present their research at National meetings. For more information, contact Dr. Rosemary Gray at (801) 581-5013 or BioURP@bioscience.utah.edu.

DESCRIPTION (provided by applicant): A comprehensive RNAi screen for genes effecting neuron degeneration and regeneration in C. elegans Neuronal degeneration and regeneration has been studied in humans and other vertebrate model systems for over 100 years and yet we still do not have a comprehensive molecular model nor an effective treatment to prevent degeneration or induce regeneration. Surprisingly, the powerful genetic model systems used so successfully to study body pattern formation, programmed cell death, neural development and many other important biological problems have not been used to study neuronal degeneration and regeneration. This is because it has been difficult to devise a robust screening assay for neural regeneration in either D. melanogaster or C. elegans. Recently, Hammarlund and myself made an observation that now makes it possible to screen for genes required for regeneration in C. elegans. We discovered that embryonic neurons lacking [unreadable]-spectrin develop normally (normal growth cone motility, pathfinding, and target recognition), but after hatching undergo a movement-induced axotomy followed by regeneration. This is a robust phenotype, with most commissural axons in each animal breaking and regenerating before the animal reaches adulthood. There is a progressive failure of regeneration as each cycle of axotomy and regeneration takes place so that the adult displays a severely abnormal nervous system. This well-characterized regeneration phenotype in C. elegans mimics the phenotype of mammalian neurons in response to axotomy. I propose to use RNAi knockdown to assay the function of every gene in the worm genome in the process of neuronal degeneration and regeneration. Recently, two genetic mutations were identified that sensitize neurons to RNAi. This sensitized genetic background has been used and validated in a successful large scale RNAi screen for genes that function in synaptic transmission. There is every reason to believe that this technique can be used to screen for genes that function in degeneration and regeneration using the [unreadable]-spectrin mutant phenotype as the basis for the assay. If successful, it would be the first unbiased "genetic" screen for genes functioning in neuron degeneration and regeneration and should identify novel genes as entry points for theurapies targeting neuronal degeneration and regeneration. This proposal represents the first unbiased "genetic" screen for genes functioning in neuron degeneration and regeneration in C. elegans. If successful it will identify novel genes as entry points for therapies aimed at the prevention neuronal degeneration and the induction of neuronal regeneration.

Brain and spinal cord neurons in mammals, including humans, do not regenerate when they are broken due to injury or disease. Yet, neurons have a tremendous capacity for regeneration in mammalian embryos and in some adult animals. The puzzle of why adult mammalian neurons lose their capacity to regenerate can be solved by using the simple model organism, C. elegans. A genetic mutation was discovered in C. elegans that causes axons to break spontaneously. In response to these spontaneous axon breaks, the neurons successfully regrow new axons back to their target muscles. This spontaneous axon break, followed by axon regeneration, will be used to test the function of each gene in the C. elegans genome for its effect on axon regeneration. This would be the first genetic screen to identify all genes involved in axon regeneration in any organism and should lead to a comprehensive and fundamental understanding of the signaling pathways controlling axon regeneration. It should also allow identification of the signaling changes that occur with age that prevent axon regeneration in most adult mammalian neurons. The similarity between genes in C. elegans and humans means that any knowledge gained from the screen should be applicable to human nerve cell regeneration and provide rational targets for new drug therapies. This research will also provide training in cutting edge research technologies for graduate students and undergraduates, and will provide research opportunities for high school students through the University of Utah Bio-Sciences program that places students in laboratories for 7 weeks each summer.