Martin Chalfie - US grants

DESCRIPTION (provided by applicant): We will continue our study of genes needed for neuronal differentiation and function in the nematode Caenorhabditis elegans. Most of the research will center, as in the past, on the analysis of the development and activity of the six touch receptor neurons (TRNs). Previous research under this grant has identified genes needed for the generation, specification, maintenance and function of the TRNs. In particular in the last funding period we enlarged the collection of TRN-expressed genes 8-fold to approximately 200 genes, identified components that restrict TRN development to six cells, and identified the transduction complex that sense touch in these cells. This last complex is the first transduction complex to be identified in any eukaryotic mechanosensory neuron. We also developed several new methods that we will exploit in the upcoming funding period. In particular we will investigate how cell fate is determined and maintained and how the lipid bilayer and extracellular matrix affect mechanosensation. The specific aims of the proposal are: 1) to investigate the regulation of post-mitotic gene expression, particular examining how genes needed only early in development of neurons are turned off; 2) to investigate the basis of cell-type specification within the TRNs; 3) to characterize lipid- binding and modulating components of the MEC-4 channel complex and similar proteins; and 4) to investigate the role of the extracellular matrix (ECM) in touch sensitivity. A secondary consequence of the proposed experiments will be the discovery a wealth of genes needed for general development and function of the C. elegans nervous system.

Mutations causing the degeneration of specific nerve cells have been identified in many organisms. We have identified and cloned two genes that can be mutated to cause the degenerative and late-onset death of specific nerve cells in the nematode Caenorhabditis elegans. Dominant mutations in the gene mec-4 lead to the deaths of all six of the C. elegans touch receptor neurons, whereas a dominant mutation in the gene deg-1 results in a similar appearing death of a small set of different neurons. Molecular analysis reveals that both genes are members of the same gene family. We are calling the products of this family "degenerins." Suppression studies have identified the gene mec-6 as being required for the degenerations and, thus, begin to define components needed for this abnormal cell death. The proposed research will use these and other mutations to study the process of this inherited neurodegeneration in C. elegans and the nature of the degenerins and their suppressors. The specific aims of the research are: 1. to continue the molecular characterization of the deg-1 and mec-4 genes and their products; 2. to characterize molecularly the mec-6 gene; 3. to characterize the events occurring during cell degeneration in these mutants using light and electron microscopy; 4. to identify and characterize molecularly other members of the degenerin gene family in C. elegans; and 5. to identify other genes that are required for the degenerations. The health relatedness of this project stems from the nature of the mutations. We do not know whether degenerins are found in higher organisms (although crosshybridization is seen) or whether, if found, they are involved in specific disease situations. Nonetheless, the study of these genes using molecular and transmission genetics and observations on single, identified cells should highlight cell biological processes underlying inherited degenerative neurological disorders.

Research on the nematode Caenorhabditis elegans ranges from such global problems as the functioning of an entire nervous system and the evolution of form to the molecular bases of the gene structure and function. C.l elegans has becomes an important experimental organism for the study of many aspects of animal biology, particularly the genetic and molecular bases of development and behavior. Dr. Anderson is requesting funds to help support two C. elegans meetings to be held at the university of Wisconsin at Madison, WI in June of 1991 and 1993. Previous C. elegans meeting shave led to the exchange of knowledge, methods, mutants, and clones and have been vital in establishing the sense of excitement and collegiality that characterizes this field.

Mutations causing the degeneration of specific nerve cells have been identified in many organisms. We have identified and cloned two genes that can be mutated to cause the degenerative and late-onset death of specific nerve cells in the nematode Caenorhabditis elegans. Dominant mutations in the gene mec-4 lead to the deaths of all six of the C. elegans touch receptor neurons, whereas a dominant mutation in the gene deg-1 results in a similar appearing death of a small set of different neurons. Molecular analysis reveals that both genes are members of the same gene family. We are calling the products of this family "degenerins." Suppression studies have identified the gene mec-6 as being required for the degenerations and, thus, begin to define components needed for this abnormal cell death. The proposed research will use these and other mutations to study the process of this inherited neurodegeneration in C. elegans and the nature of the degenerins and their suppressors. The specific aims of the research are: 1. to continue the molecular characterization of the deg-1 and mec-4 genes and their products; 2. to characterize molecularly the mec-6 gene; 3. to characterize the events occurring during cell degeneration in these mutants using light and electron microscopy; 4. to identify and characterize molecularly other members of the degenerin gene family in C. elegans; and 5. to identify other genes that are required for the degenerations. The health relatedness of this project stems from the nature of the mutations. We do not know whether degenerins are found in higher organisms (although crosshybridization is seen) or whether, if found, they are involved in specific disease situations. Nonetheless, the study of these genes using molecular and transmission genetics and observations on single, identified cells should highlight cell biological processes underlying inherited degenerative neurological disorders.

Gain-of-function mutations in three genes in the nematode Caenorhabditis elegans (deg-1, mec-4, and mec-10) lead to the vacuolated degeneration of specific nerve cells. Most of the affected cells have mechanosensory function. all three genes encode novel proteins (degenerins) that appear to be membrane proteins. Similar mammalian proteins form the amiloride- sensitive channels that control sodium uptake in kidney, colon, and lung. The similarity with the mammalian proteins and the affects of mutations on touch sensitivity suggest that the degenerins may be components of mechanosensory receptors. By studying these proteins, we wish to gain information not only about this new class of channel proteins, but also about the molecular basis of mechanosensory transduction. The specific aims of this research are: 1. To identify functional elements within the degenerins. We will use both in vitro and in vivo mutagenesis to characterize various domains within the degenerin proteins. These domains include a putative extracellular gating domain, regions that may interact with extracellular components, glycosylation sites, and phosphorylation sites. We will also test the inter-changeability of degenerin proteins. 2. To characterize genes that enhance or suppress degenerin activity. Several genes modulate degenerin function. We will clone the modulatory genes, sequence wild-type and mutant alleles, and examine their patterns of expression. 3. To test predictions of our model for touch receptor function. we will examine the relationship of the unique touch cell microtubules with the plasma membrane and degenerins within it, test predicted protein interactions and screen for new ones using the yeast two hybrid system, and test predicted interactions between the deg-3 acetylcholine receptor and the touch cell mechanoreceptor. 4. To investigate the embryonic lethality associated with an unusual deg- 1 mutation. The u506 mutation produces a cold-sensitive lethal phenotype either because of extra cell death or from neuronal hyperactivity. We will identify the affected cell(s) and test its importance in embryonic development by laser ablation.

neural degeneration; gene mutation; gene expression; Caenorhabditis elegans; suppressor mutations; phenotype; behavioral genetics; biological models; genetic mapping; gene interaction; cell death; mutant; membrane proteins; animal genetic material tag; site directed mutagenesis; transfection; nucleic acid sequence; molecular cloning;

In nematodes an alternative third larval stage, often called the dauer stage, allows the animals to weather periods of low food availibility (if free living) or to disperse (if parasitic. Recently, studies in the nematode Caenorhabditis elegans have indicated that mutations in several genes that control the entry into and exit from the dauer stage have profound effect upon the effect upon the life span of the animal. We have recently identified a mutation affecting the expression of an unusual catalase gene, ctl-1, in C. elegans that in preliminary experiments acts as if it is downstream of the genes in the dauer pathway. Messenger RNA coding for this catalase is elevated in dauer larvae. The mutation affecting ctl-1 expression causes the early death of dauer larvae and prevents the life-span extention of the dauer (daf) mutations. The hypothesis driving the proposed research is that this catalase (along with superoxide dismutase) is an important final target (perhaps the most important target) of the dauer pathway in the control of life-span. Specific aims designed to establish this hypothesis are 1) to characterize molecularly the ctl-1 and ctl-2 (s); 2) to complete the characterization of the effect of the ctl-1 mutation on life-span; and 3) to examine the expression of ctl-1 and ctl-2 using antibodies and GFP fusions and to test the functionality of the C. elegans catalases in yeast.

musculoskeletal system; microscopy; nervous system; genetics; biomedical resource; Invertebrata;

Training Program: At the heart of the training program is the Core Course, which runs for two semesters and is required of all first-year graduate students. This intensive course has been in place for the past seven years, meets three times a week for 1.5 to 2 hours, and provides a graduate-level introduction to the basic concepts of modern cellular and molecular biology. Every member of the departmental faculty currently teaches in the core course. The format is primary papers; there is currently no textbook. In addition to the core course, students are required to take 3 elective courses at the graduate level within the department. All of these courses are conducted in a seminar format, giving students the opportunity to discuss recent literature and develop scientific speaking skills. The program director hopes to incorporate a unit on immunology and bioinformatics into the core course in the next few years. The department is now forming a new "graduate-exclusive" journal club, which will allow students to present recent papers relevant to their current areas of research. The core course sets this program apart from its counterpart at the Medical Center and provides cohesion between the student population. The first-year course work, teaching experience, and housing gives the students a "home base." It is envisioned that with the evolution of the program more and more of the Medical Center faculty will join in the teaching effort for the core course as well as specialty graduate courses. This will have the advantage of providing the first "real bridge" between the two campuses as well as increasing mentor visibility. There is some concern that without a method to synchronize course times on both campuses, effective cross talk will not take place. Many of the current students are taking courses at the Medical Center. It is the hope of the current program that the students themselves will extend and support effective links between the two campuses, forcing faculty to be more cognizant of classes and teaching. It is hoped that the outcome of the proposed program will be better networking, forcing more competitive/interactive research programs. Currently, students recruited for the graduate program in the department can arrive at Columbia the summer before they enter their first year. This may serve as a rotation and sometimes becomes the thesis research laboratory. Students are strongly encouraged to do 2-3 rotations. Students can meet faculty through a multiplicity of pathways, including one-on-one advising, the recruitment brochure, the interview process, and finally the planned retreat. The retreat will involve, as much as possible, faculty on both campuses. Students are also introduced to faculty and their respective research interests within the department through a pre-research seminar series wherein each faculty member in the department, along with some allied faculty outside of the department, discuss research programs in detail. This appears to be well attended by faculty and students alike. During the previous fall semester, a total of 26 presentations were given, with 19 from within the department and 7 from Medical Center departments. Students generally start teaching in their first year. The department/program is committed to training in the area of teaching. Training in this area is not merely in the form of "on-the-job" but rather is overseen by a specific faculty member who functions a s a department "master teacher." Each student is required to complete 12 credits of teaching, usually by assisting in one course for three semesters. Preparation for the experience is well orchestrated with tutorials, practice teaching sessions, and feedback on classroom performance. The qualifying exam is loosely structured as the successful completion of the first-year coursework and a research proposal written at the end of the second year. The research proposal consists of the student's proposed thesis research and is defended before a committee of two faculty members, neither of whom is the student's mentor. Students effectively prepare for this hurdle throughout the duration of their second year, having chosen a thesis laboratory after finishing rotations. Resources and Environment: The research and training environment are outstanding. The Department of Biological Sciences has a mandate to hire six new faculty members. Over the past five years the department has hired 2 structural biologists, 2 mouse biologists, 2 cell biologists, and 1 neurobiologist. The new hires have been primarily at the senior level, and the department is finished with the initial hiring plan. The new hires will be junior and will be in areas that are targeted as national initiatives. The proposed training program represents a significant paradigm shift in its support of student mobility/training between the two campuses. The Dean lends his strong support for the directional shift not only in philosophy but also financially. Departmental monies will support students during the duration of their rotations until they enter a thesis laboratory. There is considerable support and conviction that the connectivity between the two campuses is critical. Not being without precedent, however, the Neurobiology and Behavior as well as the Biophysics programs are currently highly successful inter-campus programs and therefore good models for the current proposal. Institutional support for the students is over a two-year period and the summer in between and serves as a quid pro quo for the three semesters of that the students are required to teach. This money is not withheld if the students are successful in garnering outside fellowship support. Part of the institutional support comes in the form of subsidized housing.

Nematodes (roundworms) are the most abundant multicellular animals on earth, colonizing virtually every terrestrial, marine and freshwater habitat. While the nematode laboratory model C. elegans is the focus of intense, technically sophisticated research, much less is known about other nematodes, despite their biological importance. Of particular significance are parasitic nematodes, which comprise more than half of the estimated 28,000 nematode species; these parasites have important effects on the biology of their plant and animal hosts, threatening agricultural crops, and causing debilitating human diseases that affect over a billion people worldwide. By promoting interactions and collaborations between C. elegans researchers and investigators of parasitic nematodes, this project will increase awareness among C. elegans scientists of the issues and problems that parasitic nematode researchers face, and will pave the way for application of the powerful molecular and cell techniques developed in C. elegans research to investigations of parasitic nematodes. The planned symposium at the International C. elegans Meeting (Los Angeles June 26-30th, 2013) will provide an exceptional opportunity to reach the majority of C. elegans researchers. Six internationally recognized experts on plant, animal and human parasitic nematodes will speak on the life history and unique biology of parasitic species and on the outstanding issues in their field. In addition to formal education of the C. elegans community, this session will provide time for researchers in the two communities to interact more informally as well. The symposium aims to stimulate C. elegans researchers to think about problems facing the study and control of parasitic nematodes and to apply their expertise toward solving these problems. This session will be particularly valuable for undergraduate and graduate students and post-docs by exposing them to new possible career paths. Funds will also be provided for under-represented minority graduate students to attend the session.

DESCRIPTION (provided by applicant): Funds are requested for partial support of the Society for Developmental Biology's (SDB) annual meetings for the next five years (2014-2018). The Society has organized the major meeting in developmental biology for the past 75 years (missing two during World War II). Since the annual meeting changed its format from a single theme symposium to an open, inclusive meeting in 1993, the meeting size has grown three-fold (from 263 in 1994 to an average of 760 in the last 10 years). By allowing presentations by students and postdoctoral fellows, we have increased the number of abstract submissions from less than 100 to an average of 600's in the last few years. The expansion to include cutting edge themes in developmental biology and related areas such as stem cell and regenerative biology, together with the fast technical advances that allowed investigators to find explanations to questions unanswered before also contributed to this growth in attendance, as well as its standing today in biomedical research and public interest. SDB was founded in 1939 and is today the major society devoted to this field, with over 2200 members worldwide, including undergraduate and graduate students, postdoctoral fellows, junior and established investigators, many of whom hold NIH grants and fellowships. The proposed meetings will continue the tradition of mixing poster presentations, plenary sessions, concurrent symposia, the Hilde Mangold Postdoctoral Symposium, the awards lectures, an education symposium and workshops on new technologies, education and current issues. In all sessions, a special effort is made to have a diversity of invited speakers in terms of: model organisms, experimental approaches, career stages, gender and racial/ethnic background. For the concurrent symposia at least four short talk slots are reserved per session for abstracts selected from submissions, with preference given to qualified junior investigators. The education symposium and workshop focus on topics such as effective teaching and mentoring strategies at university and pre-college levels, outreach to the lay public, as well as career-related issues. They also address bioethics of research, which is highly relevant to establishing national policies for research on stem cells and cloning, as well as public understanding of science, including evolution. Additional activities that enrich the annual meetings include: On alternating years we hold a Boot Camp for New Faculty or a Reboot Camp for Mid-career Faculty the day preceding and on the first day of the meeting for professional development of untenured and mid-career faculty, respectively. SDB's Professional Development and Education Committee is responsible for these workshops. Call for up to two SDB member organized Satellite Symposia are made and will continue to be made yearly to SDB members. These Symposia complement the offerings of the annual meeting and are held on the opening day of the meeting. These pre-meeting events have been well-attended throughout the last ten years and will continue during this proposal period. The next meeting, to be held at the University of Washington, Seattle, WA is a return to our previous tradition of meetings on university campuses, a practice no longer possible because only a few campuses have facilities large enough for our growing meeting needs. It will also celebrate the 75th anniversary of founding of the Society and as such, sessions highlighting history and breakthroughs will be included in the program. The opening Presidential Symposium will address What have genetic model organisms taught us, where experts will give us an overview of what each organism has contributed to advancing our understanding of developmental processes. The two plenary and nine concurrent sessions will bring reports on the latest discoveries in developmental mechanisms, such as gene regulation, genomics, cell polarity, migration and differentiation, invasion and cancer, mechanical influences, tissue engineering, morphogenesis, organogenesis, evo-devo, organismal interactions, neural development. This meeting will be followed by meetings in Snowbird, Utah, Central region, West and East coast, as rotation throughout the various regions eases the travel burden for participants in the different zones of the country. Efforts will be continually made to find venues of easy access and as economical as possible, including keeping the tradition of university campuses as venue for their collegial atmosphere so cherished by our members.