This Major Research Instrumentation grant supports the acquisition of a fluorescence stereomicroscope and an imaging system to enhance the research programs of Dr. Darrell J. Killian and Dr. Nancy N. Huang in the Biology Department at Colorado College. The real-time visualization of fluorescent molecules in living model organisms has become an indispensable technique because it allows the direct observation and documentation of changes in gene activity, the localization of proteins within a cell, and the tracking of cells and tissues during development. An important question in biology is how changes in gene activity determine sex-specific development. The Killian Lab is interested in the identification and characterization of genes that affect sex-specific development in the popular model organism Caenorhabditis elegans (C. elegans), a soil-dwelling roundworm. C. elegans populations contain males and hermaphrodites, which have some obvious morphological differences. However, sex-specific cell type differences in the nervous system are not easy to see. Fluorescent markers for sex-specific cell types have emerged as powerful and sensitive tools for the identification of defects in sex-specific development. Mutants have been isolated that are morphologically normal males or hermaphrodites, but with neurons that are normally only found in the opposite sex. The identification of the genes affected by these mutations and their subsequent characterization will provide new insights into the genetic basis of sex-specific development. Fluorescence stereomicroscopy and imaging are essential for the observation and documentation of sexual defects that are detected in vivo using fluorescent markers for sex-specific cell types. The Huang Lab is interested in how cellular identity, or cell fate specification, is established. MEX-3 is a protein that plays an important role in cell fate specification during C. elegans embryonic development. The MEX-3 protein is initially present in all cells of the embryo but becomes depleted from the posterior cells by the four-cell stage. This change in MEX-3 distribution is required for the establishment of the anterior/posterior axis of the embryo, thus it is important to understand how MEX-3 is localized in the embryo. Preliminary evidence suggests that MEX-3 is actively degraded in a manner that is dependent upon two different types of protein modifications termed phosphorylation and ubiquitination. The identification of the specific sites of these modifications within the MEX-3 protein and the identification of genes that regulate these modifications are of great interest. Fluorescence microscopy will enable an in vivo study of the stability of fluorescently tagged MEX-3 when specific sites in MEX-3 are mutated or when other genes in the C. elegans genome are disrupted.

Colorado College is the only nationally ranked liberal arts college in the Rocky Mountain region of the United States and enrolls approximately 2000 undergraduates from all over the United States and internationally. The Biology Department has a strong commitment to conducting original research with undergraduate collaboration and the fluorescence microscope and imaging system will provide important research and training opportunities for approximately 6-10 undergraduates working in the Killian and Huang labs each year. In addition, many courses in the Biology curriculum feature inquiry-based research lab experiences. Students with such experiences are best equipped to handle any postgraduate challenges that they might face. The equipment will augment and improve laboratory activities, demonstrations, and experiments associated with several courses and will impact approximately 200 undergraduates per year. The imaging system will be a particularly valuable teaching tool because it will enable the instructor and a group of students to simultaneously observe the same sample on a computer monitor. The Biology Department also plays a strong service role in the college by offering non-majors courses that foster a strong appreciation for basic biology research and empower students to make informed decisions and opinions on many popular topics in biology such as stem cell research, gene therapy, genetically modified organisms, and environmental issues. The fluorescence stereomicroscope will provide non-majors with unique and exciting opportunities to observe cutting-edge science first-hand. Finally, Colorado College actively seeks to enhance diversity on campus and the availability of state-ofthe- art instrumentation, such as a fluorescence stereomicroscope and imaging system, will help attract underrepresented minorities and women to participate in biology research.

Project Report

This MRI grant supported the purchase of a research quality fluorescence stereomicroscope and imaging system to improve research opportunities for Colorado College faculty and students, increase training opportunities for research students, and enhance labs associated with several courses including those for non-science majors. One focus of the Killian Lab is to understand the genetic pathways that specify the development of sex-specific characteristics using the popular genetic model organism Caenorhabditis elegans, a soil worm. This grant supported research that implicates a protein called RFX as a regulator of sex-specific development in the nervous system. RFX, in worms and in humans, directs the formation of a cellular structure called a sensory cilium, which is required for the function of some types of nerve cells. Since defects in RFX and the genes it controls are associated with human diseases known as ciliopathies, this study, which reveals new information about how RFX works, has the potential to lead to new ways to diagnose or treat these diseases. The Huang Lab is focused on understanding how a single cell, the fertilized egg, divides to form different cells with different patterns of gene expression. This grant supported research into how the cell fate determinant MEX-3, an RNA binding protein, is regulated in the early C. elegans embryo. We found that two different genes, zif-1 and zyg-11, are required for MEX-3 degradation and appear to work at different points in development. Homologs, or relatives of MEX-3 in other organisms including humans, contain a RING finger domain that is missing in C. elegans MEX-3. RING finger domains function in the same process of regulated protein degradation as zif-1 and zyg-11. MEX-3 homologs are involved in a variety of important processes including regulation of gene expression in human intestinal epithelial cells, control of growth and differentiation in stem cells, and regulation of programmed cell death. Better understanding of how MEX-3 is regulated in the early C. elegans embryo may provide insight into how these other processes are regulated. Throughout the course of this grant, a total of 11 undergraduate research students were trained to use research grade equipment such as the fluorescence stereomicroscope purchased with this grant. Such training makes these students competitive for jobs and graduate programs in the sciences and several students are now working as lab technicians, attending graduate programs in biology, and attending medical school. The fluorescence stereomicroscopes purchased with this grant were also used to enhance several courses taught at Colorado College to biology, neuroscience, and biochemistry majors as well as non-science majors. In total 249 students were impacted in five different courses over two years. In addition to research, student training, and course enhancement, the grant supported several community outreach activities with perhaps 200 participants that stimulated community interest and awareness of science.

National Science Foundation (NSF)
Division of Biological Infrastructure (DBI)
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Robert Fleischmann
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Colorado College
Colorado Springs
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