Teaching Genomics at Small Colleges > Inquiry-based Integrated Instructional Units > Expression of gerontogenes in neurons: A comparative genomic approach to studying the role of the nervous system in lifespan/aging

Expression of gerontogenes in neurons: A comparative genomic approach to studying the role of the nervous system in lifespan/aging

Kathleen Susman, Vassar College
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A representative student-generated protein similarity tree for the gerontogene, spe-26

This 4-5 week laboratory exercise is ideal for an intermediate or more advanced undergraduate level course in neuroscience and behavior or even evolutionary biology. This laboratory module has students explore the role of the nervous system in aging and lifespan using genetic mutants of C. elegans. Gerontogenes are genes that influence lifespan in many organisms, including nematodes, insects and mammals. An intriguing question in evolutionary biology is what function is served by gerontogenes. Are these genes actually regulating aging? Or, are they involved in other cellular or physiological processes and influence aging/lifespan only indirectly (via pleiotropic effects)?

Students use a bioinformatic approach to identify candidate gerontogenes in C. elegans. They select a gene to become "expert" on based on primary research articles that we have discussed in class. They then take a comparative genomic approach by identifying orthologs of candidate genes in other organisms and explore evolutionary relationships by sequence alignment and phylogenetic tree construction (making a protein sequence similarity tree). Students design and carry out a behavioral experiment, such as a thermotolerance test, based on literature-based exploration, that tests aspects of candidate gene function in behavior and conduct a behavioral screen of mutant nematodes. The laboratory culminates in a presentation, along with a scientific manuscript, that integrates the students' behavioral data, their literature-based work, as well as their protein sequence similarity analysis.

Learning Goals

Summary of Learning Goals

  1. Critical thinking and experimental design
  2. Statistical analysis of behavioral data
  3. Use/limitations of model organisms in studying complex physiological phenomena like aging.
  4. Gain confidence in the use of computational and bioinformatics approaches to explore evolutionary relationships at the gene and protein level.
  5. Integration and synthesis across taxa and level of analysis.
  6. Distinguish basic relationships among genes and protein sequences like paralogy and orthology.
  7. Integration, synthesis and presentation of behavioral, bioinformatic and comparative genomic approaches.

This module introduces students to the powerful tools and resources available to learn about genes involved in behavior and neural systems. Bioinformatic and comparative genomics approaches are relatively new to the field of neuroscience. Introducing students to this approach to studying important biological questions, when combined with the more familiar behavioral experimentation and the powerful nematode model organism enhances the learning of current issues in neuroscience and behavior.
The course focuses on developing critical thinking and reading skills. This module enhances those skills by having students become expert on a particular gene- learn what is known about that gene in a number of different organisms and relate their findings back to what is known in the field more generally. In addition, students develop and expand their skills in experimental design and statistical analysis of behavioral data. Another goal of this module is for students to grasp both the strengths and the limitations of using simpler model organisms to study complex physiological phenomena like aging. At the end of the module, students present their work to the class and thereafter the class discusses how the independent investigations expand the general knowledge of the different genes of interest, as well as inform the broader field of aging. The comparative genomic exploration allows students to study a system from multiple levels of analysis (a key goal of the entire course)–from cellular/molecular to organismal to behavioral to comparative to genomic to evolutionary.

Context for Use

This multi-week laboratory module is part of the laboratory component of an intermediate-level course in Neuroscience and Behavior.
The class usually consists of sophomores and juniors who have completed introductory biology, introductory psychology and an intermediate-level psychology course in physiological psychology. The students who take this course have already been introduced to rudimentary bioinformatics approaches in their introductory biology course. Thus, this module builds upon those skills. The module is closely tied to the material being covered in the rest of the class time.The course format includes two weekly 75 minute lecture/discussion meetings that focus on primary experimental literature of the neuroscience of aging, comparative neuroanatomy, the neuroscience of development, stress, social behavior and environmental toxicology. The laboratory module occurs within the middle portion of the course, after students have been introduced to the model organism with a behavioral chemotaxis lab series earlier in the course.

This modular format would adapt very well to any course that uses a model organism for which simple behavioral studies are available, such as Caenorhabditis elegans, Drosophila melanogaster or even mice.

Description and Teaching Materials

The laboratory module can be downloaded here.Gerontogenes laboratory module (Microsoft Word 284kB Jun22 09)

Week 1: Gerontogenes I: Exploration of candidate gene and Experimental Design

Preceding this laboratory period, students read and discuss in class several articles introducing students to the major theories of aging. In addition, they have read a recent article about using C. elegans as a model organism to explore the genes involved in aging and lifespan. A small set of genes, called gerontogenes, are involved in aging in C. elegans; several are expressed in the nervous system. Many of these genes seem to confer thermotolerance, suggesting a potential experimental assay. Students, working in pairs, choose a candidate gerontogene from a selection of mutants that we have available for them to become expert on over the ensuing weeks in the laboratory module.
Some example mutants, all available from the Caenorhabditis elegans Genome Center (http://www.cbs.umn.edu/CGC/) are: daf-2, daf-16, spe-26.

In the first laboratory, the pairs of students explore what is known about their candidate gene using WormBase, WormAtlas and the NCBI MapViewer databases. They use the database resources to justify their choice of gerontogene. Why is this gene interesting to consider? Where is this gene expressed in C. elegans? Another goal of this week is for the students to download, in FASTA format, both the genomic and the protein sequence for their genes, save the sequences in separate text files in preparation for the third week of the laboratory module. A third goal of this week's laboratory is for the students to design an experiment, based on the thermotolerance response described in one of the papers the class discussed the previous week. The students select a strain of worm (from a provided selection) that has a mutated version of their candidate gene.

Week 2: Independent Experiments

Students carry out the experiments that they designed the previous week.
Prior to carrying out the experiment, the student groups have met with the instructor for a consultation session to refine and improve the design, to discuss appropriate controls and statistical analyses.
They collect behavioral data for wild type and the mutant worms they have chosen and analyze the data statistically. This document contains some suggestions for independent experiments that can be performed.Experiment ideas for gerontogenes (Microsoft Word 28kB Jun23 09) For the experiments, it is important to have adequate plates of worms and the materials students need to conduct their experiments. It is also important to have computers outfitted with appropriate statistical software.

Weeks 3 and 4: Comparative genomics and protein sequence similarity tree construction

In these laboratory sessions, students download the protein sequence for their candidate gene using NCBI MapViewer. They use BLASTp for 6-10 different animal taxa (one taxon at a time) and select the "best hit" for each organism that has an ortholog of the gene. They also note those organisms that lack an ortholog. They collect the hits into a properly formatted text file and use either MEGA or ClustalX (which works with Macs and is free) to align the protein sequences. A protein sequence similarity tree of their aligned sequences is constructed using any of a number of different packages like MEGA. We use ClustalX and TreeViewer, and compare the output we generated with CLC SequenceViewer. In addition to the phylogenetic tree construction, students use literature databases and other links through NCBI to explore what is known about their candidate genes in other organisms.
Students during this period also do a homework assignment to reinforce their skills. The problem set has them perform a similar analysis using a set of bacterial rRNA sequences with a strong phylogenetic signal. In class, we discuss the different underlying uses of phylogenetic tree construction, from phylogenies to protein sequence evolutionary comparison.

Week 5: Student presentations and class discussion

This laboratory session is in the format of a research symposium.
An example of a student slide presentation
The groups of students present their work to the class. Following the presentations, we have a class discussion relating the findings to the more general concepts about aging and the role of the nervous system in aging. This is a very satisfying aspect of the module for the students because all of the experiments are different, which generates lots of discussion. An aspect of peer-review can be incorporated into this session as well.

Teaching Notes and Tips

  • Students need to have access computers that have MEGA software installed and operational. If you do not have access to that software, you can also use ClustalX and TreeViewer or CLC SequenceViewer, which are all free programs that work pretty well.
  • The mutant worm strains are available from the CGC http://www.cbs.umn.edu/CGC/ for a nominal fee.
  • Petri plates with NGM (Nematode Growth Medium) agar and E. colifood will need to be available. Some experience with C. elegans is very useful. However, it is possible to develop a similar laboratory module using other model organisms, including Drosophila or mice.
    The worms need to be maintained for several weeks prior to the lab and for the month of labs. The behavioral experiments the students design may require multiple plates to have enough replicates for statistical analysis. Attention to the developmental stage also requires careful planning to have enough worms at the right developmental stage.

  • Students need to have access to statistical software for behavioral data analysis. This laboratory module can be expanded to include a real-time PCR analysis of changes in gene expression resulting from the thermotolerance assay in week 2.
    Students can extract mRNA from their samples using Trizol reagent and convert to cDNA. Click this link for information about this kind of laboratory experience if you want to incorporate this into your course http://serc.carleton.edu/genomics/units/33694.html Then, the following week, students can perform real-time PCR using provided primer sets developed by the instructor prior to the lab and assay changes in gene expression using Sybr green. Addition of this wet-lab component will add 3-4 weeks to the laboratory module.


Assessment materials:

  • Skills assessment survey given before the beginning of the module and after the completion of the module to gauge changes in perception of confidence working with bioinformatic, genomic and computer-based tools. Skills assessment questionnaire (Microsoft Word 35kB Jun22 09)
  • Problem sets and small assignments to gauge knowledge and skills mastery. These assignments are designed to assess performance of skills like BLASTp, sequence alignment, tree construction and interpretation, use of databases to gather information. Another problem set assesses mastery of skills like statistical analysis and experimental design.
  • Student presentation to gauge knowledge mastery and level of success.
  • Likert scale administration at the the end of the course to assess changes in attitudes/beliefs toward this approach to neuroscience and behavior. Attitude questionnaire for gerontogenes (Microsoft Word 49kB Jun22 09)

The assessment strategies employed stress knowledge, skills and attitudes/beliefs.
The knowledge that we hope students gain is a familiarity with the use of major databases, particularly NCBI, WormBase and others. We also expect them to gain substantial knowledge about their chosen gene, as well as ways to design experiments, use of appropriate statistics. We assessed this knowledge by having problem set style assignments. For example, students submitted experimental designs and then had faculty/student conferences to go over the designs and discuss other details important for planning and conducting a good experiment (issues like appropriate controls, sample size, types of statistical analysis, etc). Another assignment included a sequence alignment and phylogenetic tree using bacterial 16S rRNA sequence data from a database provided by the Joint Genome Institute 16S rRNA phylogenic homework assignment (Microsoft Word 196kB Jun21 09). Another assessment of student knowledge and mastery is the student presentation.

Initial skills level was assessed with a brief questionnaire. In addition, skills of database mining, construction of phylogenetic tree using MEGA, protein sequence alignment were assessed by the problem set assignment as well as the student presentation. To demonstrate mastery of the skills and knowledge covered in the laboratory module, the quality of the student presentation was an important feature.

The final category of assessment is that of attitudes and beliefs. There are several aspects we are interested in assessing. How does this approach to a neurobiological question enhance the learning of complex behaviors or mechanisms of neuron function? How does using a computer to explore protein sequence relationships affect student interest in neuroscience and behavior? How does this inquiry-based laboratory module affect the student attitude toward neuroscience and behavior? To try to assess these beliefs and attitudes, a Likert-based scale questionnaire was developed and handed out at the beginning of the module. To assess changes in attitudes, the questionnaire is handed out again at the end of the course, allowing several weeks to pass between the laboratory module and the reapplication of the scale.

Summary of overall assessments we conducted

  • Knowledge: based on scores on problem sets and other assignments, we are confident that all students showed substantial increases in knowledge items we were interested in.
  • Skills: When the pre-skills questionnaire and the post-skills questionnaires were compared, we find a substantial improvement in the level of skills confidence, as well as skills acquisition, measured by performance on assignments. A more complete analysis will be published and will be referenced here.
  • Attitudes/beliefs: More than 75% of the students remarked in the Liekert survey that they would be interested in taking another course that included these bioinformatic/genomic approaches. In addition, the vast majority of students surveyed felt that the inclusion of this laboratory module enhanced their understanding of and appreciation for these kinds of approaches in neuroscience and behavior.

References and Resources

  1. Computer resources: ClustalXhttp://www.clustal.org/, TreeView X https://code.google.com/p/treeviewx/, MEGA, CLC Sequence Viewer http://www.clcbio.com/index.php?id=28. All of these are freeware and are easily downloaded and used.
  2. Websites: NCBI (including MapViewer) and WormBase http://www.wormbase.org
  3. Students read and discussed various primary research articles about their chosen gene of interest. These will vary with the student.
  4. For protocols and information about maintaining C. elegans, see WormBook http://www.wormbook.org Another site of potential interest is WormClassroom http://www.wormclassroom.org