Senior Research Proposal
Gap junctions are intercellular channels that allow direct transfer of electrical currents or small signaling molecules between coupled cells, and they consist of two hemichannels, one in each of the coupled cells. Each hemichannel is composed of six subunits, and in vertebrates, these subunits are connexin or pannexin membrane proteins while in invertebrates they are composed of innexins, invertebrate homologs of pannexins. Each of the six subunits can be made from the same or different gap junction proteins, thus producing many different types of gap junctions from just few proteins. The size and charge of the protein subunits determine channel permeability. Along with determining channel permeability, subunit proteins also determine hemichannel specificity. Gap junction proteins consist of two extracellular loops, one intracellular loop domain, and four trans-membrane domains. It has been hypothesized that extracellular loops confer the specificity between two hemichannels to form gap junctions while intracellular domains are involved in channel permeability.1
Gap junctions are widely expressed in the developing nervous system of vertebrates and invertebrates including Caenorhabditis elegans. However, the biological significance of gap junctions is only partly understood. It has been a challenge to directly address biological roles of gap junctions in the complex nervous system of vertebrates. I propose to use the simple nervous system in the model organism C. elegans, composed of just 302 neurons, to uncover fundamental mechanisms that are likely to be used in the human brain. Also, C. elegans are a good model system because normally they are hermaphrodites ( <1% of the populations are male) , which makes for easy maintenance, but they can be engineered to produce a higher percentage of males. The hermaphrodites will preferentially mate with males rather than self-cross, thus, C. elegans are also good candidates for genetic manipulation.
Dr. Chiou-Fen Chuang has recently shown that the innexin gap junction protein NSY-5 establishes asymmetric gene expression in the left and right amphid wing cells (AWC), which are a pair of olfactory neurons.2 During embryogenesis, only one AWC neuron expresses the gene str-2, which encodes a olfactory receptor, but mutations in nsy-5 leads to the loss of str-2 expression in that AWC neuron. This result uncovered novel roles of gap junctions in neuronal network formation and function. nsy-5 is expressed in many other neurons, and though the two AWC neurons do not form gap junctions with each other, they are indirectly connected via two other pairs of neurons: the amphid finger cells (AFD) and the amphid neurons with single ciliated endings (ASH), which also express nsy-5. AFD is a thermosensory neuron that helps control locomotion in response to temperature and ASH is a chemosensory neuron that helps control avoidance behavior in response to acidity. It is via AFD and ASH that nsy-5 establish left-right asymmetric gene expression in AWC. It is probable that nsy-5 is not the only gap junction protein for establishing left-right asymmetry of the C. elegans nervous system. I hypothesized that other innexins also contribute to the establishment of left-right neuronal asymmetry in the C. elegans. As the first step in testing this hypothesis, I previously identified additional innexins that are expressed in the C. elegans nervous system and then determine their spatial and temporal expression patterns. I identified twelve candidates based on the fact that they are expressed in the head region, which is where AWC is located, thus, since the remaining innexins present in the genome are not expressed in the head region, they can not forming gap junctions with AWC and thus could not be collaborating with nsy-5 in this process. In previous experiments, I was unable to to detect expression of any of the twelve candidate innexins in AWC, and the final step in testing this hypothesis is to determine whether any these innexins are expressed in AFD or ASH.
I will determine whether any of the twelve candidate innexins are expressed in the neurons AFD or ASH, by taking transgenic worms that express green fluorescent protein (GFP) with the promoters of each of the innexins (these strains were kindly provided by Dr. Zhao-Wen Wang, University of Connecticut Health Center) and cross them with other transgenic worms that express red fluorescent protein (DsRed) with a nsy-5 promoter (kindly provided by Dr. Chuang, Cincinnati Children's Hospital). Thus, a percentage of the progeny of those each crosses will express its respective innexin-GFP marker and the nsy-5-DsRed marker. Since nsy-5 is expressed in AFD and ASH and the expression pattern of nsy-5 as well as the morphologies of AFD and ASH have been mapped, I will be able to identify whether any of the candidate innexins are expressed in AFD and ASH by looking for co-localization between the two markers.3 My positive control would be to cross the nsy-5-DsRed transgenic worms with worms that have AWC labeled with GFP (also provided by Dr. Chuang's lab), and my negative control would be to cross the nsy-5-DsRed transgenic worms with wild type worms.
Statement of Relevance
In addition to contributing to the current project on left-right asymmetric gene expression in the AWC olfactory neurons in C. elegans by indicating other possible participants, this project could contribute in a small way to human medicine. Mutations in connexins, the homoplastic counterparts of innexins in vertebrates, have been linked to the following humans diseases and disorders: deafness; skin disorders; Charcot Marie Tooth Disease (CMTX), a disease characterized by the demyelination of peripheral neurons; cataracts; and oculodentodigital dysplasia (ODDD), characterized by the developmental abnormalities of the face, eyes, and limbs.4 Due to the simplicity as well as the large amount of knowledge that we have on the structure of the C. elegans nervous system using these worms could give us insight as to what we should look for and study in vertebrate models and eventually in humans. Also, we have very little information about pannexins, and since pannexins are actually homologous with innexins (that is in fact how they were discovered), unlike connexins, gaining knowledge about innexins could give us clues into the roles of pannexins.
Since some my work will be done in Dr. Costi D. Sifri's laboratory at the University of Virginia and I currently have no realistic means of transportation until next semester, my goals for this semester are to establish effective means of maintaining the worm at Mary Baldwin College and to write up the research that I did this summer in the appropriate format. As for next semester, I plan to set up my crosses in groups of three to four thus making four groups. The worms' life cycle is approximately three days long at 22°C, thus I would set up my crosses during one trip to UVA and then return three days later (and possibly again on the fourth day to get more results) to analyze my results for co-localization. I would repeat this for all groups.
Expertise/ Institutional Support
I learned how to care for and handle C. elegans this summer at an internship at Cincinnati Children's Hospital in Dr. Chiou-Fen Chuang's laboratory. I also learned how to cross males and hermaphrodites, as well as basic techniques for identifying neurons based on morphology. I would utilize some of the facilities in Dr. Costi D. Sifri's laboratory at UVA, as well as a corner of the back bench in the physiology lab at MBC. I hope to complete this thesis under the guidance of Dr. Lundy Pentz.