Damian Marlee et al. (2005) International Worm Meeting "Expression Profiling of the Excretory Cell"

Steve Von Stetina, David M Miller, Kathie Watkins, Alfred L George, Clay Spencer, Damian Marlee

[International Worm Meeting, 2005]

The excretory cell in C. elegans is critical for osmoregulation. This H-shaped cell is made up of a large ectodermal cell body, located beneath the posterior pharyngeal bulb, and bilateral tubular canals that run the length of the animal directly beneath the hypodermis. We propose to define a gene expression fingerprint of the excretory cell as a first step toward identifying genes with key roles in excretory cell development and function. These studies will exploit available Affymetrix microarray gene 'chips' representing most genes in the nematode genome coupled with a molecular genetic strategy for isolating enriched populations of mRNAs from single cell populations.1 We have used microparticle bombardment to create transgenic animals for expression localization studies and for mRNA tagging. Transgenic lines expressing GFP driven by the clh-4 promoter of, a chloride channel previously shown to be expressed solely in the excretory cell2, were created and, as expected, GFP expression was localized to the excretory cell. Next, we used the clh-4 promoter to drive expression of an epitope tagged poly-A-binding protein (3XFLAG-PAB-1); anti-FLAG-staining detects excretory-specific expression in this transgenic line. Co-immunoprecipitation with anti-FLAG antibody enables capture of excretory cell-specific mRNAs cross-linked to FLAG-PAB-1. These mRNAs will be amplified, biotin labeled, and applied to the C. elegans Affymetrix array. A comparison to microarray profiles obtained from all cells should reveal excretory enriched transcripts. We intend to apply this strategy to identify excretory cell transcripts as the excretory cell develops in embryonic and larval stages as well as to define homeostatic excretory mRNAs in the adult. We expect that this information will identify homologs in vertebrate/mammalian osmoregulatory systems that can be tested in RNAi experiments to define their roles in the differentiation and function of nematode excretory cell. 1. Roy PJ, Stuart JM, Lund J, Kim SK. Nature 2002; 418:975-979. 2. Nehrke K, Begenisich T, Pilato J, Melvin JE. Am J Physiol Cell Physiol. 2000 279(6):C2052-66

Joseph Watson et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Toward gene expression profiling of rare C. elegans cells"

Joe Don Heath, David Miller, Damian Marlee, Joseph Watson, Stephen Von Stetina, Shenglong Wang

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

With its sequenced genome and simple, well-defined nervous system, C. elegans is ideally suited for generating a high-resolution gene expression map of the nervous system. However, the limited amounts of mRNA that can be easily obtained from rare cell types is a barrier to the realization of this goal. WT-OvationTM, a new, more efficient method for amplifying mRNA should faciliate this effort. To test this approach, the mRNA tagging strategy was used to profile gene expression in all neurons. A T7 RNA polymerase dependent method (IVT) required at least 25ng of template mRNA whereas robust microarray profiles were obtained from 2ng of starting mRNA amplified with WT-Ovation. Enriched genes from both methods showed marked neuronal expression (~90%) and together identify ~1100 common transcripts. Interestingly, each method also identified unique transcripts not detected by the alternative strategy. To test the applicability of this approach to a single cell, we have selected the excretory cell, one of the largest cells in C. elegans. The excretory cell uses neuronal guidance cues and receptors to elaborate an H-shaped network of tubes with essential osmoregulatory functions. The clh-4 promoter was used to drive exclusive expression of 3XFLAG-PAB-1 in the excretory cell. WT-OvationTM amplification of mRNA (2ng) immunoprecipiated from this line detects known excretory cell genes on the Affymetrix microarray. For example, ceh-6, a homeodomain protein that specifies excretory cell differentiation is enriched whereas transcription factors that regulate neuronal development (e.g. unc-86) are not elevated in the excretory cell microarray data. The preponderance of genes encoding ion channels and transporters (~20%) is consistent with the osmoregulatory function of the excretory cell. We conclude that WT-OvationTM affords a reliable mRNA amplification method that should faciliate ongoing efforts in our lab to profile gene expression in single types of C. elegans neurons.

Joseph Watson et al. (2007) International Worm Meeting "Gene expression profiling using limited quantities of mRNA."

Joe Don Heath, Nurith Kurn, Steve Von Stetina, David Miller, Shenglong Wang, Sarah Anthony, Joseph Watson, Shawn Levy, Damian Marlee, W. Clay Spencer

[International Worm Meeting, 2007]

With its sequenced genome and simple, well-defined nervous system, the nematode C. elegans is ideally suited to generate a high-resolution gene expression map of the nervous system. However, the limited amounts of RNA that can be obtained from rare cell types are a barrier to the realization of this goal. WT-Ovation PicoTM, a novel method for amplifying RNA that can use as little as 2ng of starting material, may make individual cell profiling possible. To test this approach, an epitope-tagged mRNA binding protein was used to immunoprecipitate RNA from all neurons. A T7 RNA polymerase dependent method (IVT) required at least 25ng of mRNA whereas robust profiles were obtained from 2ng using WT-Ovation Pico. Enriched genes from both methods showed marked neuronal specificity (~90%) and the intersection of these two datasets identified ~1100 transcripts (68% overlap). Interestingly, each method also identified a unique set of transcripts not detected by the other. These alternative lists appear to be equally valid, however, because both contain transcripts with defined functions in neurons. For example, the E3 ubiquitin ligase, rpm-1, which is expressed throughout the nervous system and is known to regulate synaptic morphology, is only enriched in the WT-Ovation Pico dataset. In contrast, the synaptic vesicle priming protein, unc-13, is found only in the IVT-enriched dataset. Additionally, we have now confirmed neuronal expression for transcripts exclusive to either WT-Ovation Pico or IVT through the creation of representative GFP reporters. Thus, a more complete picture of gene expression in the nervous system can be obtained by using a combination of amplification methods. Following these proof of principle experiments, we used WT-Ovation Pico to profile gene expression in a limited subset of neurons. To that end, we have now generated microarray data from larval GABAergic motor neurons (Poster by S. Anthony). Preliminary data indicate that established GABAergic transcripts are enriched in this data set and thus WT-Ovation Pico will be useful for profiling other specific classes of C. elegans neurons.