Rebecca M Fox et al. (2002) West Coast Worm Meeting "Microarray experiments to identify unc-4 target genes in FACS-isolated unc-4::GFP embryonic motor neurons."

David M Miller III, Azza Gasmelseed, Rebecca M Fox

[West Coast Worm Meeting, 2002]

The UNC-4 homeoprotein is expressed in A-type motor neurons (DA, VA, SAB) where it functions to maintain normal levels of synaptic vesicles. UNC-4 is also required in VA motor neurons to regulate presynaptic specificity. In unc-4 mutants, VAs show reduced numbers of synaptic vesicles and are miswired with inputs normally reserved for their lineal sister cells, the VB motor neurons. We have proposed that these defects arise from the failure of UNC-4 and its transcriptional cofactor, UNC-37/Groucho to repress B-motor neuron specific genes. Here we describe a new strategy that relies upon physical isolation of unc-4::GFP cells to identify these genes.

Rebecca M Fox et al. (2003) International Worm Meeting "Gene expression profiling of specific C. elegans cells."

David M Miller, Stephen E Von Stetina, Azza Gasmelseed, Susan J Ruff, Kellen Olszewski, Rebecca M Fox

[International Worm Meeting, 2003]

Individual C. elegans cells are easily visualized in vivo but are tantalizingly inaccessible: the small size of the animal as well as its tough outer cuticle have largely prevented dissection of specific cells for molecular analysis. To circumvent this problem, we have now developed methods for isolating GFP-marked cells from primary cultures grown in vitro. Conditions for culturing C. elegans embryonic cells have been previously described. GFP-marked neurons and muscle cells differentiate in vitro where they appear to express normal morphological, molecular and physiological traits.1 These GFP cells can be isolated by FACS to generate highly enriched (~90% GFP) preparations. In principle, mRNA from these cells could be used to interrogate the Affymetrix C. elegans microarray to detect cell-specific gene expression. To test this idea, we isolated body muscle cells from dissociated myo-3::GFP embryos. Total RNA (~100 ng) from these cells (~100,000) was amplified (2x) and labeled. This experiment detected over 1300 genes that are > 2x elevated (p < 0.05) in myo-3::GFP muscle cells relative to all embryonic cells. This data set includes most known muscle-specific genes (e.g. myo-3, unc-15, unc-27) and also shows substantial overlap with an independently derived expression profile of body muscle cells in the intact animal.2 We have now extended this approach to specific classes of motor neurons. unc-4::GFP is expressed in 13 embryonically-derived cholinergic motor neurons (9 DAs, 3 SABs, 1 I5). unc-4::GFP cells are not evident in freshly prepared blastomeres but appear to differentiate normally and approach the expected frequency (~2%) after one day in culture1. These cells are loosely attached to a poly-L-lysine substrate from which they can be readily dissociated for FACS. Over 400 genes are > 2x enriched (p < 0.05) in unc-4::GFP neurons. These include synaptic vesicle components (snb-1, snt-1, unc-17) as well as other genes known to be neuronally expressed (unc-129, unc-5, acr-2). As expected, unc-4 mRNA is highly enriched (30x) in unc-4::GFP neurons. We will report the results of similar experiments for motor neurons marked with unc-25::GFP (DD) and acr-2::GFP (DA,DB). In the future, we will use this approach to identify genes that are normally repressed by the UNC-4 homeodomain transcription factor in A-class motor neurons. unc-4 regulates the specificity of synaptic input as well as the strength of synaptic output for A-class motor neurons. Therefore, UNC-4 target genes that we uncover are likely to control key events in motor neuron differentiation and function. 1Christensen, M., et al. Neuron 33, 503-14. (2002). 2 Roy, P. J., et al. Nature 418, 975-9. (2002)