Genome sequences lack information about when and where genes are expressed. To fill this gap, we are working on methods to determine the expression profiles of individual cells, especially neurons. We harvest single, GFP-labeled neurons (using the same techniques used to record from neurons in vivo) and use the method of C. Dulac for single-cell RT-PCR. Successful amplification is verified by secondary PCR with primers directed against adjacent 3' exons of GFP and of known cell-specific genes. Transcripts encoding GFP (expressed from a multi-copy array) are detected in most GFP+ neurons (41/62) and few unlabelled neurons (2/18). Endogenous transcripts encoding known cell-specific genes are detected in ~1/5 neurons examined. We tested for the following genes:
gcy-8 in AFD cells (9/47);
gcy-5 in ASER cells (2/10); and
odr-10 in AWA cells (1/3). Amplified material is used for further analysis only when both GFP and at least one endogenous, cell-specific transcript are detected by secondary PCR. Verified cellular cDNAs can be used either to screen for new genes by secondary PCR or to construct cell-specific cDNA libraries. For example, we screened AFD cDNAs for expression of two gene families implicated in sensory transduction: transient receptor potential channels ( trp ) and transmembrane guanylyl cyclases ( gcy ). We found three new AFD genes: the C. elegans trp ortholog (R06B10.4) and two gcy genes (
gcy-23 and
gcy-26 ). We are currently expanding our search for new genes by constructing and screening AFD, ASER, AWA and AWC-specific cDNA libraries. We will conduct differential screens with these libraries to identify genes expressed specifically by each sensory neuron. These experiments are a first step toward determining the expression profiles of individual neurons and mapping the genome onto the nervous system.