Identification of C. elegans sensory ray genes using whole-genome expression profiling

1. Identification of C. elegans sensory ray genes using whole-genome expression profiling Douglas S. Portman and Scott W. Emmons

2. Aim • Identify genes involved in the regulatory network controling C. elegans sensory ray development and function

3. What are sensory rays • Tail sensilla, likely both mechano- and chemosensory, used by males during copulation • Each ray is composed of two distinct neurons, and a structural cell • Adult males have 18 rays, and are the most numerous neural subtypes in these animals

4. Approach • Using microarray analysis to compare total RNA between two mutant strains that differ widely in the number of rays • EM673 males contain wild type set of 18 rays, and several ectopic rays, resulting from a mutation in lin-22. • EM672 males are essentially rayless as a result of mutations in the bHLH genes lin-32 and hlh-2.

5. Isolate RNA from synchronized young adult males of these strains. • Fluorescently labelled cDNA derived from these samples was used for seven microarray hybridizations. • For each spot on the array an intensity ratio measuring the average change between the EM673 and EM672 hybridization signal intensities was calculated. • Restricted their analysis to genes that gave significant results in at least four of the seven arrays.

6. Identified 13,305 unique genes, and generated an average EM673 to EM 672 ratio for each of these. • Removed 3975 potential germline genes from consideration as candidate ray genes. • This leaves a data set of 9330 unique somatic genes, which would be a group more highly enriched for genes relevant to sensory ray development.

7. How effective is this approach • Measuring expression levels in the context of whole animal mRNA, so the contribution of RNA from other cells is likely to be significant compared to the relatively low amount expected from sensory rays. • Ray expressed genes with significant expression outside the ray may show little change in expression levels. • Mutations present in these strains will affect gene expression in other cells as well.

8. Estimating Effectiveness • Examined the scores generated by their experiments for 8 genes whose expression is known to be reasonably specific to rays. • None of theses genes were missed by removing potential germline genes. • Six of these genes were in the top seventh percentile, one in the top 12th, and one did not show a change in expression.

10. Identification of ray-expressed genes • Chose 25 previously unstudied genes, and constructed at least one GFP or YFP reporter, for each gene. • For each reporter several transgenic lines were inspected for ray fluorescence. • Identified expression in rays for 13 genes. • Described 11 of these in some detail.

12. Four novel genes associated with polycystins • The ray RnB neurons, as well as male specific CEM and HOB sensory neurons, use a polycystin signalling pathway to sense mechanosensory cues. • Microarray data identified 4 novel genes that share expression profiles characteristic of polycystin genes, named these cwp (coexpressed with polycystins)

13. Summary • This approach has allowed the identification of many sensory ray expressed genes, but there are inherent problems in the comparison of total RNA between two mutant strains. • The data set may also yield a significant number or additional ray-expressed genes.