Identifying Cell-Specific Transcripts
The C. elegans genome is completely sequenced yet many predicted genes lack direct evidence of transcription and a substantial number of cryptic protein-coding genes and ncRNAs are likely to have been missed by gene prediction software. We are participating in the modENCODE (model organism Encyclopedia of DNA Elements) consortium project to identify all C. elegans genes (The Transcriptome). To identify these transcripts, we are isolating RNA from specific C. elegans cells and tissues for tiling array analysis. This approach ensures detection of rare RNAs from small populations of cells while also providing clues to their in vivo functions.
Embryonic and Larval Expression Profiling
We use specific promoters to mark cells for isolation by FACS (Fox et al., 2005) or for mRNA extraction by the mRNA tagging method (Von Stetina & Watson et al., 2007). The small amount of RNA obtained by these methods (< 25 ng) is amplified to generate a labeled ds cDNA target for hybridization to the Affymetrix C. elegans Tiling 1.0R array. We have produced tiling array profiles of major tissues (nervous system, muscle, intestine, skin) and from specific embryonic and larval cell types (e.g., coelomocytes, excretory cell, GABA neurons, PVD nociceptor neurons, AVA command interneurons). Threshold analysis detects transcripts from established gene models as well as from candidate transcriptionally active regions (TARs) in intergenic and intronic domains. Biased detection ofknown tissue and cell-specific transcripts validates these data sets and suggests that other differentially expressed TARs may exercise cell-specific functions. In addition to detecting novel transcripts, our approach is producing a gene expression map that matches the single cell resolution of the C. elegans anatomy. The capacity to produce robust expression profiles from nanogram quantities of RNA should be generally useful for other applications in which RNA is limiting (Gerstein et al., 2010, Lu et al., 2011, Spencer, Zeller et al., 2011).
Please use this link to download cell-specific gene expression data sets and to access tools for visualizing transcripts (Genome Browser) and for quantifying expression levelsfor specific genes across cell types and developmental stages (WormViz).
We have recently developed methods for generating cell-specific RNA-Seq profiles from C. elegans larvae and adults (Spencer et al., 2014). In the SeqCel (RNA Seq of C. elegans cells) method, we use FACS to isolate fluorescently labeled cells for RNA-seq analysis. In our intial studies, we have focused on neurons because the nervous system contains the largest variety of specific cell types in C. elegans. In addition to cataloging the gene expression fingerprint of each type of profiled neuron, SeqCel should be highly useful for more focused applications. For example, we are now using SeqCel to identify the targets of transcription factors that regulate regulate key mechanisms in neural development such as synaptic specificity (UNC-4), circuit remodeling (UNC-55, IRX-1) and dendritic branching (MEC-3).
Specific sensory and motor neurons are accessible to isolation by FACS from multiple larval stages
Currently working on the Project...
Andrea Cuentas Condori