Identification of DVA interneuron regulatory sequences in Caenorhabditis elegans.

Carmie Puckett Robinson,Erich M Schwarz,Paul W Sternberg

doi:10.1371/journal.pone.0054971

Abstract

BackgroundThe identity of each neuron is determined by the expression of a distinct group of genes comprising its terminal gene battery. The regulatory sequences that control the expression of such terminal gene batteries in individual neurons is largely unknown. The existence of a complete genome sequence for C. elegans and draft genomes of other nematodes let us use comparative genomics to identify regulatory sequences directing expression in the DVA interneuron.Methodology/Principal FindingsUsing phylogenetic comparisons of multiple Caenorhabditis species, we identified conserved non-coding sequences in 3 of 10 genes (fax-1, nmr-1, and twk-16) that direct expression of reporter transgenes in DVA and other neurons. The conserved region and flanking sequences in an 85-bp intronic region of the twk-16 gene directs highly restricted expression in DVA. Mutagenesis of this 85 bp region shows that it has at least four regions. The central 53 bp region contains a 29 bp region that represses expression and a 24 bp region that drives broad neuronal expression. Two short flanking regions restrict expression of the twk-16 gene to DVA. A shared GA-rich motif was identified in three of these genes but had opposite effects on expression when mutated in the nmr-1 and twk-16 DVA regulatory elements.Conclusions/SignificanceWe identified by multi-species conservation regulatory regions within three genes that direct expression in the DVA neuron. We identified four contiguous regions of sequence of the twk-16 gene enhancer with positive and negative effects on expression, which combined to restrict expression to the DVA neuron. For this neuron a single binding site may thus not achieve sufficient specificity for cell specific expression. One of the positive elements, an 8-bp sequence required for expression was identified in silico by sequence comparisons of seven nematode species, demonstrating the potential resolution of expanded multi-species phylogenetic comparisons.

Highlights

Neurons express a largely overlapping set of genes required for their general function as a neuron
The total number of animals scored is in parentheses with YFP expressing animals shown as a percentage of the total under the corresponding regions of the nervous system
In WT53 produced the most consistent neuronal expression (p,0.0001) (Table 3; Figure 6M–N), consistent with sequences in the Mut5 and Mutant 2 (Mut2) regions acting to repress expression

Summary

Introduction

Neurons express a largely overlapping set of genes required for their general function as a neuron. The specific identity of each individual neuron, in turn, requires the expression of distinct sets of genes comprising terminal gene batteries [1,2,3]. The regulatory sequences determining the expression of sets of genes comprising the terminal gene battery have been identified, but most remain obscure. Identifying these regulatory sequences remains a challenging problem due to the complexity of the nervous system [3]. The ability to identify neurons by Nomarski optics and to examine cell-specific gene expression by transgenic reporters makes C. elegans useful to investigate pertinent regulatory sequences. The existence of a complete genome sequence for C. elegans and draft genomes of other nematodes let us use comparative genomics to identify regulatory sequences directing expression in the DVA interneuron

Methods

Results

Discussion

Conclusion