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. 2013;8(1):e54971.
doi: 10.1371/journal.pone.0054971. Epub 2013 Jan 28.

Identification of DVA Interneuron Regulatory Sequences in Caenorhabditis Elegans

Free PMC article

Identification of DVA Interneuron Regulatory Sequences in Caenorhabditis Elegans

Carmie Puckett Robinson et al. PLoS One. .
Free PMC article


Background: The 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 findings: Using 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/significance: We 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Schematic images of C. elegans tail ganglion and dorsal rectal ganglion neurons.
The labeled ganglion are the Pre-anal ganglion (PA), Lumbar ganglion (LG) and Dorsal Rectal Ganglion (DRG). The individual neurons comprising the DRG are DVB, DVA and DVC in the black box. The gut is in pink and the rectum shown in darker brown. The images were derived from, by Christopher Grove (Caltech).
Figure 2
Figure 2. A. Conserved regions analyzed for DVA expression.
Relative location of conserved regions identified by MUSSA from the acr-15, fax-1, nmr-1 and twk-16 genes. Conserved regions are depicted as red boxes below the corresponding gene and denoted as cs1-cs4 based on their relative position from the first exon in black. The intergenic regions are shown as a black line with the size (kb) above. Neuronal expression is shown in the vertical oriented box as+or - under DVA or Broad. The parentheses and asterisk (53 bp*) following the 73 bp twk-16 cs1 region denotes that the 53 bp fragment of the 73 bp cs1 region expressed in DVA and Broadly. The 73 bp twk-16 cs1 region does not show expression. B. Expression vector. The features of the PCR expression vector are denoted by colors: experimental sequences (red); Δpes-10 (purple); nuclear localization signal (NLS) (blue); YFP (yellow); and derived from the Fire Vector pPD122.53. The unc-119 promoter and unc-119 mini-gene are in green. Experimental sequences were fused by PCR to this expression vector to form a single PCR product.
Figure 3
Figure 3. Conserved regions driving expression in DVA.
Panels A–D are photomicrographs of the tail region of transgenic L4-adult C. elegans. DVA expression is denoted by a yellow line identifying the DVA neuron. The gene name is followed by the conserved region numbered by its position relative to the first exon. A. DVA expression of the 190 bp conserved region 2 of nmr-1 (nmr-1.cs2). B. DVA expression of the 308 bp fragment containing conserved region 1 of twk-16 (twk-16.cs1). C. DVA expression of the 180 bp conserved region 3 of fax-1 (fax-1.cs3). D. DVA expression of the 322 bp conserved region 4 of fax-1 (fax-1.cs4). Scale bar = 20 µm.
Figure 4
Figure 4. Sequence analyses.
A. Alignment of conserved twk-16 sequences. Sequence level comparison of the MUSSA alignment of the 73 bp conserved regions of twk-16.cs1 and orthologous twk-16 regions from C. elegans, C. briggsae, C. brenneri and C. remanei. WT85 is shown at the top with the two sub-fragments of WT53 shown as WT29 in blue and WT24 in red type. WT53 is shown above in brackets. Conserved sequences in the four species MUSSA comparison using a window of 20 and threshold of 17 are in red type with red lines between the orthologs. B. The consensus GA-rich motif identified by MEME. The sequences representing the GA-rich motif in each fragment are highlighted in color type with flanking regions in black type. The strand is indicated as either+or – and start site of the GA-rich motif is indicated in each of the four genes used in the analysis. The respective genes and conserved regions used in the MEME analysis: fax-1.cs3 (180 bp), fax-1.cs4 (322 bp), nmr-1.cs2 (190 bp) and twk-16.cs1 (308 bp). The respective fragments contained 3 GA-rich motifs in fax-1.cs3, 4 GA-rich motifs in fax-1.cs4, 3 GA-rich motifs in nmr-1.cs2 and 3 GA-rich motifs in the 308 bp fragment containing twk-16.cs1. The 144 base start of the GA-rich motif in 308 bp twk-16.cs1 fragment corresponds to position 17 in the 53 bp WT53 and is shown in Figure 8B. The strand, start site, p-value and sequences were identified by MEME.
Figure 5
Figure 5. Mutation analysis. A. Analysis of the twk-16 intron and enhancer.
Deletion analysis of the twk-16 intron with 73 bp twk-16.cs1 (cs1) and 259 bp twk-16.cs2 (cs2) denoted in red with flanking sequences in black and not to scale. The approximate sizes of the wild-type (WT) sequences are denoted by numbers from WT2000 to WT53. WT2000 was a plasmid construct and contains 500 bp 5′ of exon 1, exon 1 and 1.4 kb of the first intron containing both cs1 and cs2. WT700 contains 38 bp of flanking sequences 5′ to cs1 and 244 bp of flanking sequences 5′ to cs2 and 102 bp of 3′ flanking sequences. WT350 contains 55 bp of flanking sequences 5′ to cs2 and 45 bp of 3′ flanking sequences. WT500 contains 202 bp of flanking sequences 5′ to cs1 and 223 bp of 3′ flanking sequences. W300 contains 114 bp of flanking sequences 5′ to cs1 and 121 bp of 3′ flanking sequences. WT195 contains 114 bp of 5′ flanking sequence to cs1 and 8 bp of 3′ flanking sequence. WT113 contains 23 bp of 5′ flanking sequences to cs1 and 17 bp of 3′ flanking sequence. WT85 contains 4 bp of 5′ flanking sequences to cs1 and 8 bp of 3′ flanking sequence. WT85 contains WT53 with 17 bp of 5′ flanking sequence and 15 bp of 3′ flanking sequence. WT53 contains 53 bp of the 73 bp twk-16.cs1 region. B. Mutational analysis of WT53. The wild-type sequence is denoted by black type with the mutations of WT53 shown in red type. Conserved sequences identified by the seven species MUSSA comparison are in blue type with blue underlining. WT29 and WT24 are generated by cleavage of WT53 within the Mut3 region. DVA or broad neuronal expression is denoted by+or – in the box to the right of each construct.
Figure 6
Figure 6. Photomicrographs of the expression of twk-16 constructs in transgenic lines.
The photomicrographs are arranged from left to right in three columns of six photomicrographs. DVA expression of wild-type twk-16 intron constructs. Panels A–F are photomicrographs of the tail region of transgenic L4-adult C. elegans generated with the experimental sequences shown in Figure 5A. Yellow lines indicate DVA neurons expressing YFP. The constructs used to generate the transgenics in each panel were: A. WT2000∶500 bp of the 5′region of twk-16 gene, the first exon and entire 1.4-kb first intron with twk-16.cs1 and twk-16.cs2. B. WT500: twk-16.cs1 and flanking sequence producing both DVA and DVC expression. C. WT300: twk-16.cs1 and flanking sequence D. WT195: twk-16.cs1 and flanking sequence. E. WT113: twk-16.cs1 and flanking sequence. F. WT85: twk-16.cs1 and short flanking sequences. Expression of wild-type twk-16 .cs1 constructs. Panels G–L are photomicrographs of the tail of transgenic L4-adult C. elegans animals with the following constructs: G. WT85∶53 bp of twk-16.cs1 with 17 bp 5′ and 12 bp 3′ of flanking sequence. H. WT53∶53-bp fragment of twk-16.cs1 in wild-type orientation. I. WT53R in reverse orientation (3′-5′) to the expression vector. J. WT29: the 5′ 29 bp of WT53. K. WT24: the 3′ 24 bp of WT53 L. Vector: no experimental sequence and PCR expression vector Δpes-10::4X NLS::YFP::unc-54::unc-119. Expression of mutated twk-16 .cs1 constructs. Panels M–R are photomicrographs of the tail region of transgenic L4-adult C. elegans animals made with the following constructs containing mutations (Mut2-Mut8) of the 53-bp fragment (WT53) of the twk-16.cs1 region: M. Mut5. N. Mut2. O. Mut2. P. Mut3. Q. Mut6. R. Mut8. L4 Vulva expression in Mut2 transgenic is shown in Panel O with a yellow line identifying the vulva. Scale bars are specific to each column of six photomicrographs and = 20 µm.
Figure 7
Figure 7
A. Mutation of WT24 in the context of larger fragments. The sequences of WT53 and sub-fragments WT29 and WT24 are shown in black type. WT53 is the black box in the diagrams of WT195 and WT85. Mutations of the bases of WT24 are in red type in Mut24. Mut24 is shown as a red box in the diagrams of Mut195 and Mut85. Neuronal expression in transgenic lines derived from the four experimental sequences is shown in the box as either+or – expression under DVA or Broad. B. Mutation of the flanking sequences of WT53. The WT53 sequence is shown in black type at top. WT53 sequence is represented by the black box with flanking wild-type sequences found in WT85 in black type. Mutations of the flanking sequences are shown in red type and labeled: 5′ Mut85 (mutation of the 17′bp 5′ of WT53); 3′ Mut85 (mutation of the 14 bp 3′ of WT53); and 5′3′ Mut85 (mutation of the 5′ and 3′ flanking sequences). Expression in DVA or Broad neuronal expression is denoted as+or - in the box to the right of the experimental sequences.
Figure 8
Figure 8
A. Seven species comparison of twk-16 enhancer and model. The WT53 element is in black type and highly conserved bases identified by seven species MUSSA analysis in blue type and underlined. The experimental sequences of Mut2-Mut8 (Figure 5B) containing mutations of WT53 sequence are shown in red type and wild-type sequences of WT29 and WT24 in black type. Neuronal expression is denoted by+or – under DVA or Broad. MUSSA comparison of 3′-ward WT53 or WT53-like sequences from the twk-16 genes or homolog’s of seven nematode species: C. elegans, C. briggsae, C. remanei, C. brenneri, C. japonica, C. angaria and Heterorhabditis bacteriophora. WT53 and WT53-like sequences are in uppercase; adjacent 3′-ward residues are in lowercase. Conserved bases shared by all seven species are in blue type. B. Model of the 85 bp twk-16 enhancer. The WT85 sequence showing the A-D regions: A region 17 bp (purple); the D region 15 bp (purple); B region 29 bp (green); and C region 24 bp (orange). UniPROBE predicted TF binding sites for homeodomain TF’s (denoted by HOX) and ETS family TF’s (ETS) are shown below as colored sequence corresponding to the WT85 sequence. The GA-rich motif (green) in the B region with green arrow denoting the GA-rich motif is on the minus strand. Below the A-D regions is a summary of effects on neuronal expression as+or - of the four regions in rows labeled as DVA or Broad. The model diagram shows the A-D regions as letters with the same color scheme as the above WT85 sequence. Lines with arrowheads designate a positive effect on expression and the lines ending with a vertical line designate a negative effect on DVA or Broad neuronal expression.

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