The molecular signature and cis-regulatory architecture of a C. elegans gustatory neuron

Abstract

Taste receptor cells constitute a highly specialized cell type that perceives and conveys specific sensory information to the brain. The detailed molecular composition of these cells and the mechanisms that program their fate are, in general, poorly understood. We have generated serial analysis of gene expression (SAGE) libraries from two distinct populations of single, isolated sensory neuron classes, the gustatory neuron class ASE and the thermosensory neuron class AFD, from the nematode Caenorhabditis elegans. By comparing these two libraries, we have identified >1000 genes that define the ASE gustatory neuron class on a molecular level. This set of genes contains determinants of the differentiated state of the ASE neuron, such as a surprisingly complex repertoire of transcription factors (TFs), ion channels, neurotransmitters, and receptors, as well as seven-transmembrane receptor (7TMR)-type putative gustatory receptor genes. Through the in vivo dissection of the cis-regulatory regions of several ASE-expressed genes, we identified a small cis-regulatory motif, the "ASE motif," that is required for the expression of many ASE-expressed genes. We demonstrate that the ASE motif is a binding site for the C2H2 zinc finger TF CHE-1, which is essential for the correct differentiation of the ASE gustatory neuron. Taken together, our results provide a unique view of the molecular landscape of a single neuron type and reveal an important aspect of the regulatory logic for gustatory neuron specification in C. elegans.

Figure 1.

SAGE analysis of isolated ASER neurons. (A) Schematic representation of amphid chemosensory neurons. Gustatory neurons revealed by laser ablation studies (Bargmann and Horvitz 1991) are colored. (B) ASER-specific ntIs1 transgene (gcy-5^prom∷gfp) used to isolate ASER from embryos. (C) Number of genes found in the raw and filtered ASE SAGE libraries. See Materials and Methods for more details and Supplementary Tables 1 and 3A–C for a list of genes. (D) Filtering of the ASE SAGE library by comparing it with an AFD SAGE library. The success of the filtering is assessed through the use of control data sets (Supplementary Table 2) that contain genes whose expression pattern was previously determined. It should be kept in mind that expression patterns in these control data sets are almost exclusively inferred from reporter gene analysis, which may not provide an accurate reflection of endogenous expression as they may be lacking relevant regulatory information and/or may not be sensitive enough to detect low-level, “leaky” expression. Previously known, ASE-expressed genes in the control data set include genes that at post-embryonic stages become restricted to ASEL (Supplementary Table 2); their representation in the ASE library confirms that the isolation of ASER from embryos indeed provides the gene expression profiles of both ASEL and ASER. (E) Summary of genes with specific predicted function in the ASE > AFD library. See Supplementary Tables 5–7 for more detail on the genes. [¹An additional 30 singletons raises the total number of ASE-expressed 7TMRs to 41. ²A 10th neuropeptide/hormone, ins-1, is expressed in ASE (Kodama et al. 2006), but is not represented in any SAGE library, due to the absence of a restriction site for the tagging enzyme used to generate the SAGE library.] (F) NATs in the ASE transcriptome. See Supplementary Table 8 for a list of genes and more details. [¹In eight of 20 cases, an overlap in transcript has been explicitly demonstrated by EST analysis (http://www.wormbase.org).]

Figure 2.

Expression patterns of genes from the SAGE library as assessed by gfp reporter gene technology. Only a selected and representative number of examples are shown in this figure. All analyzed reporter strains, including those shown here, are shown in Supplementary Figures 1–3. Green boxes indicate gfp coding sequences (rfp in the case of dop-3 and yfp in the case of hlh-10). If extrachromosomal arrays were generated, multiple lines were analyzed for each reporter construct, as indicated below each panel of micrographs. Each data set of micrographs shows expression of the reporter construct under investigation in a high-magnification (400×) image, the overlap with a red fluorescent marker for ASEL/ASER (otIs151 transgene; “ASE-RFP”), and a low-magnification (160×) overview of expression of the reporter construct under investigation throughout the whole worm. In the low-magnification images, the asterisk (*) indicates expression of the gut-specific injection marker elt-2∷gfp. (A) Expression patterns of two selected TFs from the SAGE data set. (B) Expression patterns of two selected peptidergic signaling proteins from the SAGE data set. (C) Expression patterns of two selected putative chemoreceptor proteins of the 7TMR family from the SAGE data set. The srg-30 7TMR-type chemoreceptor shows the most restricted expression pattern with strong expression in ASEL/R and weak expression in the chemosensory neurons ASIL/R and ADLL/R. Additional expression can be observed in a pharyngeal neuron. The srd-33 7TMR reporter gene fusion also shows a restricted expression pattern (ASEL/R, AWBL/R, ASHL/R, AVKL/R, PHAL/R, PHBL/R, and weakly in another unidentified tail neuron pair, possibly PHCL/R). The bottom panels in C demonstrate that 7TMR-type chemoreceptors localize to the dendritic ending of the ASE neurons. In order to boost expression in the ASE neurons, a 188-bp ASEL-specific regulatory element from the gcy-7 gene was appended to each locus (indicated as a black box). One transgenic line each was analyzed in detail. Yellow arrows point to the dendritic endings.

Figure 3.

Dissection of the cis-regulatory architecture of ASE-expressed genes reveals a common motif, the “ASE motif.” (A) Determining minimal regulatory elements required for ASE expression. Blue arrows indicate the presence of a conserved site, the ASE motif (detailed in C). The number of transgenic lines scored is indicated in parentheses next to each construct. (B) Scanning mutational analysis of the regulatory elements of the ASE-expressed genes gcy-5 (ASER), gcy-7 (ASEL), and lim-6 (ASEL). The ASE motif is indicated in red. The number of transgenic lines scored is indicated in parentheses next to each construct. Only cases in which a loss of gfp expression (rather than ectopic expression) is observed are indicated here. In the case of the gcy-5 gene, the deletion of a single 25-bp region causes loss of expression in ASER (“del 6”). “del 6.2” and “del 6.3” are deletions that eliminate sequences directly adjacent to the ASE motif. In the case of the gcy-7 gene, deletions of two neighboring 25-bp regions affect the ASE motif and cause loss of expression in ASEL. In the case of the lim-6 gene, the deletion of four 25-bp regions each causes loss of expression in ASEL. Two of these regions each contain a single copy of the ASE motif (ASE motif score: 0.64 and 0.57). Deletion of another 25-bp element specifically causes a failure to maintain ASE expression. Since we previously showed that lim-6 genetically autoregulates its own expression (Johnston et al. 2005), we presume that these 25 bp contain a lim-6 autoregulatory element. The 25-bp region contains a GAATAAA motif that is conserved in three nematode species. When this motif is deleted alone, similar maintenance defects are observed (data not shown). The fourth 25-bp region that is required for ASEL expression also contains a phylogenetically conserved motif; however, its precise excision has no impact on ASE expression (data not shown). We note that the presence of multiple sites required for ASE expression is consistent with our previous genetic analysis, which points to several regulatory inputs into the lim-6 locus (see final model figure). (C) Alignment of a conserved motif present in ASE-expressed regulatory elements defined by reporter gene analysis. Shown here are only those ASE motifs whose binding to the CHE-1 TF have been confirmed by EMSA, as shown in Figure 6. Other previously known ASE-expressed genes also harbor ASE motifs (data not shown), whose relevance was not further tested. Gray shading indicates that the functional relevance of this motif was explicitly confirmed by deleting the motif in a reporter gene construct and observing a loss of gfp expression in ASE, as shown in Figure 4. The sequence alignment defines a position weight matrix (PWM) that is represented by a sequence logo; the quality of the match of each individual ASE motif with the sequence logo is assigned what we term an “ASE motif score” (see Materials and Methods). ASE motifs in genes defined by SAGE analysis are represented in the form of ASE motif scores in Table 1. (D) ASE motifs preferentially cluster within the first ∼1 kb upstream of the ATG start codon of a gene. This graph contains all genes shown in C.

Figure 4.

Functional characterization of the ASE motif. (A,B) Requirement of the ASE motif for gene expression in ASE. A shows representative animals expressing a reporter construct for the ceh-36 gene with the normal ASE motif either present (left panel) or deleted (right panel). B shows a schematic representation of normal or ASE motif-mutated/deleted reporter constructs and their expression. “No ASE expression” indicates a complete loss of detectable gfp expression in ASE that we observed upon deletion of all motifs (representative example is shown in A), with the exception of flp-13, where some weak gfp expression persists in ASE after the ASE motif mutation. The number of transgenic lines analyzed is indicated in parenthesis. All lines for each construct show similar expression patterns. Note that besides its functional ASE motif (with an ASE motif score of 0.66; see text and Materials and Methods for explanation of how an ASE score is calculated), several low-scoring ASE motifs (ASE motif score: 0.54, 0.56, and 0.53) can be found in the flp-13 promoter. None of these low-scoring ASE motifs is required for expression in ASE (last panel). (C) Orientation independence of the ASE motif. A 306-bp regulatory element from the gcy-5 locus drives expression in ASER in either orientation. (D) Distance independence of the ASE motif. The 306-bp regulatory element from the gcy-5 gene is functional when artificially separated by several kilobases from the start of the gfp reporter. Separation was achieved through the addition of the sra-6 promoter, which is active in ASH, among other cells (Troemel et al. 1995). (E–I) Sufficiency of the ASE motif. ASE expression is observed upon insertion of 31 bp, containing the ASE motif, either upstream of an AWC-specific promoter fragment from the ceh-36 gene (E), or upstream of a fragment of the ttx-3 promoter, in which the AIY motif, normally required for expression of this motif in AIY, is deleted (Wenick and Hobert 2004) (F). (G) A 24-bp element (12 bp of ASE motif + 6 bp on either side) also drives ASE expression; additional gfp expression can be observed in a few other neurons. Eight multimerized copies of an 18-bp element that contains the gcy-5 ASE motif (plus 3 bp of flanking sequences on either side) direct gfp expression in only the ASE neurons, in either the forward (H) or reverse (I) orientation. In all panels, “ASE-RFP” indicates the presence of the otIs151 array, which labels the ASE neurons with dsRed2. The vector backbone for all constructs is pPD95.75. The number of transgenic lines analyzed is indicated.

Figure 5.

che-1 controls the expression of various reporter constructs in vivo. che-1 regulates a variety of ASE-expressed genes, as inferred by crossing reporter gene arrays from a wild-type background into a che-1(ot27) mutant background. All white arrows point to the location of the ASE neuron. (A) Control of the “ASE motif-only” construct from Figure 4G by che-1. (B) Control of the ASE motif-containing che-1 promoter by che-1, demonstrating that che-1 autoregulates its expression. Note that in che-1 mutants, the ASE neuron is aberrantly taking up the dye DiI, thereby demonstrating that even in the absence of che-1, ASE remains a sensory neuron (Uchida et al. 2003). The analysis of several cell fate markers has, however, failed to show any specific identity to which the aberrant ASE neuron may have switched in che-1 mutants (Uchida et al. 2003). In contrast to Uchida et al. (2003), we were able to demonstrate that che-1 positively regulates its own expression. We have no explanation for these differences in results. (C) Regulation of an ASE motif-containing promoter of a TF from the SAGE data set. (D) Regulation of an ASE motif-containing promoter of a 7TMR gene from the SAGE data set. (E) che-1 controls the expression of flp-4, which does not contain a functional ASE motif (see below). Apart from the genes shown here, che-1 had been shown previously to regulate several additional genes (tax-2, gcy-5, gcy-6, gcy-7, ceh-23, flp-6, F55E10.7, R13H7.2, ceh-36, and cog-1) (Chang et al. 2003; Uchida et al. 2003).

Figure 6.

The ASE motif is a binding site for the CHE-1 Zn finger TF. (A) Comparison of the ASE motif consensus motif (represented as a sequence logo) with the predicted binding site of CHE-1 determined by C2H2-enoLOGOS and with the experimentally determined binding site of the Drosophila CHE-1 ortholog GLASS in the Lozenge and Rhodopsin promoters. (B) EMSA of bacterially produced CHE-1 and the ASE motif from the gcy-5 locus. In order to compare binding affinities and to avoid potential problems with probe-labeling efficiencies, binding to ASE motifs was determined by competition assays in which binding to the radiolabeled gcy-5 probe was competed with unlabeled ASE motifs from other genes. As specificity controls, the same unlabeled oligos were used in which the core GAANCC motif is mutated; those probes are not able to compete for binding. Supplementary Table 9 contains sequences of probes. (C) Alignment of the Zn fingers of CHE-1 and the Drosophila ortholog GLASS with predicted DNA-contacting residues. Predictions are according to Pavletich and Pabo (1991). The DNA-contacting residues mutated in the constructs used in D and E are indicated. (D) EMSA with wild-type CHE-1 and point-mutated CHE-1, as indicated. (E) Ectopic expression of CHE-1 in other sensory neurons under control of the gpa-10 promoter results in ectopic activation of the ASE motif-containing gcy-5 promoter, as previously reported (Uchida et al. 2003) and as assayed by counting of ectopically gfp-expressing cells, using the ntIs1 Is[gcy-5∷gfp] transgene. This activity largely depends on the last two Zn fingers (#3 and #4) of CHE-1. For each experiment, four transgenic lines were scored, except for mutated Zn finger 2, in which three transgenic lines were scored. For the wild-type control, ntIs1 Is[gcy-5∷gfp] was scored in an otherwise wild-type background.

Figure 7.

Genome-wide distribution and context dependency of the ASE motif. (A) Distribution of the ASE motif in the genome and various SAGE data sets. The occurrence of ASE motifs in various data sets are shown as a cumulative distribution function, in which the highest-scoring match to the ASE motif in a particular promoter (X-axis) is plotted against the fraction of all promoters (in this particular set) that have site scores up to and including this particular score (Y-axis). The promoter size was chosen as 1.5 kb since most experimentally verified ASE motifs locate within this region (Fig. 3D). The distribution of ASE motif scores in the ASE > AFD library (blue line) is slightly different (P < 0.0597) from the distribution of the ASE motifs in all genes in the genome (black line). If one considers genes with progressive stringency in the ASE > AFD data set (5×, 7× enriched tag number in ASE vs. AFD, or >4 or >6 tags only in ASE, not in AFD) one observes a further increase in the statistical significance of enrichment of high-scoring ASE motifs and a depletion of low-scoring ASE motifs when compared with the genome-wide occurrence of ASE motifs. One-hundred sets of 100 genes were drawn randomly from the “all genes” set to form a graphical negative control in thin cyan lines. Kolmogorov-Smirnov statistics were calculated for all sets to determine the probability that each set was drawn from the same distribution of scores as that of all genes in the genome. See the Supplemental Material for more details. (B) The presence of an ASE motif is not a sufficient predictor of ASE expression. ASE motif scores from genes analyzed in the course of the SAGE data validation are shown. The difference between light and dark blue is that the relevance of the ASE motifs in the latter category was explicitly confirmed through deletion of the motifs. See Supplementary Tables 5–7 for reporter gene fusions, expression in ASE, and ASE motif score. (C) Manipulations of the context of ASE motifs demonstrate the context dependency of the ASE motif. Colored boxes represent ASE motifs as shown in the bottom panel. The swaps were conducted by mutating the dark-gray-shaded residues from one ASE motif to that of another ASE motif. The primary data for the “ASE motif-alone” construct from the gcy-5 promoter is shown in Figure 4G. The “equidistant” srt-63 ASE motif is a construct in which the ASE motif of srt-63 was positioned at exactly the same distance from the gfp start site as the ASE motif in the gcy-5 construct. “ASE expression” indicates reporter gene expression in ASE neurons.

Figure 8.

CHE-1 in the context of ASE development and gene regulatory networks. All interactions involving the ASE motif and CHE-1 are shown in green. (A) Neuronal gene expression programs are defined by parallel gene regulatory pathways that determine general and cell-type-specific features and can be classified as “differentiation subroutines.” The N1 motif is a functionally relevant motif found in many pan-neuronal genes (Ruvinsky et al. 2006). We described here that che-1 regulates three aspects of ASE neuron differentiation: It regulates scores of bilaterally symmetric, terminal differentiation markers, such as ion channels, chemoreceptors, neurotransmitters, etc.; it triggers the expression of components of a bistable feedback loop that eventually controls left/right asymmetric terminal differentiation features of the ASE neurons, such as the gcy genes (in whose regulation che-1 also directly participates; see B); and it controls the expression of newly identified TFs that control as-yet-unknown aspects of ASE development and/or function. (B) che-1 appears to be directly responsible for inducing the previously described hybrid precursor state in the embryo, characterized by the bilateral expression of genes that later in embryogenesis and larval development become restricted to either ASEL or ASER. This restriction is mediated by a bistable feedback loop composed of TFs and miRNAs, each of which contain a che-1-responsive ASE motif. The left/right differential activity of the bistable feedback loop appears to be programmed into the system by as-yet-unknown means at an early embryonic stage (Poole and Hobert 2006).