Expression Profiling of GABAergic Motor Neurons in Caenorhabditis elegans

Current Biology, Vol. 15, 340–346, February 22, 2005, ©2005 Elsevier Ltd All rights reserved.

DOI 10.1016/j .c ub . 20 05 . 02 .0 2 5

Expression Profiling of GABAergic Motor Neurons in Caenorhabditis elegans Hulusi Cinar,1 Sunduz Keles,3,4 and Yishi Jin1,2,* 1 Department of Molecular, Cell and Developmental Biology 2 Howard Hughes Medical Institute University of California, Santa Cruz Santa Cruz, California 95064 3 Division of Biostatistics, School of Public Health University of California, Berkeley Berkeley, California 94720

examined. To monitor contamination from other motor neurons and muscle cells in the ventral cord, we performed reverse transcriptase polymerase chain reaction (RT-PCR) on unc-25, unc-4, and unc-49 genes. unc-25 PCR product indicated an enrichment for GABAergic neurons (Figure 1F), whereas transcripts of unc-4 and unc-49 genes, markers for cholinergic A-type motor neurons [14] and body wall muscle cells [15], respectively, had no significant change as compared to the reference.

Summary Neurons constitute the most diverse cell types and acquire their identity by the activity of particular genetic programs [1]. The GABAergic nervous system in C. elegans [2] consists of 26 neurons that fall into six classes [3, 4]. Animals that are defective in GABAergic neuron function and development display “shrinker” movement [5], abnormal foraging and defecation [4, 6]. Among the known shrinker genes, unc-25 and unc47 encode the GABA biosynthetic enzyme glutamic acid decarboxylase and vesicular transporter, respectively [7, 8]. unc-30 encodes a homeodomain protein of the Pitx family and regulates the differentiation of the D-type GABAergic neurons [9]. unc-46 probably functions in presynaptic GABA release [6], but its identity has not been reported. By cell-based microarray analysis, we identified over 250 genes with enriched expression in GABAergic neurons. The highly enriched gene set included all known genes. In vivo expression study with computational predictions further identified six new genes that are potential transcriptional targets of UNC-30. Behavioral studies of a deletion mutant implicate a function of a nicotinic receptor subunit in D-type neurons. Our analysis demonstrates the utility of neuron-specific genomics in identifying cell-specific genes and regulatory networks. Results and Discussion Recent innovations in cell and RNA purification in the worm enabled cell-based expression profiles [10, 11]. We used an integrated Punc-25-GFP reporter to label the six DD and four RME neurons in the developing embryo [12] (Figure 1A). Embryos were dissociated, and the cells were cultured in vitro for 1 day. Dissociated GFP-labeled cells assumed neuron-like morphology [11, 13] (Figure 1B) and were purified by fluorescence-activated cell sorting (FACS) (Figures 1C–1E). A total cell fraction depleted in GFP signal was collected to be used as reference in microarray analysis. mRNA from the FACS-collected cells was linearly amplified, and representation of known marker genes was *Correspondence: [email protected] 4 Present address: Department of Statistics and Biostatistics and Medical Informatics, University of Wisconsin, Madison, K6/440 CSC, 600 Highland Avenue, Madison, Wisconsin 53792.

Genome-Wide Subtractive Gene Expression Analysis of GABAergic Neurons To identify GABAergic neuron specific genes, we hybridized cRNA samples of enriched GABAergic neurons (n ⫽ 5 replicates) and total cells (n ⫽ 6) to oligonucleotide microarrays (Affymetrix). Focusing on genes primarily active in GABAergic neurons with background-level signal intensities in the reference, we calculated differential log2 ratios of average signal intensities per gene and ranked them to indicate expression in GABAergic neurons. These criteria yielded a list of ⵑ260 genes with a threshold of 3.0-fold differential expression (see Table S1 in the Supplemental Data available with this article online). Among the genes that displayed highly enriched expression were genes known to be expressed in GABAergic neurons (the genes unc-25, unc-47, and unc-30) (Table 1). The top-ranking C04F5.3 gene is mapped on chromosome V, near unc-46. We confirmed it to be unc46 (K. Schuske and E. Jorgensen, personal communication) and did not pursue further analysis. This result confirms the gene-cloning utility of microarray analysis [11]. In addition, several identified genes such as syd-1, jnk-1, and jkk-1 exhibit specific functions in D neurons, defining the acquisition of axonal identity and trafficking of synaptic components [16, 17]. Lastly, the detected ionotropic channels may underlie the physiological processes in the GABAergic neurons (see below). Thus, this initial analysis indicates that GABAergic neuron-based expression profiling detected a significant number of genes that might function in these neurons. Proper selection of reference is crucial in high throughput applications. For example, we used total cells as the reference, and this may explain the identification of genes with common synaptic function and widespread neural expression including synaptotagmin (snt-1), synaptogyrin (sng-1), and synapsin (snn-1) [18]. To improve the sensitivity of gene detection, we used purified non-GABAergic neurons labeled by a Pacr-2GFP reporter as the reference (n ⫽ 3). About half of the GFP-labeled neurons in this strain are the DA and DB cholinergic motor neurons in the ventral cord. We observed little change in the differential log2 rankings of the known “shrinker” genes in the subtractive analysis, whereas the ranks of pan-neurally expressed genes (e.g., snt-1, snn-1, and sng-1) and known muscleexpressed genes demoted (Table 1, Rank1 versus

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Figure 1. Isolation of GABAergic Motor Neurons for Expression Profiling (A) unc-25-GFP reporter strain. RME neurons, asterisk; DD neurons, arrowheads. Anterior to the left, ventral to the bottom. Bar, 10 ␮m. GABAergic neurons fall into six classes by their innervation pattern and behavioral output [3, 4]. The four RME neurons innervate head muscles to control foraging, AVL and DVB innervate enteric muscles and regulate defecation, and RIS has not been assigned a function yet. The remaining 19 class D neurons, the six DD and 13 VD, provide inhibitory input to the body muscles. (B–D) Morphology of the dissociated GFP-labeled cells. Bar, 2 ␮m. Phase-contrast (C) and fluorescence microscopy (D) images show unsorted fraction of GFP-labeled cells. (E) Sorted GFP cells. (F) RT-PCR analysis on linearly amplified mRNA from GFP or total cell sorts. Results of a representative sample are shown. Right triangles on the top indicates cDNA dilution series. Transcript markers: unc-25, GABA neurons; unc-4, cholinergic neurons; unc-49, muscle cells.

Rank2; Table S2). Notably, this analysis detected the ahr-1 gene which encodes the C. elegans ortholog of aryl hydrocarbon receptor and regulates the fate of RMEL/R neurons [19] (Table 1). These findings suggest that subtractive expression profiling with related neurons as reference enhanced detection of cell specific genes. In Vivo Expression Analysis of MicroarrayIdentified Genes To further verify the microarray list, we assessed expression patterns of high-ranking candidate genes by GFP reporter transgene analysis (Table S3). Nine genes out of 18 (50%) candidates showed expression in D-type GABAergic neurons. The expression pattern of these nine genes fell into two groups. Five genes showed expression not only in D neurons but also in other neu-

rons, frequently the other types of ventral cord motor neurons (Figure 2A). The second group showed specific expression in D neurons of the L1 ventral cord with varying additional neuronal expression elsewhere and contained the genes C55F2.2, oig-1, flp-13, and acr-14 (Figure 2B). The ventral cord expression pattern was reminiscent those of unc-25 (Figure 2B) and unc-47 [7, 8]. C55F2.2 encodes a protein similar to a secreted enzyme from medicinal leech that can stimulate neurite extension in spinal cord ganglion cultures [20, 21]. flp13 encodes a FMRFamide-like neuropeptide that may modulate synaptic transmission [22]. oig-1 is a small secreted protein with a single immunoglobulin domain and may function in cell-cell interactions [23, 24]. The protein identities suggest possible roles in GABAergic neuron physiology and development. However, no mutations have been reported for these genes, and doublestranded RNA interferences (in N2 or rrf-3 strains) also

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Table 1. Candidate Genes Expressed in GABAergic Neurons Gene

Locus

Log2 (Fold)

Rank1a

Rank2b

Y37D8A.23 C09E7.3 C04F5.3 T20G5.6 R13A5.9 C18H9.7 R01E6.4 F31E8.2 Y38C1BA.2 C55F2.2 F33D4.3 F53B6.2 F36A2.4 C41G11.3 T05C12.2 W03F8.1 F58G4.1 T08A9.3 C04G2.1 F35D2.5 B0564.10 B0478.1 Y71D11A.5 F21D12.3 F35C8.3 C41G7.5

unc-25 oig-1 unc-46 unc-47

5.66 5.02 4.37 4.07 3.60 3.40 3.35 3.15 3.06 3.04 2.79 2.69 2.64 2.50 2.49 2.36 2.29 2.25 2.22 2.17 2.11 2.03 1.95 1.82 1.67

1 2 3 5 9 10 11 15 18 19 39 44 47 68 71 95 105 123 130 147 164 191 221 299 408 1608

6 2 10 9 10180 97 5646 2763 443 1055 44 142 9853 113 27 3295 8539 5077 423 73 16 1743 1459 1873 58 49

rpy-1 acr-12 snt-1 snn-1 flp-13 twk-30 acr-14 tni-4 sng-1 syd-1 unc-30 jnk-1

jkk-1 ahr-1

D Neuron Expression

unc-30 Regulation

PCR Ratioc

Description

⫹

⫹ ⫹

3.9 4.0

Glutamic acid decarboxylase Small Ig-containing protein

⫹

1.7

Vesicular GABA transporter

⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹

1.7 2.0

⫹

1.5

⫹

⫹

3.4

⫹ ⫹

⫹

0.7

Postsynaptic protein (RAPSYN) Acetylcholine receptor protein Synaptotagmin Synapsin Destabilase FMRFamide-like peptide 13 Thrombospondin-like repeats Potassium channel protein RGS16 (regulator of G-protein signaling 16) Neuronal acetylcholine receptor protein Troponin Myosin Synaptogyrin Transthyretin-like protein Transcr. activator HAP4 Homeobox protein (otd subfamily) MAP kinase Ion channel protein Amino acid transporter MAPK kinase Aryl hydrocarbon receptor

Genes were selected from Table S1 according to their annotations. a Rank1 represents differential log2 rankings derived from subtraction of total cells from GABAergic neurons. b Rank2, differential log2 rankings derived from subtraction of acr-2-GFP-labeled neurons from GABAergic neurons. Both rankings were obtained from the raw data without data processing except reference signal intensities were set to 50 for background correction. c Fold differences in the level of endogenous transcripts between total RNA from N2 and unc-30(e191).

did not reveal detectable abnormality (data not shown), which could be due to insensitivity of neurons to RNAi [25, 26] or may imply that impairing the function of these genes might not lead to detectable behavioral or morphological abnormalities with standard assays. Identification of Potential UNC-30 Target Genes by In Vivo Expression Analysis In unc-30 mutant animals, D neurons lack GABA and have defects in axonal pathfinding and synaptic connections, whereas other types of GABAergic neurons are unaffected [9]. UNC-30 directly controls the expression of unc-25 and unc-47 in D neurons to specify their transmitter identity [27]. Further extent of gene regulation by UNC-30 in GABAergic motor neurons is poorly understood. Considering their protein identities and expression patterns, we investigated whether acr-14, oig-1, flp-13, and C55F2.2 are regulated by UNC-30. To test this, we examined the effect of unc-30 mutations on expression pattern of each gene. In unc-30 animals, fluorescence produced by unc-25-GFP in the ventral cord D neurons abolishes (Figure 2B, first row). Similarly, the GFP reporter transgenes of the four genes lost their D neuron activity in unc-30 mutants (Figure 2B). These results suggest that these genes may represent potential regulatory targets of UNC-30 in D-type GABAergic neurons. UNC-30 Binding Site Sequence Is Enriched in Promoter Regions of UNC-30 Target Genes Next, we examined the 5⬘ upstream regions of the six UNC-30 target genes for regulatory elements and used

sequence conservation in C. briggsae cognate regions to improve analysis [28]. The consensus sequence 5⬘TAATCC-3⬘ was previously reported as a common core of UNC-30 binding site in unc-25 and unc-47 promoters [27]. Similarly, we observed an enrichment of the 5⬘TAATC(C)-3⬘ consensus sequence in the promoter regions of UNC-30 target genes (Figure 3A). To filter out nonconserved occurrences, we aligned cognate promoter regions from C. briggsae and identified the consensus WNTAATCHH (Figure 3B; Table S4). This consensus is significantly enriched in promoter regions of UNC-30 target genes (p ⬍ 0.005 for 500 bp). These observations are consistent with a nonrandom sequence feature presumably defining the UNC-30 binding motif and support the identification of these novel genes as potential UNC-30 regulatory targets. Computational Predictions Revealed Additional Potential UNC-30 Target Genes To detect additional UNC-30 targets, we scored the promoter regions of the microarray-identified genes for WNTAATCHH instances with a position weight matrix and predicted potential UNC-30 target genes by C. briggsae cognate comparisons (Figure 3C). Subsequent transgene analysis revealed at least two of the predicted genes, F21D12.3 and C04G2.1 (data not shown), as potential targets of UNC-30. F21D12.3 encodes a putative amino acid transporter protein, and C04G2.1 belongs to a gene family of transthyretin-like proteins in the worm. To independently verify the regulation by UNC-30, we quantitated the endogenous transcripts of the target

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Figure 2. Genes Expressed in GABAergic Motor Neurons (A) Expression patterns of group A promoterGFP reporter constructs in L1 larval animals. Orientation as in Figure 1A. This group of genes showed expression in the D-type GABAergic neurons as well as other neurons. The commissural pattern illustrated by arrowheads is diagnostic for DD neurons in L1 larvae (upper left). The ventral cord is indicated by an arrow in all panels. Arrowheads mark GFP-expressing neuronal cell bodies where some of them are the D-type neurons (bottom panels). Reporter constructs are based on the promoter sequences of F53B6.2a, a conserved member of immunoglobulin superfamily; acr-12, an acetylcholine receptor subunit; F36A2.4/twk-30, potassium channel protein; and rpy-1, rapsyn. Bar, 10 ␮m. (B) Expression of group B genes and their regulation by UNC-30. Expression patterns of promoter-GFP reporter strains in L1 larval wild-type (left panels) and unc-30 mutant backgrounds (right panels) were shown for unc-25 [7], acr-14, C55F2.2, flp-13, and oig-1. Orientation as in Figure 1A. Bar, 10 ␮m.

genes by a real-time RT-PCR assay with total RNA from wild-type and unc-30 mutant embryos. Seven of eight target genes including the shrinkers unc-25 and unc-47 seemed to be downregulated in unc-30 mutant background (Table 1). Altogether, these results suggested that a combination of microarray and computational analysis enhanced the identification of UNC-30 target genes.

Potential Functions of a Nicotinic Acetylcholine Receptor in GABAergic D-Type Neurons D-type motor neurons receive cholinergic synaptic input [3], and we identified three nAChR subunit genes, acr-9, acr-12, and acr-14, whose expressions were enriched in D neurons. acr-14 encodes a non-␣ nicotinic acetyl choline receptor (nAChR) subunit that shares extensive homology with vertebrate ␣-7-10 nAChR subunits [29].

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Figure 3. Computational Predictions of UNC-30 Target Genes from Microarray Data (A and B) Description of a signature sequence representing UNC-30 consensus binding site. (A) Comparison of TAATC occurrences in 500 bp promoter regions of C. elegans UNC-30 target genes and their C. briggsae cognates. (B) A graphical display of consensus binding site WNTAATCHH derived from the sequences with conserved relative positions as shown in (A) (Table S4). (C) UNC-30 target gene predictions by phylogenetic footprinting. 1 Kb promoter regions of microarray-identified C. elegans genes are scanned for instances of WNTAATCHH with a position weight matrix and compared to their C. briggsae cognates. Shown are genes with similar consensus occurrences in 500 bp promoter regions.

acr-12 encodes a protein that is homologous to vertebrate ␣-type nAChR subunits [30]. By transcriptional GFP reporter analysis, acr-14 and acr-12 appear to be expressed in D neurons (Figures 2A and 2B). These observations raise the possibility that they may mediate synaptic input to these neurons.

We next assessed the function of acr-14 with mutant animals. acr-14(ok1155) is a 965 bp deletion within the open-reading frame, which removes all transmembrane domains and thus is likely to be a null allele (Figure 4A). The mutant animals were largely normal in gross appearance and in cellular and synaptic morphology of

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the physiological state of specific neurons. This approach complements genetic methods by providing a direct examination of genetic networks in a cell type. Genetic screens do not favor the detection of genes with redundant function or genes that give subtle phenotypes when mutated. Screens for mutants with behavioral defects specific to loss of GABA function are conversely biased against genes with pleiotropic and/or essential functions. Cell-based subtractive microarray analysis enhances assignment of function to genes by integrating into traditional genetic methods, gene-based anatomy, and computational analysis. It provides a valuable tool to crack the regulatory code underlying cellular identity. Supplemental Data Supplemental Data includes five tables and Supplemental Experimental Procedures and can be found with this article online at http:// www.current-biology.com/cgi/content/full/15/4/340/DC1/. Acknowledgments

Figure 4. acr-14 Is a Potential nAChR Subunit That May Function in D-type Neurons (A) Gene structure of acr-14. Underlying black line denotes deleted segment in acr-14(ok1155). Predicted transmembrane domains are indicated by black boxes. The acr-14(ok1155) mutation would result in a nonfunctional protein truncated at amino acid 227. Scale bar, 200 bp. (B) Behavioral analysis of acr-14(ok1155) animals. 1-day-old animals are scored for time to cross tracks after initial mid-lawn placement and free crawl (Experimental Procedures). The y axis shows the percentage of animals that cross their tracks within 2 min, and the data are the mean of three independent tests ⫾ SEM. Multiple transgenic lines were observed, and data are shown for acr-14(ok1155); juEx881[Psur-5-GFP] [31] and acr-14(ok1155);juEx884[T05C12.5; Psur-5-GFP]. Asterisk, p ⬍ 0.03; double asterisk, p ⬍ 0.01 (Student’s t test).

D neurons as judged by morphology assays based on GFP reporter genes (data not shown). We detected a subtle behavioral impairment in acr-14(ok1155) animals as they tended to verve to their dorsal side and left knottier tracks on bacterial lawn than wild-type animals did (Figure 4B). When we injected a PCR-amplified genomic fragment containing acr-14(T05C12.5) into acr14(ok1155) animals, we observed reproducible and significant partial rescue in the behavioral phenotype in comparison to transgenic lines expressing a coinjection marker alone (Figure 4B). This analysis reveals a role of acr-14 in the control of body movement, likely through modulating the motor output of the ventral cord neural circuitry. It also reassures that neuron-specific microarray analysis facilitate the identification of genes that would otherwise evade detection because of subtle, complex or redundant functions in GABAergic neurons. Conclusions By comparing isolated wild-type and mutant cells, Zhang et al. used cell-based expression profiling to identify targets of the MEC-3 transcriptional program that regulates touch neuron differentiation in the worm [11]. Here, we used wild-type cells to infer genes that reflect

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