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A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans

Abstract

The stereotyped mating behaviour of the Caenorhabditis elegans male is made up of several substeps: response, backing, turning, vulva location, spicule insertion and sperm transfer. The complexity of this behaviour is reflected in the sexually dimorphic anatomy and nervous system1. Behavioural functions have been assigned to most of the male-specific sensory neurons by means of cell ablations; for example, the hook sensory neurons HOA and HOB are specifically required for vulva location2. We have investigated how sensory perception of the hermaphrodite by the C. elegans male controls mating behaviours. Here we identify a gene, lov-1 (for location of vulva), that is required for two male sensory behaviours: response and vulva location. lov-1 encodes a putative membrane protein with a mucin-like, serine–threonine-rich amino terminus3 followed by two blocks of homology to human polycystins, products of the autosomal dominant polycystic kidney-disease loci PKD1 and PKD2 (ref 4). LOV-1 is the closest C. elegans homologue of PKD1. lov-1 is expressed in adult males in sensory neurons of the rays, hook and head, which mediate response, vulva location, and potentially chemotaxis to hermaphrodites, respectively2,5. PKD-2, the C. elegans homologue of PKD2, is localized to the same neurons as LOV-1, suggesting that they function in the same pathway.

Main

We examined the mating behaviour of existing mutants that are defective in sensory behaviours including mechanosensation, osmotic avoidance and chemotaxis to soluble and volatile odorants. Only males with severe defects in all sensory neuron cilia (daf-10, osm-5, osm-6 and che-3) were defective of vulva location (Table 1); all cilia in C. elegans are in the dendritic endings of sensory neurons5,6. Only ciliated neurons express osm-6::gfp, with male-specific expression in four CEM head neurons and neurons of the rays and copulatory spicules7. Expression of osm-6::gfp begins at the L4 stage in neuronal cell bodies and extends to dendrites as neuronal outgrowth proceeds. The RnA and RnB neurons of each ray (rays 1–9), both HOA and HOB hook neurons, the spicule neurons SPV and SPD, and the PCB postcloacal sensilla neurons accumulate green fluorescent protein (GFP). OSM-6 may be required for the structure and function of ciliated neurons in the tail of the adult male, just as it is for neurons involved in many sensory behaviours8,9.

Table 1 Vulva-location behaviour of wild-type and mutant males

By screening for mutants defective in vulva location, we identified lov-1(sy552), which results in specific response and vulva-location defects. When a lov-1(+) male encounters a hermaphrodite, it responds by placing its tail flush on the hermaphrodite, then backs along her body, and turns at her ends until it stops at her vulva. lov-1 mutant males frequently fail to respond to hermaphrodite contact. Compared with the 88% responsiveness of wild-type males (n = 82), sy552 male responsiveness is 24% (n = 37, P < 0.0001). When responsive is initiated, lov-1(sy552) mutants back and turn normally but frequently pass the vulva (Table 1), although spicule insertion and sperm transfer are unaffected. lov-1(sy552) males exhibit high mating efficiency with severely paralysed unc-52 hermaphrodites, but sire few progeny with active, moving dpy-17 hermaphrodites, presumably because a paralysed partner is an easy target, whereas an active partner evades the lov-1 mutant male. lov-1(sy552) mutants appear normal for movement, egg-laying, nose touch, tap, mechanosensation and osmotic avoidance.

We cloned the lov-1 gene on the basis of its genetic map position (Fig. 1). sy552 is recessive. mnDf21/sy552 and sy552/sy552 males are phenotypically indistinguishable, so it is likely that sy552 decreases the function of lov-1. lov-1 falls between the breakpoints of eDf21 and mnDf21. Cosmids in this region were injected and only ZK945 rescued lov-1 defects (4 out of 5 stable lines). A 16.9-kilobase (kb) subclone (plov-1.1) rescued both the response and vulva-location defects of sy552, but shorter subclones did not (Fig. 1). Both a 6.7-kb (plov-1::gfp1) and a plov-1.1 frameshirt clone (plov-1.3) failed to rescue sy552 defects, yet dominantly inhibited vulva location but not response (Fig. 1).

Figure 1: lov-1 and pkd-2 genomic structures, constructs, rescue data and expression patterns.
figure 1

The line above the lov-1 gene indicates the 1,055-bp deletion in lov-1(sy582Δ). Numbers in parentheses indicate the ratio of rescuing stable lines to the number of total stable lines examined. DN, dominant negative.

lov-1 encodes a predicted 3,125-amino-acid membrane-bound protein with a serine–threonine-rich, potential extracellular domain similar to mucins3, a polycystin homology block 1 (26% identity), and a carboxy-terminal polycystin block 2 with 20% identity to polycystin proteins 1, 2 and 2L, encoded by the PKD1, PKD2 and PKDL loci, respectively (Fig. 2). A Kyte–Doolittle hydropathy plot predicts several transmembrane domains. No signal peptide is predicted in LOV-1. Mutations in PKD1 or PKD2 account for 95% of autosomal dominant polycystic disease4. There is similarity between LOV-1, the polycystins and the TRP and voltage-activated calcium and potassium family of channels in the transmembrane spanning region10,11,12,13,14. LOV-1 lacks the Ca2+-binding EF-hand of polycystins 2 and L, the characteristic pore region of channels, and a cytoplasmic coiled-coil tail of all three polycystins (Fig. 2), which mediate hetero- and homotypic interactions between polycystin 1 and polycystin 2 (refs 15, 16). However, truncation of 58 C-terminal amino acids of LOV-1 (LOV-1::GFP5) destroys its function (Fig. 1), indicating that the LOV-1 cytoplasmic tail may be important for LOV-1 action. LOV-1 possesses a potential nucleotide-binding domain (Fig. 2) not present in the human polycystins. Polycystins have recently been implicated in signal transduction17,18,19. Consistent with a signalling function, PKD2 and the TRPC1 channel interact in vitro, as do PKD1 and TRPC family members20.

Figure 2: LOV-1 structural features and homologies.
figure 2

The LOV-1 N terminus is serine/threonine rich with several potential glycosylation sites followed by an ATP/GTP-binding domain (asterisk) and two polycystin blocks of homology. Block 2 also shows homology with voltage-activated Ca2+-channel and TRP-channel families. The number of identical amino acids between LOV-1 and a particular polycystin is indicated. A coiled-coil is predicted in the middle of LOV-1, using the most stringent criteria for the COILS program. PKD-2 was identified in a BLAST search of unpublished sequences available through the Sanger Centre. A partial corresponding cDNA, yk219e1, was sequenced and aligned to the Genefinder ORFs Y73F8a.b and Y73F8a.a, changing the predicted size of PKD-2 from 913 to 815 amino acids. PKD-2 is more similar to PKD2 (33% identity, 52% similarity, 8% gaps over 681 amino acids) than LOV-1 (22% identity, 42% similarity, 13% gaps over 530 amino acids). Additional similarity between PKD2, PKD2L and PKD-2 is denoted by a hatching. Overall identity between polycystins, LOV-1 and PKD-2 is indicated.

We isolated sy582Δ, a genomic deletion of lov-1 that encodes a truncated protein lacking the polycystin/channel homology domain (Fig. 1). lov-1(sy582Δ) has a similar genetic and phenotypic profile to sy552 (Table 1). sy552/sy582Δ males are phenotypically indistinguishable from sy552/Df males, consistent with sy582Δ being a null allele. Thus, the polycystin block 2 is required for LOV-1 activity.

We examined the expression pattern of LOV-1::GFP reporters. A 6.7-kb fusion (LOV-1::GFP1) and a 15.4-kb fusion lacking only 58 amino acids (LOV-1::GFP5) direct expression in male-specific sensory neurons (Figs 1 and 3). Shorter versions of lov-1::gfp1 are not expressed in the same set of male-specific neurons, nor do they act as dominant negatives (Fig. 1). lov-1::gfp1 and lov-1::gfp5 are expressed in male-sensory neurons, specifically the CEM neurons, the hook neuron HOB, and the sensory ray neurons. Expression begins during late L4 lethargus and peaks in the adult male. LOV-1::GFP1 is localized at high levels in the cell body (punctate cytoplasmic expression) and ciliated endings (Fig. 3), whereas LOV-1::GFP5 is found exclusively in cell bodies. The temporal and spatial regulation of lov-1 is concordant with its role in adult male mating behaviour. Rays mediate responses to contact with a hermaphrodite2, the hook mediates vulva location2, and the CEMs may be involved in chemosensation5.

Figure 3: LOV-1::GFP1 and PKD-2::GFP2 are colocalized to adult male sensory-neuron cell bodies and dendrites.
figure 3

The spicules, hook structure and posteriormost fan region autofluoresce. Arrows, neuronal cell bodies; arrowheads, dendrites or ciliated endings. Images (merged DIC and fluorescence) were obtained using confocal microscopy. ac, lov-1::gfp1. a, HOB and ray cell bodies (arrows), HOB dendritic process (arrowhead). b, HOB and ray process 5 (arrowheads). c, Ciliated endings in nose tip from male-specific cephalic CEM neurons (cell bodies not shown). df, pkd-1::gfp2. d, Ray cell bodies (arrow) and ray process 2 (arrowhead). e, Ray process 5 (arrowhead). f, Male-specific cephalic CEM ciliated endings (arrow). Scale bar, 20 µm.

The functions of the polycystins and the molecular basis of kidney cystogenesis are not known, but polycystin 1 and polycystin 2 have been proposed to function in concert4. The C. elegans genome contains a PKD2 homologue with 27% overall identity (Fig. 2). Like PKD2, PKD-2 possesses six membrane-spanning domains, a positively charged fourth membrane-spanning segment, a pore region and the coiled-coil domain of all polycystins. PKD-2 is localized to the same male-specific sensory neurons as LOV-1 (Figs 1 and 3). Rarely, very faint non-sex- and non-stage-specific expression of pkd-2::gfp1 is observed in nerve-ring cell bodies. Expression of pkd-2::gfp1 (which lacks transmembrane domains) is uniform throughout the HOB, ray and CEM neurons. In contrast, the basodendritic localization of PKD-2::GFP2 (which contains membrane-spanning regions) is identical to that of LOV-1::GFP1 (Figs 1 and 3). If the expression patterns of lov-1::gfp1 and pkd-2::gfp2 do indicate protein localization, then the basodendritic localization of LOV-1 and PKD-2 is consistent with plasma-membrane localization and a role for polycystins in maintaining cell polarity21. Although GFP localization might not reflect protein localization, and subcellular protein localization remains ambiguous, LOV-1 and PKD-2 are colocalized to male-specific sensory neurons and so might function in the same pathway.

Although we cannot rule out a subtle developmental defect, the following lines of evidence suggest that LOV-1 has a sensory function. The rays of many cilium-structure mutants ectopically fill with FITC9 but this defect is not observed in lov-1(sy552). By using a functional OSM-6::GFP fusion protein to image sensory neurons, we found that lov-1 sensory endings appear structurally normal. Also, LOV-1::GFP1 and PKD-2::GFP2 are not mislocalized in lov-1 mutants suggesting that their neuronal structure is intact. Expression of lov-1::gfp peaks in the adult male, as opposed to expression of osm-6::gfp, which begins about the time of neuronal outgrowth. Furthermore, the CEMs are formed during embryogenesis, whereas expression of lov-1::gfp in the CEMs is strictly restricted to L4 lethargus and adulthood. Finally, scanning electron microscope and Normarski images show that hook and ray structures are normal.

Chemosensation and mechanosensation are probably involved in vulva location (M.B. and P.S., manuscript in preparation). Because mechanosensory mutant males are not defective in response or vulva location, these behaviours may be mediated by a set of gene products other than those involved in body-touch mechanosensation. LOV-1 might act in a sensory signalling pathway, coupling voltage-activated signalling or store-operated conductance in a similar way to hair-cell mechanosensation22 and touch resonse in C. elegans23 or light-induced cation conductance in Drosophila photoreceptor cells24, respectively. Alternatively, LOV-1 might function as a molecular scaffold for other molecules, such as PKD-2, in establishing or maintaining neuronal cell polarity, or ultrastructurally in the assembly of male-specific cilia.

Methods

Mating efficiency and mating behaviour

Standard assays were performed using hermaphrodite strains N2, unc-52 (e444), dpy-17(e164), and unc-31(e169) with him-5(1490) or heat-shock-generated males2,25,26. Responsiveness reflects the percentage of males successfully responding to hermaphrodite contact within 10 min. An individual male's vulva-location ability was calculated as the number of positive vulva locations divided by the total number of vulva encounters. Vulva-location efficiency indicates the average behaviour of a genotypic population. Pairwise comparisons were made using Mann-Whitney nonparametric and two-sided t-tests.

Genetic screen for and mapping of lov-1

An F2 clonal screen of EMS mutagenized PS1395 hermaphrodites [plg-1(e2001d); him-5]27 (K. Liu and P.S., unpublished) identified lov-1(sy552). Mapping was performed using standard methods. From unc-4(e120) let-25(mn25) crosses, the sy552 genotype was screened by complementation. Of 12 Unc non-Let recombinants, 2 segregate the lov-1 mutant phenotype.

Transformation rescue of lov-1(sy552) mutants

Cosmids and plasmids (15–100 ng µl-1) in the region from the right breakpoint of eDf21 to the right breakpoint of mnDf21 and PHA-1 (100 ng µl-1 pBX) was injected into lov-1(sy552); pha-1(e2123ts); him-5. Stable lines were selected at either 19 or 25 °C (ref. 28). A 16.9-kb HindIII fragment of ZK945 was cloned into pBS(SK+) (plov1.1). A frameshift in plov1.1 was created at nucleotide 17724 of ZK945 by inserting a BssHII GFP fragment from plasmid pPD95.02 (A. Fire, personal communication) out of frame into the StuI site of plov-1.1, creating plov-1.3.

PCR screen for genomic deletion of lov-1

We followed the procedure of G. Moulder and R. Barstead (personal communication) and screened approximately 315,000 haploid genomes by using primers designed to delete the PKD/channel domain. lov-1(sy582Δ) deletes nucleotides 16972–18027 of ZK945.

DNA-sequence analysis

DNA sequence analysis of cDNA clones generated by RT-PCR from him-5(e1490) RNA revealed three exons in the junction between ZK945.10 and ZK945.9: one from 25742–25195, a second from 25151–25071, and a third initiating at position 25021, corresponding to exons I, J and K, in Fig. 1, respectively. Sequencing of the partial pkd-2 cDNA yk219e indicates coding regions of Y73F8A.b and Y73F8A.a at 4932–4480, 3813–3562, 3400–3147, 2100–1972, and 1692–1606.

Expression analysis

GFP29 was used as a marker for lov-1 and pkd-2 gene expression. plov-1::GFP1 was constructed by cloning a 6.7-kb HindIII–BamH1 fragment of plov-1.1 into the vector pPD95.81, plov-1::GFP2 a HindIII-HpaI fragment. plov-1::GFP3 and plov-1::GFP4 are SacI and HindIII–HpaI (Klenow filled-in and relegated) deletions of plov-1::GFP1, respectively. plov-1::gfp5 was constructed by cloning a 15.4-kb HindIII–AfeI fragment of plov-1.1 into the HindIII–SmaI site of pPD95.79. ppkd-2.1, ppkd-2::gfp1 and ppkd-2::gfp2 were constructed by cloning PCR-amplified 8.9-kb, 2.0-kb and 5.9-kb fragments into the vectors pPD.95.97, pPD95.75 and pPD95.77, respectively. Cells were identified by comparing Nomarski and fluorescent or confocal images of the same animals to determine cell-body position30. HOB assignment was confirmed by laser ablation of precursor cells.

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Acknowledgements

We thank J. Copeland for help isolating lov-1(sy582Δ); L. Jiang, R. Garcia and R. Ballester for discussions and constructive criticisms; members of our lab, S. Myers and M. Snow for experimental suggestions; D. Sherwood and B. Smith for assistance with confocal microscopy; A. Fire for vectors; and R. Herman for osm-6::gfp. The Caenorhabditis Genetics stock Center and Sanger Center provided numerous strains, cosmids and sequencing data. This work was supported by the Howard Hughes Medical Institute, with which P.W.S. is an investigator and M.M.B. is an associate, and by the Seaver Institute.

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Correspondence to Paul W. Sternberg.

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Barr, M., Sternberg, P. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature 401, 386–389 (1999). https://doi.org/10.1038/43913

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