Many cells require cilia to receive environmental signals. Mutations in the ciliary gene fam149b1 result in the ciliopathy Joubert syndrome. The C. elegans homolog of fam149b1, xbx-4, is required for normal cilium structure. We found that loss of XBX-4 hinders multiple cilium-mediated behaviors. xbx-4 mutants display mild defects in male mating and nose touch behaviors and dramatic changes in social feeding. Unexpectedly, xbx-4 mutants increased reversal behavior in response to ethanol, a control stimulus used in olfactory assays. Variation in the magnitude of phenotypes for different neurons is consistent with known cell-specific impacts of XBX-4 loss on cilium structure.
","acknowledgements":"Schematics were created in BioRender and can be found at https://BioRender.com/ku6aocf. We thank the CGC for providing strains, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Ravi Nath for help in identifying the strain of interest. We thank Gloria Androwski for excellent technical assistance and the Barr lab for constructive feedback on this project.
","authors":[{"affiliations":["Rutgers, New Brunswick, NJ, US"],"departments":["Genetics Department"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kaitlin.ching@rutgers.edu","firstName":"Kaitlin","lastName":"Ching","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-0517-2421"},{"affiliations":["California Institute of Technology, Pasadena, CA, US"],"departments":["Division of Biology and Biological Engineering"],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"pws@caltech.edu","firstName":"Paul W.","lastName":"Sternberg","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7699-0173"},{"affiliations":["Rutgers, New Brunswick, NJ, US"],"departments":["Genetics Department"],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"mmbarr@hginj.rutgers.edu","firstName":"Maureen M.","lastName":"Barr","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4483-2952"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"This work was supported by National Institutes of Health (NIH) DK059418, DK116606, and NS120745 (M.M.B) and the Howard Hughes Medical Institute (147-101) (P.W.S, 2015); P.W.S. is a Bren Professor of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/b795a5692ad7f2d9201f6abac2440a65.png"},"imageCaption":"(A) Schematic and plot of mating response efficiency for wild-type (WT) and xbx-4 mutant males. Each dot represents one worm. n = 9 worms per genotype across N = 3 experiments. p = 0.0063 by Mann-Whitney U test. (B) Schematic and plot of nose touch responses for WT and xbx-4 mutant hermaphrodites. Each dot represents one experiment. n = 40 worms per genotype across N = 5 experiments. Z = 3.9 by two-portion Z test. (C) Schematic and plot of olfactory avoidance in response to ethanol (EtOH control) or octanol diluted in ethanol (oct in EtOH, aversive stimulus) for WT and xbx-4 mutant hermaphrodites. Each dot represents one worm. n = 16 worms per genotype across N = 3 experiments. p = 0.00090 (WT EtOH vs. WT oct) and 0.0049 (WT EtOH vs. xbx-4 EtOH) by Mann-Whitney U test. (D) Schematics for two social feeding behaviors in WT (negative control), xbx-4 mutant, and osm-5 mutant (positive control) hermaphrodites and table for portion of each population displaying that behavior. n = number of worms for given genotype across N = 2 plates. Border dwelling: Z = -11.6 (WT vs. xbx-4), -5.7 (WT vs. osm-5), -6.8 (osm-5 vs. xbx-4). Contact: Z = -8.7 (WT vs. xbx-4), -3.8 (WT vs. osm-5), -4.2 (osm-5 vs. xbx-4) by two-portion Z test. For all plots, lines show mean values, and error bars show 95% confidence intervals. Asterisks indicate statistical significance (p < 0.05 or Z < -1.96 or > 1.96), including multiple hypothesis correction for D (Z < -2.4).
","imageTitle":"Behavioral assays with worms lacking XBX-4
","methods":"Strains and conditions
Animals were cultured at 20°C on standard nematode growth medium (NGM) with the OP50 strain of E. coli bacteria as a food source (Brenner, 1974). For strains lacking a him-5 mutation, males were initially obtained by heat shocking L4 hermaphrodites (30°C for 5.25-7.5 hours), picking male progeny, and maintaining plates by crossing males and hermaphrodites. Note that, due to reduced mating efficiency, xbx-4 mutants generally required more males, relative to N2, to maintain enough male progeny for experiments. For each experimental replicate, an equal number of wild type or mutant animals were assayed on plates from the same batch of NGM and OP50. This study used the strains listed in Table 1.
Table 1: strains used in this study
genotype | source, strain code, and reference |
wild type | Caenorhabditis Genetics Center (CGC) (N2) |
xbx-4(ok635) | CGC (OE3003) |
osm-5(sa126); him-5(e1490) | Barr lab (PT40 (Qin et al., 2001)) |
unc-31(e169) | Sternberg lab (CB169 (Avery et al., 1993)) |
Male mating
L4 unc-31 mutant hermaphrodites were selected and grown overnight. Wild-type and mutant L4 males were selected and assayed the next day (24-27 hours later) with the observer blinded to the genotype. Assay plates were standard NGM plates seeded with 10 µL of fresh OP50 grown for 2 hours at 37°C and then cooled to room temperature. unc-31 mutant hermaphrodites were allowed to settle on the plate for at least ten minutes prior to adding males. A new plate of hermaphrodites was used after assaying approximately four males. A mating response was scored if the tail of the male flattened against the hermaphrodite and the male swam in reverse, scanning the hermaphrodite (Liu and Sternberg, 1995). The number of mating responses was normalized to the number of times the male’s tail contacted a hermaphrodite, resulting in a response efficiency score for each animal.
Nose touch
Wild-type and mutant L4 hermaphrodites were selected and assayed the next day as adults with the observer blinded to the genotype (22-24 hours later) on standard NGM plates that had been seeded with 120 µL OP50 grown overnight. Each worm was assayed individually by placing a clean eyebrow pick in front of the forward-swimming worm and waiting for contact. A reversal response was scored if the animal immediately reversed upon contact, and a failure was scored if regions at or posterior to the pharyngeal bulb made contact before reversal, typically occurring if the hermaphrodite swam over, under, or along the side of the eyebrow.
Olfactory avoidance
Wild-type and mutant L4 hermaphrodites were selected and assayed the next day (24 hours later) with the observer blinded to the genotype. Each animal was assayed on a standard NGM plate that had been seeded with 120 µL of OP50 grown overnight or briefly at 37°C, resulting in a thin lawn. Each animal was scored first with the ethanol (control) stimulus and then with 10% 1-octanol (Tokyo Chemical Industry Products #O003625ML) dissolved in ethanol. For each test, an eyebrow pick was dipped in the solution for approximately 5 seconds and then immediately held in front of the forward-swimming animal located in the center of the field of view. The stimulus was kept in front of the animal until it swam in reverse, defined as at least one body bend in the posterior direction. For animals that did not reverse, time to reversal was recorded as the time the animal swam out of the field of view. Robustness of the octanol response in wild-type worms varied greatly between replicates, possibly due to environmental conditions (e.g. temperature or air flow). Therefore, data from an experimental replicate were only included if a majority of wild-type worms responded to the octanol stimulus (determined after data were unblinded).
Social behaviors: border dwelling
Wild-type and mutant hermaphrodite L4 animals were selected, genotypes blinded, and grown for two days. Assay plates were made by seeding standard NGM plates with 120 µL of fresh OP50, incubating at 37°C for one hour, and then incubating at room temperature (20.5°C) overnight. Border thickness was visually determined to be similar for all plates used. For each genotype, a similar number of animals (50 for one replicate, 80 for the other) were picked onto each assay plate and incubated at 20°C for 2.5-3.5 hours. Number of animals in contact with the border, defined as the dark edge of the OP50 lawn, and the total number of live animals remaining on the NGM (i.e. not dead on the plastic surfaces, as is common for osm-5 mutants, which leave the NGM and desiccate on the walls and lids of the Petri dishes) were scored for each plate.
Social behaviors: contact
The same plates used for the border dwelling assay were used to score body contact. At the end of incubation, the amount of the hermaphrodite’s body in contact with another hermaphrodite was estimated visually. The number of animals with at least 50% of the body in contact with another animal was scored and normalized to the total number of animals remaining on the NGM.
Statistical methods
For the male mating, nose touch, and olfactory avoidance assays, a small pilot data set was collected and used for a power analysis (power = 0.8) to determine the final size of the data set. For social behaviors, both replicates were conducted on the same day, and no additional data were collected. Plots were generated and Mann-Whitney U tests were conducted in GraphPad Prism (version 10.4.2). Two-portion Z tests were computed in Microsoft Excel.
Knowledge Bases
WormBase was used throughout experimental planning and interpretation of results (Sternberg et al., 2024).
","reagents":"","patternDescription":"Cilia are hair-like organelles with a microtubule core that can serve as cellular antennae to receive environmental signals. Diseases resulting from ciliary dysfunction are known as ciliopathies. Mutations in ciliary protein FAM149B1 correspond with elongated cilia and disrupted Sonic Hedgehog (SHH) signaling, resulting in the ciliopathy Joubert syndrome (Shaheen et al., 2019). Whether changes to ciliary signaling are due to specific defects in SHH signaling or broader defects in ciliary function remains unclear. Nematodes lack a canonical Hedgehog-Patched-Smoothened signaling pathway (Bürglin and Kuwabara, 2005). Therefore, we sought to determine if the Caenorhabditis elegans (C. elegans) fam149b1 homolog is involved in ciliary signaling.
The C. elegans fam149b1 homolog xbx-4 regulates cilium shape and length in AWA amphid wing sensory neurons (Maurya and Sengupta, 2021). xbx-4 is expressed in dye filling ciliated amphid and phasmid sensory neurons in the head and tail (Maurya and Sengupta, 2021). The only ciliated cells in the C. elegans body are sensory neurons, and many of these neurons mediate well-characterized behaviors. To test the role of FAM149B1/XBX-4, we used the null allele xbx-4(ok635) (Maurya and Sengupta, 2021). In this study, we assessed ciliary functions in xbx-4 mutant animals using a variety of behavioral assays. We found that cilium function was, indeed, defective, though the severity of the sensory defect varied between the neuron types tested.
The first cilium-mediated behavior we assayed was mating. C. elegans populations include two sexes: self-fertile hermaphrodites and males, which can transfer sperm to hermaphrodites for reproduction. Male mating consists of a series of specific behaviors, which are mediated by specific sets of neurons (Liu and Sternberg, 1995). The first step, the mating response, occurs when the male tail contacts the hermaphrodite body. The tail flattens, and the male swims in reverse to scan the hermaphrodite body. Response behavior requires nine pairs of ray sensory neurons in the tail and requires functional cilia on these ray neurons (Barr and Sternberg, 1999; Barr et al., 2018). CeNGEN single-cell RNAseq data (Hammarlund et al., 2018) show that ray neurons contain high levels of xbx-4 RNA.
To determine if XBX-4 is required for male mating behavior and ray cilium function, we placed males with unc-31(e169) uncoordinated hermaphrodites, which move very little. We quantified male mating responses and normalized the number of responses to the number of times the male tail contacted a hermaphrodite. We found that xbx-4 mutant males were significantly less efficient in their mating response compared to wild-type males (mean efficiency = 0.78 for wild type vs. 0.38 for xbx-4 mutants, n = 9 worms per genotype across N = 3 experiments, p = 0.0063 by Mann-Whitney U test) (figure panel A). Most xbx-4 mutant males exhibited a mating response and located the hermaphrodite vulva within 5 minutes (n = 8 out of 9), in contrast to severe cilium structure mutants (Barr and Sternberg, 1999). This reduced response efficiency suggests that the ciliated ray neurons are partially defective, but signaling is not completely abolished in the absence of XBX-4.
To determine if cilium defects extend beyond ray neurons, we assayed the nose touch response because 79% of the behavior relies on the ciliated neurons ASH, FLP, and OLQ (Kaplan and Horvitz, 1993). We found that xbx-4 mutant hermaphrodites had a reduced nose touch response (95% of wild type respond vs. 58% xbx-4 mutants respond, n = 40 worms per genotype across N = 5 experiments, Z = 3.9 by two-portion Z test) (figure panel B). A subset of worms was retested three times in succession, and all worms were able to reverse at least once (n = 6). The contribution of non-ciliated neurons (ALM, AVM) accounts for only a small portion of the nose touch response (Kaplan and Horvitz, 1993), suggesting that the function of ciliated neurons ASH, FLP, and/or OLQ is defective in worms that lack XBX-4.
To test a cilium-mediated behavior that is not mechanosensory, we assayed octanol avoidance. Octanol is an aversive olfactory stimulus that relies primarily on ciliated ASH neurons for short-range chemosensation (Troemel et al., 1995). Wild-type hermaphrodites reversed more quickly when presented with 10% octanol than with the ethanol control (27 seconds for ethanol vs. 7.4 second for octanol, n = 16 worms per genotype across N = 3 experiments, p = 0.0090 by Mann-Whitney U test). Surprisingly, xbx-4 mutants reversed significantly faster than wild-type animals when presented with the ethanol control stimulus (27 seconds for wild type vs. 8.2 seconds for xbx-4 mutants, p = 0.0049 by Mann-Whitney U test) (figure panel C). Whether the decreased response time is due to increased ethanol sensation, which may be mediated by IL2 ciliated neurons (Johnson et al., 2017), or simply a higher baseline rate of reversal requires further investigation. The high reversal rate of xbx-4 mutants in the ethanol control condition made it difficult to determine if xbx-4 mutants had a defect in olfactory avoidance. However, the high rate of reversal suggests that the defective mating and nose touch responses, which both require worms to swim backward, were not due to a decreased ability to reverse. Instead, the high reversal rate confirms that decreased mating and nose touch responses were specific to the function of the ciliated neurons.
While conducting other behavioral assays, we noted that xbx-4 mutant worms had an increased tendency to reside in groups at the edge of the bacterial lawn, so we assayed social feeding behaviors. Social feeding is observed in some isolates of C. elegans; however, the lab strain Bristol N2 isolate typically does not exhibit this behavior due to the inhibitory function of ciliated AQR and PQR neurons, as well as the non-ciliated URX neuron (de Bono and Bargmann, 1998; Coates and de Bono, 2002). xbx-4 mutant hermaphrodites were significantly more likely to dwell on the border of the bacterial lawn on which they feed (27% of 121 wild-type animals, 98% of 130 xbx-4 mutants across N = 2 plates each, Z = -11.6 by two-portion Z test). xbx-4 mutants were also more frequently in contact with at least one other worm along the majority of the body (4% of wild type vs. 55% of xbx-4 mutants, Z = -8.7 by two-portion Z test) (figure panel D). Because the phenotype was so dramatic, we also included worms lacking the well-characterized cilium structure gene osm-5/IFT88. We found that xbx-4 mutant worms had stronger social feeding behaviors than osm-5 mutant worms, which have defective cilium structure and function (64% on border and 29% in contact of 115 osm-5 mutant animals, Z = -4.2 by two-portion Z test) (figure panel D). These strong social feeding behaviors are consistent with a severe defect in AQR and PQR cilia in worms that lack XBX-4, with the caveat that function of the non-ciliated URX neuron in xbx-4 mutants is not known.
Taken together, results from our behavioral assays demonstrate that worms lacking XBX-4 are defective in multiple cilium-mediated sensory behaviors to varying degrees. xbx-4 mutants exhibited mild defects in male mating and nose touch responses, a striking increase in cilium-opposing social feeding behaviors, and an unexpected increase in reversal rate in our control olfactory condition (ethanol). These results suggest that ciliary signaling defects observed in cells from Joubert syndrome patients with mutated fam149b1 may not be specific to the SHH pathway and may extend to ciliary functions more broadly. One model consistent with our findings is that sensory neurons rely on XBX-4 in a cell-type specific manner, depending on the structure, environment, or composition of the cilium. This model is supported by the finding that the wing-shaped cilia of AWA neurons is dramatically changed in xbx-4 mutants, whereas the rod-shaped cilia of ASH amphid channel neurons exhibit a subtle but significant change in length (Maurya and Sengupta, 2021). It is not known if ciliary signaling changes in xbx-4 mutants are due to ciliary ultrastructural defects, specific cellular environments, and/or specific composition of different cilia. Further resolving the function of xbx-4 may shed light on the connection between cilium structure and signaling and the underlying disease mechanism for Joubert patients with disease-causing variants of fam149b1.
","references":[{"reference":"Avery L, Bargmann CI, Horvitz HR. 1993. The Caenorhabditis elegans unc-31 gene affects multiple nervous system-controlled functions. Genetics 134(2): 455-64.
","pubmedId":"8325482","doi":""},{"reference":"Barr MM, García LR, Portman DS. 2018. Sexual Dimorphism and Sex Differences in Caenorhabditis elegans Neuronal Development and Behavior. Genetics 208(3): 909-935.
","pubmedId":"29487147","doi":""},{"reference":"Barr MM, Sternberg PW. 1999. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature 401(6751): 386-9.
","pubmedId":"10517638","doi":""},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
","pubmedId":"4366476","doi":""},{"reference":"Coates JC, de Bono M. 2002. Antagonistic pathways in neurons exposed to body fluid regulate social feeding in Caenorhabditis elegans. Nature 419(6910): 925-9.
","pubmedId":"12410311","doi":""},{"reference":"de Bono M, Bargmann CI. 1998. Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans. Cell 94(5): 679-89.
","pubmedId":"9741632","doi":""},{"reference":"Hammarlund M, Hobert O, Miller DM 3rd, Sestan N. 2018. The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System. Neuron 99(3): 430-433.
","pubmedId":"30092212","doi":""},{"reference":"Johnson JR, Edwards MR, Davies H, Newman D, Holden W, Jenkins RE, et al., Barclay JW. 2017. Ethanol Stimulates Locomotion via a G(αs)-Signaling Pathway in IL2 Neurons in Caenorhabditis elegans. Genetics 207(3): 1023-1039.
","pubmedId":"28951527","doi":""},{"reference":"Kaplan JM, Horvitz HR. 1993. A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans. Proc Natl Acad Sci U S A 90(6): 2227-31.
","pubmedId":"8460126","doi":""},{"reference":"Liu KS, Sternberg PW. 1995. Sensory regulation of male mating behavior in Caenorhabditis elegans. Neuron 14(1): 79-89.
","pubmedId":"7826644","doi":""},{"reference":"Maurya AK, Sengupta P. 2021. xbx-4, a homolog of the Joubert syndrome gene FAM149B1, acts via the CCRK and RCK kinase cascade to regulate cilia morphology. Curr Biol 31(24): 5642-5649.e5.
","pubmedId":"34731674","doi":""},{"reference":"Qin H, Rosenbaum JL, Barr MM. 2001. An autosomal recessive polycystic kidney disease gene homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons. Curr Biol 11(6): 457-61.
","pubmedId":"11301258","doi":""},{"reference":"Shaheen R, Jiang N, Alzahrani F, Ewida N, Al-Sheddi T, Alobeid E, et al., Alkuraya FS. 2019. Bi-allelic Mutations in FAM149B1 Cause Abnormal Primary Cilium and a Range of Ciliopathy Phenotypes in Humans. Am J Hum Genet 104(4): 731-737.
","pubmedId":"30905400","doi":""},{"reference":"Sternberg PW, Van Auken K, Wang Q, Wright A, Yook K, Zarowiecki M, et al., Stein L. 2024. WormBase 2024: status and transitioning to Alliance infrastructure. Genetics 227(1): 10.1093/genetics/iyae050.
","pubmedId":"38573366","doi":""},{"reference":"Bürglin TR, Kuwabara PE. 2006. Homologs of the Hh signalling network in C. elegans. WormBook: 1-14.
","pubmedId":"18050469","doi":""},{"reference":"Troemel ER, Chou JH, Dwyer ND, Colbert HA, Bargmann CI. 1995. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83(2): 207-18.
","pubmedId":"7585938","doi":""}],"title":"Joubert syndrome gene fam149b1 homolog, xbx-4, is required for multiple sensory behaviors.
","reviews":[{"reviewer":{"displayName":"Jeff Barclay"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]},{"id":"d0dd6d78-2491-4e37-a6d3-fcf5e07794aa","decision":"accept","abstract":"Many cells require cilia to receive environmental signals. Mutations in the ciliary gene fam149b1 result in the ciliopathy Joubert syndrome. The C. elegans homolog of fam149b1, xbx-4, is required for normal cilium structure. We found that loss of XBX-4 hinders multiple cilium-mediated behaviors. xbx-4 mutants display mild defects in male mating and nose touch behaviors and dramatic changes in social feeding. Unexpectedly, xbx-4 mutants increased reversal behavior in response to ethanol, a control stimulus used in olfactory assays. Variation in the magnitude of phenotypes for different neurons is consistent with known cell-specific impacts of XBX-4 loss on cilium structure.
","acknowledgements":"Schematics were created in BioRender and can be found at https://BioRender.com/ku6aocf. We thank the CGC for providing strains, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Ravi Nath for help in identifying the strain of interest. We thank Gloria Androwski for excellent technical assistance and the Barr lab for constructive feedback on this project.
","authors":[{"affiliations":["Rutgers, New Brunswick, NJ, US"],"departments":["Genetics Department"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kaitlin.ching@rutgers.edu","firstName":"Kaitlin","lastName":"Ching","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-0517-2421"},{"affiliations":["California Institute of Technology, Pasadena, CA, US"],"departments":["Division of Biology and Biological Engineering"],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"pws@caltech.edu","firstName":"Paul W.","lastName":"Sternberg","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7699-0173"},{"affiliations":["Rutgers, New Brunswick, NJ, US"],"departments":["Genetics Department"],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"mmbarr@hginj.rutgers.edu","firstName":"Maureen M.","lastName":"Barr","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4483-2952"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health (NIH) DK059418, DK116606, and NS120745 (M.M.B) and the Howard Hughes Medical Institute (147-101) (P.W.S, 2015); P.W.S. is a Bren Professor of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/b795a5692ad7f2d9201f6abac2440a65.png"},"imageCaption":"(A) Schematic and plot of mating response efficiency for wild-type (WT) and xbx-4 mutant males. Each dot represents one worm. n = 9 worms per genotype across N = 3 experiments. p = 0.0063 by Mann-Whitney U test. (B) Schematic and plot of nose touch responses for WT and xbx-4 mutant hermaphrodites. Each dot represents one experiment. n = 40 worms per genotype across N = 5 experiments. Z = 3.9 by two-portion Z test. (C) Schematic and plot of olfactory avoidance in response to ethanol (EtOH control) or octanol diluted in ethanol (oct in EtOH, aversive stimulus) for WT and xbx-4 mutant hermaphrodites. Each dot represents one worm. n = 16 worms per genotype across N = 3 experiments. p = 0.00090 (WT EtOH vs. WT oct) and 0.0049 (WT EtOH vs. xbx-4 EtOH) by Mann-Whitney U test. (D) Schematics for two social feeding behaviors in WT (negative control), xbx-4 mutant, and osm-5 mutant (positive control) hermaphrodites and table for portion of each population displaying that behavior. n = number of worms for given genotype across N = 2 plates. Border dwelling: Z = -11.6 (WT vs. xbx-4), -5.7 (WT vs. osm-5), -6.8 (osm-5 vs. xbx-4). Contact: Z = -8.7 (WT vs. xbx-4), -3.8 (WT vs. osm-5), -4.2 (osm-5 vs. xbx-4) by two-portion Z test. For all plots, lines show mean values, and error bars show 95% confidence intervals. Asterisks indicate statistical significance (p < 0.05 or Z < -1.96 or > 1.96), including multiple hypothesis correction for D (Z < -2.4).
","imageTitle":"Behavioral assays with worms lacking XBX-4
","methods":"Strains and conditions
Animals were cultured at 20°C on standard nematode growth medium (NGM) with the OP50 strain of E. coli bacteria as a food source (Brenner, 1974). For strains lacking a him-5 mutation, males were initially obtained by heat shocking L4 hermaphrodites (30°C for 5.25-7.5 hours), picking male progeny, and maintaining plates by crossing males and hermaphrodites. Note that, due to reduced mating efficiency, xbx-4 mutants generally required more males, relative to N2, to maintain enough male progeny for experiments. For each experimental replicate, an equal number of wild type or mutant animals were assayed on plates from the same batch of NGM and OP50. This study used the strains listed in Table 1.
Table 1: strains used in this study
genotype | source, strain code, and reference |
wild type | Caenorhabditis Genetics Center (CGC) (N2) |
CGC (OE3003) | |
Barr lab (PT40 (Qin et al., 2001)) | |
Sternberg lab (CB169 (Avery et al., 1993)) |
Male mating
L4 unc-31 mutant hermaphrodites were selected and grown overnight. Wild-type and mutant L4 males were selected and assayed the next day (24-27 hours later) with the observer blinded to the genotype. Assay plates were standard NGM plates seeded with 10 µL of fresh OP50 grown for 2 hours at 37°C and then cooled to room temperature. unc-31 mutant hermaphrodites were allowed to settle on the plate for at least ten minutes prior to adding males. A new plate of hermaphrodites was used after assaying approximately four males. A mating response was scored if the tail of the male flattened against the hermaphrodite and the male swam in reverse, scanning the hermaphrodite (Liu and Sternberg, 1995). The number of mating responses was normalized to the number of times the male's tail contacted a hermaphrodite, resulting in a response efficiency score for each animal.
Nose touch
Wild-type and mutant L4 hermaphrodites were selected and assayed the next day as adults with the observer blinded to the genotype (22-24 hours later) on standard NGM plates that had been seeded with 120 µL OP50 grown overnight. Each worm was assayed individually by placing a clean eyebrow pick in front of the forward-swimming worm and waiting for contact. A reversal response was scored if the animal immediately reversed upon contact, and a failure was scored if regions at or posterior to the pharyngeal bulb made contact before reversal, typically occurring if the hermaphrodite swam over, under, or along the side of the eyebrow.
Olfactory avoidance
Wild-type and mutant L4 hermaphrodites were selected and assayed the next day (24 hours later) with the observer blinded to the genotype. Each animal was assayed on a standard NGM plate that had been seeded with 120 µL of OP50 grown overnight or briefly at 37°C, resulting in a thin lawn. Each animal was scored first with the ethanol (control) stimulus and then with 10% 1-octanol (Tokyo Chemical Industry Products #O003625ML) dissolved in ethanol. For each test, an eyebrow pick was dipped in the solution for approximately 5 seconds and then immediately held in front of the forward-swimming animal located in the center of the field of view. The stimulus was kept in front of the animal until it swam in reverse, defined as at least one body bend in the posterior direction. For animals that did not reverse, time to reversal was recorded as the time the animal swam out of the field of view. Robustness of the octanol response in wild-type worms varied greatly between replicates, possibly due to environmental conditions (e.g. temperature or air flow). Therefore, data from an experimental replicate were only included if a majority of wild-type worms responded to the octanol stimulus (determined after data were unblinded).
Social behaviors: border dwelling
Wild-type and mutant hermaphrodite L4 animals were selected, genotypes blinded, and grown for two days. Assay plates were made by seeding standard NGM plates with 120 µL of fresh OP50, incubating at 37°C for one hour, and then incubating at room temperature (20.5°C) overnight. Border thickness was visually determined to be similar for all plates used. For each genotype, a similar number of animals (50 for one replicate, 80 for the other) were picked onto each assay plate and incubated at 20°C for 2.5-3.5 hours. Number of animals in contact with the border, defined as the dark edge of the OP50 lawn, and the total number of live animals remaining on the NGM (i.e. not dead on the plastic surfaces, as is common for osm-5 mutants, which leave the NGM and desiccate on the walls and lids of the Petri dishes) were scored for each plate.
Social behaviors: contact
The same plates used for the border dwelling assay were used to score body contact. At the end of incubation, the amount of the hermaphrodite's body in contact with another hermaphrodite was estimated visually. The number of animals with at least 50% of the body in contact with another animal was scored and normalized to the total number of animals remaining on the NGM.
Statistical methods
For the male mating, nose touch, and olfactory avoidance assays, a small pilot data set was collected and used for a power analysis (power = 0.8) to determine the final size of the data set. For social behaviors, both replicates were conducted on the same day, and no additional data were collected. Plots were generated and Mann-Whitney U tests were conducted in GraphPad Prism (version 10.4.2). Two-portion Z tests were computed in Microsoft Excel.
Knowledge Bases
WormBase was used throughout experimental planning and interpretation of results (Sternberg et al., 2024).
","reagents":"","patternDescription":"Cilia are hair-like organelles with a microtubule core that can serve as cellular antennae to receive environmental signals. Diseases resulting from ciliary dysfunction are known as ciliopathies. Mutations in ciliary protein FAM149B1 correspond with elongated cilia and disrupted Sonic Hedgehog (SHH) signaling, resulting in the ciliopathy Joubert syndrome (Shaheen et al., 2019). Whether changes to ciliary signaling are due to specific defects in SHH signaling or broader defects in ciliary function remains unclear. Nematodes lack a canonical Hedgehog-Patched-Smoothened signaling pathway (Bürglin and Kuwabara, 2005). Therefore, we sought to determine if the Caenorhabditis elegans (C. elegans) fam149b1 homolog is involved in ciliary signaling.
The C. elegans fam149b1 homolog xbx-4 regulates cilium shape and length in AWA amphid wing sensory neurons (Maurya and Sengupta, 2021). xbx-4 is expressed in dye filling ciliated amphid and phasmid sensory neurons in the head and tail (Maurya and Sengupta, 2021). The only ciliated cells in the C. elegans body are sensory neurons, and many of these neurons mediate well-characterized behaviors. To test the role of FAM149B1/XBX-4, we used the null allele xbx-4(ok635) (Maurya and Sengupta, 2021). In this study, we assessed ciliary functions in xbx-4 mutant animals using a variety of behavioral assays. We found that cilium function was, indeed, defective, though the severity of the sensory defect varied between the neuron types tested.
The first cilium-mediated behavior we assayed was mating. C. elegans populations include two sexes: self-fertile hermaphrodites and males, which can transfer sperm to hermaphrodites for reproduction. Male mating consists of a series of specific behaviors, which are mediated by specific sets of neurons (Liu and Sternberg, 1995). The first step, the mating response, occurs when the male tail contacts the hermaphrodite body. The tail flattens, and the male swims in reverse to scan the hermaphrodite body. Response behavior requires nine pairs of ray sensory neurons in the tail and requires functional cilia on these ray neurons (Barr and Sternberg, 1999; Barr et al., 2018). CeNGEN single-cell RNAseq data (Hammarlund et al., 2018) show that ray neurons contain high levels of xbx-4 RNA.
To determine if XBX-4 is required for male mating behavior and ray cilium function, we placed males with unc-31(e169) uncoordinated hermaphrodites, which move very little. We quantified male mating responses and normalized the number of responses to the number of times the male tail contacted a hermaphrodite. We found that xbx-4 mutant males were significantly less efficient in their mating response compared to wild-type males (mean efficiency = 0.78 for wild type vs. 0.38 for xbx-4 mutants, n = 9 worms per genotype across N = 3 experiments, p = 0.0063 by Mann-Whitney U test) (figure panel A). Most xbx-4 mutant males exhibited a mating response and located the hermaphrodite vulva within 5 minutes (n = 8 out of 9), in contrast to severe cilium structure mutants (Barr and Sternberg, 1999). This reduced response efficiency suggests that the ciliated ray neurons are partially defective, but signaling is not completely abolished in the absence of XBX-4.
To determine if cilium defects extend beyond ray neurons, we assayed the nose touch response because 79% of the behavior relies on the ciliated neurons ASH, FLP, and OLQ (Kaplan and Horvitz, 1993). We found that xbx-4 mutant hermaphrodites had a reduced nose touch response (95% of wild type respond vs. 58% xbx-4 mutants respond, n = 40 worms per genotype across N = 5 experiments, Z = 3.9 by two-portion Z test) (figure panel B). A subset of worms was retested three times in succession, and all worms were able to reverse at least once (n = 6). The contribution of non-ciliated neurons (ALM, AVM) accounts for only a small portion of the nose touch response (Kaplan and Horvitz, 1993), suggesting that the function of ciliated neurons ASH, FLP, and/or OLQ is defective in worms that lack XBX-4.
To test a cilium-mediated behavior that is not mechanosensory, we assayed octanol avoidance. Octanol is an aversive olfactory stimulus that relies primarily on ciliated ASH neurons for short-range chemosensation (Troemel et al., 1995). Wild-type hermaphrodites reversed more quickly when presented with 10% octanol than with the ethanol control (27 seconds for ethanol vs. 7.4 second for octanol, n = 16 worms per genotype across N = 3 experiments, p = 0.0090 by Mann-Whitney U test). Surprisingly, xbx-4 mutants reversed significantly faster than wild-type animals when presented with the ethanol control stimulus (27 seconds for wild type vs. 8.2 seconds for xbx-4 mutants, p = 0.0049 by Mann-Whitney U test) (figure panel C). Whether the decreased response time is due to increased ethanol sensation, which may be mediated by IL2 ciliated neurons (Johnson et al., 2017), or simply a higher baseline rate of reversal requires further investigation. The high reversal rate of xbx-4 mutants in the ethanol control condition made it difficult to determine if xbx-4 mutants had a defect in olfactory avoidance. However, the high rate of reversal suggests that the defective mating and nose touch responses, which both require worms to swim backward, were not due to a decreased ability to reverse. Instead, the high reversal rate confirms that decreased mating and nose touch responses were specific to the function of the ciliated neurons.
While conducting other behavioral assays, we noted that xbx-4 mutant worms had an increased tendency to reside in groups at the edge of the bacterial lawn, so we assayed social feeding behaviors. Social feeding is observed in some isolates of C. elegans; however, the lab strain Bristol N2 isolate typically does not exhibit this behavior due to the inhibitory function of ciliated AQR and PQR neurons, as well as the non-ciliated URX neuron (de Bono and Bargmann, 1998; Coates and de Bono, 2002). xbx-4 mutant hermaphrodites were significantly more likely to dwell on the border of the bacterial lawn on which they feed (27% of 121 wild-type animals, 98% of 130 xbx-4 mutants across N = 2 plates each, Z = -11.6 by two-portion Z test). xbx-4 mutants were also more frequently in contact with at least one other worm along the majority of the body (4% of wild type vs. 55% of xbx-4 mutants, Z = -8.7 by two-portion Z test) (figure panel D). Because the phenotype was so dramatic, we also included worms lacking the well-characterized cilium structure gene osm-5/IFT88. We found that xbx-4 mutant worms had stronger social feeding behaviors than osm-5 mutant worms, which have defective cilium structure and function (64% on border and 29% in contact of 115 osm-5 mutant animals, Z = -4.2 by two-portion Z test) (figure panel D). These strong social feeding behaviors are consistent with a severe defect in AQR and PQR cilia in worms that lack XBX-4, with the caveat that function of the non-ciliated URX neuron in xbx-4 mutants is not known.
Taken together, results from our behavioral assays demonstrate that worms lacking XBX-4 are defective in multiple cilium-mediated sensory behaviors to varying degrees. xbx-4 mutants exhibited mild defects in male mating and nose touch responses, a striking increase in cilium-opposing social feeding behaviors, and an unexpected increase in reversal rate in our control olfactory condition (ethanol). These results suggest that ciliary signaling defects observed in cells from Joubert syndrome patients with mutated fam149b1 may not be specific to the SHH pathway and may extend to ciliary functions more broadly. One model consistent with our findings is that sensory neurons rely on XBX-4 in a cell-type specific manner, depending on the structure, environment, or composition of the cilium. This model is supported by the finding that the wing-shaped cilia of AWA neurons is dramatically changed in xbx-4 mutants, whereas the rod-shaped cilia of ASH amphid channel neurons exhibit a subtle but significant change in length (Maurya and Sengupta, 2021). It is not known if ciliary signaling changes in xbx-4 mutants are due to ciliary ultrastructural defects, specific cellular environments, and/or specific composition of different cilia. Although our findings are consistent with the predicted role of XBX-4, a caveat is that we only examined one xbx-4 mutant allele. Phenotypes observed could be due to an unrelated mutation, and examination of additional mutant lines or rescue of the xbx-4(ok635) mutant phenotypes with wild-type xbx-4(+) would be needed to rule out this possibility. Further resolving the function of xbx-4 may shed light on the connection between cilium structure and signaling and the underlying disease mechanism for Joubert patients with disease-causing variants of fam149b1.
","references":[{"reference":"Avery L, Bargmann CI, Horvitz HR. 1993. The Caenorhabditis elegans unc-31 gene affects multiple nervous system-controlled functions. Genetics 134(2): 455-64.
","pubmedId":"8325482","doi":""},{"reference":"Barr MM, García LR, Portman DS. 2018. Sexual Dimorphism and Sex Differences in Caenorhabditis elegans Neuronal Development and Behavior. Genetics 208(3): 909-935.
","pubmedId":"29487147","doi":""},{"reference":"Barr MM, Sternberg PW. 1999. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans. Nature 401(6751): 386-9.
","pubmedId":"10517638","doi":""},{"reference":"Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77(1): 71-94.
","pubmedId":"4366476","doi":""},{"reference":"Coates JC, de Bono M. 2002. Antagonistic pathways in neurons exposed to body fluid regulate social feeding in Caenorhabditis elegans. Nature 419(6910): 925-9.
","pubmedId":"12410311","doi":""},{"reference":"de Bono M, Bargmann CI. 1998. Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans. Cell 94(5): 679-89.
","pubmedId":"9741632","doi":""},{"reference":"Hammarlund M, Hobert O, Miller DM 3rd, Sestan N. 2018. The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System. Neuron 99(3): 430-433.
","pubmedId":"30092212","doi":""},{"reference":"Johnson JR, Edwards MR, Davies H, Newman D, Holden W, Jenkins RE, et al., Barclay JW. 2017. Ethanol Stimulates Locomotion via a G(αs)-Signaling Pathway in IL2 Neurons in Caenorhabditis elegans. Genetics 207(3): 1023-1039.
","pubmedId":"28951527","doi":""},{"reference":"Kaplan JM, Horvitz HR. 1993. A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans. Proc Natl Acad Sci U S A 90(6): 2227-31.
","pubmedId":"8460126","doi":""},{"reference":"Liu KS, Sternberg PW. 1995. Sensory regulation of male mating behavior in Caenorhabditis elegans. Neuron 14(1): 79-89.
","pubmedId":"7826644","doi":""},{"reference":"Maurya AK, Sengupta P. 2021. xbx-4, a homolog of the Joubert syndrome gene FAM149B1, acts via the CCRK and RCK kinase cascade to regulate cilia morphology. Curr Biol 31(24): 5642-5649.e5.
","pubmedId":"34731674","doi":""},{"reference":"Qin H, Rosenbaum JL, Barr MM. 2001. An autosomal recessive polycystic kidney disease gene homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons. Curr Biol 11(6): 457-61.
","pubmedId":"11301258","doi":""},{"reference":"Shaheen R, Jiang N, Alzahrani F, Ewida N, Al-Sheddi T, Alobeid E, et al., Alkuraya FS. 2019. Bi-allelic Mutations in FAM149B1 Cause Abnormal Primary Cilium and a Range of Ciliopathy Phenotypes in Humans. Am J Hum Genet 104(4): 731-737.
","pubmedId":"30905400","doi":""},{"reference":"Sternberg PW, Van Auken K, Wang Q, Wright A, Yook K, Zarowiecki M, et al., Stein L. 2024. WormBase 2024: status and transitioning to Alliance infrastructure. Genetics 227(1): 10.1093/genetics/iyae050.
","pubmedId":"38573366","doi":""},{"reference":"Bürglin TR, Kuwabara PE. 2006. Homologs of the Hh signalling network in C. elegans. WormBook: 1-14.
","pubmedId":"18050469","doi":""},{"reference":"Troemel ER, Chou JH, Dwyer ND, Colbert HA, Bargmann CI. 1995. Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83(2): 207-18.
","pubmedId":"7585938","doi":""}],"title":"Joubert syndrome gene fam149b1 homolog, xbx-4, is required for multiple sensory behaviors.
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":"1775853285121"}]},{"id":"c138941c-2850-4e79-8f03-fb13df8ce8fb","decision":"publish","abstract":"Many cells require cilia to receive environmental signals. Mutations in the ciliary gene fam149b1 result in the ciliopathy Joubert syndrome. The C. elegans homolog of fam149b1, xbx-4, is required for normal cilium structure. We found that loss of XBX-4 hinders multiple cilium-mediated behaviors. xbx-4 mutants display mild defects in male mating and nose touch behaviors and dramatic changes in social feeding. Unexpectedly, xbx-4 mutants increased reversal behavior in response to ethanol, a control stimulus used in olfactory assays. Variation in the magnitude of phenotypes for different neurons is consistent with known cell-specific impacts of XBX-4 loss on cilium structure.
","acknowledgements":"Schematics were created in BioRender and can be found at https://BioRender.com/ku6aocf. We thank the CGC for providing strains, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank Ravi Nath for help in identifying the strain of interest. We thank Gloria Androwski for excellent technical assistance and the Barr lab for constructive feedback on this project.
","authors":[{"affiliations":["Rutgers, New Brunswick, NJ, US"],"departments":["Genetics Department"],"credit":["conceptualization","dataCuration","formalAnalysis","investigation","writing_originalDraft","writing_reviewEditing"],"email":"kaitlin.ching@rutgers.edu","firstName":"Kaitlin","lastName":"Ching","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0002-0517-2421"},{"affiliations":["California Institute of Technology, Pasadena, CA, US"],"departments":["Division of Biology and Biological Engineering"],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"pws@caltech.edu","firstName":"Paul W.","lastName":"Sternberg","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0000-0002-7699-0173"},{"affiliations":["Rutgers, New Brunswick, NJ, US"],"departments":["Genetics Department"],"credit":["conceptualization","fundingAcquisition","supervision","writing_reviewEditing"],"email":"mmbarr@hginj.rutgers.edu","firstName":"Maureen M.","lastName":"Barr","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-4483-2952"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health (NIH) DK059418, DK116606, and NS120745 (M.M.B) and the Howard Hughes Medical Institute (147-101) (P.W.S, 2015); P.W.S. is a Bren Professor of Biology.
","image":{"url":"https://portal.micropublication.org/uploads/b795a5692ad7f2d9201f6abac2440a65.png"},"imageCaption":"(A) Schematic and plot of mating response efficiency for wild-type (WT) and xbx-4 mutant males. Each dot represents one worm. n = 9 worms per genotype across N = 3 experiments. p = 0.0063 by Mann-Whitney U test. (B) Schematic and plot of nose touch responses for WT and xbx-4 mutant hermaphrodites. Each dot represents one experiment. n = 40 worms per genotype across N = 5 experiments. Z = 3.9 by two-portion Z test. (C) Schematic and plot of olfactory avoidance in response to ethanol (EtOH control) or octanol diluted in ethanol (oct in EtOH, aversive stimulus) for WT and xbx-4 mutant hermaphrodites. Each dot represents one worm. n = 16 worms per genotype across N = 3 experiments. p = 0.00090 (WT EtOH vs. WT oct) and 0.0049 (WT EtOH vs. xbx-4 EtOH) by Mann-Whitney U test. (D) Schematics for two social feeding behaviors in WT (negative control), xbx-4 mutant, and osm-5 mutant (positive control) hermaphrodites and table for portion of each population displaying that behavior. n = number of worms for given genotype across N = 2 plates. Border dwelling: Z = -11.6 (WT vs. xbx-4), -5.7 (WT vs. osm-5), -6.8 (osm-5 vs. xbx-4). Contact: Z = -8.7 (WT vs. xbx-4), -3.8 (WT vs. osm-5), -4.2 (osm-5 vs. xbx-4) by two-portion Z test. For all plots, lines show mean values, and error bars show 95% confidence intervals. Asterisks indicate statistical significance (p < 0.05 or Z < -1.96 or > 1.96), including multiple hypothesis correction for D (Z < -2.4).
","imageTitle":"Behavioral assays with worms lacking XBX-4
","methods":"Strains and conditions
Animals were cultured at 20°C on standard nematode growth medium (NGM) with the OP50 strain of E. coli bacteria as a food source (Brenner, 1974). For strains lacking a him-5 mutation, males were initially obtained by heat shocking L4 hermaphrodites (30°C for 5.25-7.5 hours), picking male progeny, and maintaining plates by crossing males and hermaphrodites. Note that, due to reduced mating efficiency, xbx-4 mutants generally required more males, relative to N2, to maintain enough male progeny for experiments. For each experimental replicate, an equal number of wild type or mutant animals were assayed on plates from the same batch of NGM and OP50. This study used the strains listed in Table 1.
Table 1: strains used in this study
genotype | source, strain code, and reference |
wild type | Caenorhabditis Genetics Center (CGC) (N2) |
CGC (OE3003) | |
Barr lab (PT40 (Qin et al., 2001)) | |
Sternberg lab (CB169 (Avery et al., 1993)) |
Male mating
L4 unc-31 mutant hermaphrodites were selected and grown overnight. Wild-type and mutant L4 males were selected and assayed the next day (24-27 hours later) with the observer blinded to the genotype. Assay plates were standard NGM plates seeded with 10 µL of fresh OP50 grown for 2 hours at 37°C and then cooled to room temperature. unc-31 mutant hermaphrodites were allowed to settle on the plate for at least ten minutes prior to adding males. A new plate of hermaphrodites was used after assaying approximately four males. A mating response was scored if the tail of the male flattened against the hermaphrodite and the male swam in reverse, scanning the hermaphrodite (Liu and Sternberg, 1995). The number of mating responses was normalized to the number of times the male's tail contacted a hermaphrodite, resulting in a response efficiency score for each animal.
Nose touch
Wild-type and mutant L4 hermaphrodites were selected and assayed the next day as adults with the observer blinded to the genotype (22-24 hours later) on standard NGM plates that had been seeded with 120 µL OP50 grown overnight. Each worm was assayed individually by placing a clean eyebrow pick in front of the forward-swimming worm and waiting for contact. A reversal response was scored if the animal immediately reversed upon contact, and a failure was scored if regions at or posterior to the pharyngeal bulb made contact before reversal, typically occurring if the hermaphrodite swam over, under, or along the side of the eyebrow.
Olfactory avoidance
Wild-type and mutant L4 hermaphrodites were selected and assayed the next day (24 hours later) with the observer blinded to the genotype. Each animal was assayed on a standard NGM plate that had been seeded with 120 µL of OP50 grown overnight or briefly at 37°C, resulting in a thin lawn. Each animal was scored first with the ethanol (control) stimulus and then with 10% 1-octanol (Tokyo Chemical Industry Products #O003625ML) dissolved in ethanol. For each test, an eyebrow pick was dipped in the solution for approximately 5 seconds and then immediately held in front of the forward-swimming animal located in the center of the field of view. The stimulus was kept in front of the animal until it swam in reverse, defined as at least one body bend in the posterior direction. For animals that did not reverse, time to reversal was recorded as the time the animal swam out of the field of view. Robustness of the octanol response in wild-type worms varied greatly between replicates, possibly due to environmental conditions (e.g. temperature or air flow). Therefore, data from an experimental replicate were only included if a majority of wild-type worms responded to the octanol stimulus (determined after data were unblinded).
Social behaviors: border dwelling
Wild-type and mutant hermaphrodite L4 animals were selected, genotypes blinded, and grown for two days. Assay plates were made by seeding standard NGM plates with 120 µL of fresh OP50, incubating at 37°C for one hour, and then incubating at room temperature (20.5°C) overnight. Border thickness was visually determined to be similar for all plates used. For each genotype, a similar number of animals (50 for one replicate, 80 for the other) were picked onto each assay plate and incubated at 20°C for 2.5-3.5 hours. Number of animals in contact with the border, defined as the dark edge of the OP50 lawn, and the total number of live animals remaining on the NGM (i.e. not dead on the plastic surfaces, as is common for osm-5 mutants, which leave the NGM and desiccate on the walls and lids of the Petri dishes) were scored for each plate.
Social behaviors: contact
The same plates used for the border dwelling assay were used to score body contact. At the end of incubation, the amount of the hermaphrodite's body in contact with another hermaphrodite was estimated visually. The number of animals with at least 50% of the body in contact with another animal was scored and normalized to the total number of animals remaining on the NGM.
Statistical methods
For the male mating, nose touch, and olfactory avoidance assays, a small pilot data set was collected and used for a power analysis (power = 0.8) to determine the final size of the data set. For social behaviors, both replicates were conducted on the same day, and no additional data were collected. Plots were generated and Mann-Whitney U tests were conducted in GraphPad Prism (version 10.4.2). Two-portion Z tests were computed in Microsoft Excel.
Knowledge Bases
WormBase was used throughout experimental planning and interpretation of results (Sternberg et al., 2024).
","reagents":"","patternDescription":"Cilia are hair-like organelles with a microtubule core that can serve as cellular antennae to receive environmental signals. Diseases resulting from ciliary dysfunction are known as ciliopathies. Mutations in ciliary protein FAM149B1 correspond with elongated cilia and disrupted Sonic Hedgehog (SHH) signaling, resulting in the ciliopathy Joubert syndrome (Shaheen et al., 2019). Whether changes to ciliary signaling are due to specific defects in SHH signaling or broader defects in ciliary function remains unclear. Nematodes lack a canonical Hedgehog-Patched-Smoothened signaling pathway (Bürglin and Kuwabara, 2005). Therefore, we sought to determine if the Caenorhabditis elegans (C. elegans) fam149b1 homolog is involved in ciliary signaling.
The C. elegans fam149b1 homolog xbx-4 regulates cilium shape and length in AWA amphid wing sensory neurons (Maurya and Sengupta, 2021). xbx-4 is expressed in dye filling ciliated amphid and phasmid sensory neurons in the head and tail (Maurya and Sengupta, 2021). The only ciliated cells in the C. elegans body are sensory neurons, and many of these neurons mediate well-characterized behaviors. To test the role of FAM149B1/XBX-4, we used the null allele xbx-4(ok635) (Maurya and Sengupta, 2021). In this study, we assessed ciliary functions in xbx-4 mutant animals using a variety of behavioral assays. We found that cilium function was, indeed, defective, though the severity of the sensory defect varied between the neuron types tested.
The first cilium-mediated behavior we assayed was mating. C. elegans populations include two sexes: self-fertile hermaphrodites and males, which can transfer sperm to hermaphrodites for reproduction. Male mating consists of a series of specific behaviors, which are mediated by specific sets of neurons (Liu and Sternberg, 1995). The first step, the mating response, occurs when the male tail contacts the hermaphrodite body. The tail flattens, and the male swims in reverse to scan the hermaphrodite body. Response behavior requires nine pairs of ray sensory neurons in the tail and requires functional cilia on these ray neurons (Barr and Sternberg, 1999; Barr et al., 2018). CeNGEN single-cell RNAseq data (Hammarlund et al., 2018) show that ray neurons contain high levels of xbx-4 RNA.
To determine if XBX-4 is required for male mating behavior and ray cilium function, we placed males with unc-31(e169) uncoordinated hermaphrodites, which move very little. We quantified male mating responses and normalized the number of responses to the number of times the male tail contacted a hermaphrodite. We found that xbx-4 mutant males were significantly less efficient in their mating response compared to wild-type males (mean efficiency = 0.78 for wild type vs. 0.38 for xbx-4 mutants, n = 9 worms per genotype across N = 3 experiments, p = 0.0063 by Mann-Whitney U test) (figure panel A). Most xbx-4 mutant males exhibited a mating response and located the hermaphrodite vulva within 5 minutes (n = 8 out of 9), in contrast to severe cilium structure mutants (Barr and Sternberg, 1999). This reduced response efficiency suggests that the ciliated ray neurons are partially defective, but signaling is not completely abolished in the absence of XBX-4.
To determine if cilium defects extend beyond ray neurons, we assayed the nose touch response because 79% of the behavior relies on the ciliated neurons ASH, FLP, and OLQ (Kaplan and Horvitz, 1993). We found that xbx-4 mutant hermaphrodites had a reduced nose touch response (95% of wild type respond vs. 58% xbx-4 mutants respond, n = 40 worms per genotype across N = 5 experiments, Z = 3.9 by two-portion Z test) (figure panel B). A subset of worms was retested three times in succession, and all worms were able to reverse at least once (n = 6). The contribution of non-ciliated neurons (ALM, AVM) accounts for only a small portion of the nose touch response (Kaplan and Horvitz, 1993), suggesting that the function of ciliated neurons ASH, FLP, and/or OLQ is defective in worms that lack XBX-4.
To test a cilium-mediated behavior that is not mechanosensory, we assayed octanol avoidance. Octanol is an aversive olfactory stimulus that relies primarily on ciliated ASH neurons for short-range chemosensation (Troemel et al., 1995). Wild-type hermaphrodites reversed more quickly when presented with 10% octanol than with the ethanol control (27 seconds for ethanol vs. 7.4 second for octanol, n = 16 worms per genotype across N = 3 experiments, p = 0.0090 by Mann-Whitney U test). Surprisingly, xbx-4 mutants reversed significantly faster than wild-type animals when presented with the ethanol control stimulus (27 seconds for wild type vs. 8.2 seconds for xbx-4 mutants, p = 0.0049 by Mann-Whitney U test) (figure panel C). Whether the decreased response time is due to increased ethanol sensation, which may be mediated by IL2 ciliated neurons (Johnson et al., 2017), or simply a higher baseline rate of reversal requires further investigation. The high reversal rate of xbx-4 mutants in the ethanol control condition made it difficult to determine if xbx-4 mutants had a defect in olfactory avoidance. However, the high rate of reversal suggests that the defective mating and nose touch responses, which both require worms to swim backward, were not due to a decreased ability to reverse. Instead, the high reversal rate confirms that decreased mating and nose touch responses were specific to the function of the ciliated neurons.
While conducting other behavioral assays, we noted that xbx-4 mutant worms had an increased tendency to reside in groups at the edge of the bacterial lawn, so we assayed social feeding behaviors. Social feeding is observed in some isolates of C. elegans; however, the lab strain Bristol N2 isolate typically does not exhibit this behavior due to the inhibitory function of ciliated AQR and PQR neurons, as well as the non-ciliated URX neuron (de Bono and Bargmann, 1998; Coates and de Bono, 2002). xbx-4 mutant hermaphrodites were significantly more likely to dwell on the border of the bacterial lawn on which they feed (27% of 121 wild-type animals, 98% of 130 xbx-4 mutants across N = 2 plates each, Z = -11.6 by two-portion Z test). xbx-4 mutants were also more frequently in contact with at least one other worm along the majority of the body (4% of wild type vs. 55% of xbx-4 mutants, Z = -8.7 by two-portion Z test) (figure panel D). Because the phenotype was so dramatic, we also included worms lacking the well-characterized cilium structure gene osm-5/IFT88. We found that xbx-4 mutant worms had stronger social feeding behaviors than osm-5 mutant worms, which have defective cilium structure and function (64% on border and 29% in contact of 115 osm-5 mutant animals, Z = -4.2 by two-portion Z test) (figure panel D). These strong social feeding behaviors are consistent with a severe defect in AQR and PQR cilia in worms that lack XBX-4, with the caveat that function of the non-ciliated URX neuron in xbx-4 mutants is not known.
Taken together, results from our behavioral assays demonstrate that worms lacking XBX-4 are defective in multiple cilium-mediated sensory behaviors to varying degrees. xbx-4 mutants exhibited mild defects in male mating and nose touch responses, a striking increase in cilium-opposing social feeding behaviors, and an unexpected increase in reversal rate in our control olfactory condition (ethanol). These results suggest that ciliary signaling defects observed in cells from Joubert syndrome patients with mutated fam149b1 may not be specific to the SHH pathway and may extend to ciliary functions more broadly. One model consistent with our findings is that sensory neurons rely on XBX-4 in a cell-type specific manner, depending on the structure, environment, or composition of the cilium. This model is supported by the finding that the wing-shaped cilia of AWA neurons is dramatically changed in xbx-4 mutants, whereas the rod-shaped cilia of ASH amphid channel neurons exhibit a subtle but significant change in length (Maurya and Sengupta, 2021). It is not known if ciliary signaling changes in xbx-4 mutants are due to ciliary ultrastructural defects, specific cellular environments, and/or specific composition of different cilia. Although our findings are consistent with the predicted role of XBX-4, a caveat is that we only examined one xbx-4 mutant allele. Phenotypes observed could be due to an unrelated mutation, and examination of additional mutant lines or rescue of the xbx-4(ok635) mutant phenotypes with wild-type xbx-4(+) would be needed to rule out this possibility. Further resolving the function of xbx-4 may shed light on the connection between cilium structure and signaling and the underlying disease mechanism for Joubert patients with disease-causing variants of fam149b1.
","references":[{"reference":"Avery L, Bargmann CI, Horvitz HR. 1993. The Caenorhabditis elegans unc-31 gene affects multiple nervous system-controlled functions. Genetics 134(2): 455-64.
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","pubmedId":"30905400","doi":""},{"reference":"Sternberg PW, Van Auken K, Wang Q, Wright A, Yook K, Zarowiecki M, et al., Stein L. 2024. WormBase 2024: status and transitioning to Alliance infrastructure. Genetics 227(1): 10.1093/genetics/iyae050.
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","pubmedId":"7585938","doi":""}],"title":"Joubert syndrome gene fam149b1 homolog, xbx-4, is required for multiple sensory behaviors.
","reviews":[],"curatorReviews":[{"curator":{"displayName":"Gary Craig Schindelman"},"openAcknowledgement":false,"submitted":null}]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes 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