Author Summary C. elegans mutants that either fail to form or arrest development as dauer larvae, a stress-resistant lifestage, usually have defects in genes involved in evolutionarily conserved signaling pathways. In this study, we identified the gene mutated in daf-25 mutant strains, which inappropriately arrest as dauer larvae and are also defective in the sense of smell. The mammalian counter- part of DAF-25 is Ankmy2, a protein of unknown function that contains three ankyrin repeats and a zinc finger MYND domain, both of which are predicted to bind other protein(s). We show that DAF-25/Ankmy2 is required for the proper localization of a membrane-bound guanylyl cyclase—a class of protein that functions in cyclic GMP signaling—to cilia, which are conserved sensory organ- elles. We further demonstrate that mammalian Ankmy2 binds the retinal guanylyl cyclase GC1, suggesting a role for Ankmy2 in vision—which critically depends on cyclic GMP signal transduction—suggesting the potential in- volvement of Ankmy2 in human retinal disease, as well as other cilia-related diseases such as obesity. Intraflagellar transport (IFT) proteins, normally required for building cilia, are well conserved in C. elegans and several have been discovered in this organism through the identification of sensory mutants [9]. Indeed, dauer formation is a sensory behavior dependent on the balanced inputs of dauer pheromone, temperature and food signals [4]. Proteins in the olfactory component of the dauer pathway include SRBC-64 and SRBC-66 (dauer pheromone receptors), DAF-11, a guanylyl cyclase, G-proteins (gpa-2 and gpa-3), the Hsp90 molecular chaperone DAF-21, the IFT protein DAF-10, and the DAF-19 RFX-type transcription factor [10–14]. DAF-19 is strictly required for cilium formation as it regulates the expression of many cilia-related genes through a consensus sequence dubbed ‘x-box’ [15]. daf-11, daf-19 and daf-21 are Daf- c, whereas daf-6 and daf-10 are Daf-d [16]. daf-19, daf-6 and daf-10 are all dye-filling defective, indicating that their cilia (if present) are not exposed to the environment [17,10]. By contrast, daf-11 and daf-21 mutants show wild-type dye filling [18]. All five mentioned daf genes are defective in recovery from the dauer diapause, presumably because they cannot detect the bacterial food stimulus [17]. Dauer recovery defects are present for mutants with broad chemosensory defects caused by abnormal ciliogenesis or signal- ing, and for many Unc genes, such as unc-31, which encodes a dense core vesicle secretion protein [17,19,20]. Our genetic screen for C. elegans Daf mutants has uncovered a novel ciliary protein, DAF-25, which participates in cGMP-associated signaling by modulating the ciliary localization of a guanylyl cyclase, DAF-11. The mammalian ortholog of DAF-25, Ankmy2, interacts with ciliary photoreceptor guanylyl cyclase 1 (GC1), indicating that the role of the MYND domain protein in cilia function is likely to be conserved and potentially relevant to human retinal disease or other ciliopathies. Results Genetic Epistasis Analysis Places DAF-25 Function in the Amphid Cilia To identify genes potentially implicated in sensory transduction, we uncovered four alleles of daf-25 in various screens for new mutants exhibiting a temperature-sensitive Daf-c phenotype. Three alleles (m98, m137, and m362) were isolated from ethyl DAF-25/Ankmy2 Functions in Ciliary cGMP Signaling methanesulfonate (EMS) mutagenesis screens and the fourth, m488, was isolated in a screen for Daf-c mutants with transposon insertions [21,22]. Epistasis tests with the Daf-d mutants daf-12, daf-16, daf-3, daf-6 and daf-10 were used to position daf-25 into the existing genetic pathway. Mutations in the daf-12 nuclear hormone receptor gene suppress most Daf-c mutants [16,23] including daf-25 (0% dauer larva formation, n.200 for daf-25(m362); daf-12(m20) compared to 97.5%, n = 281 for daf-25(m362) at 25uC). DAF-16/FOXO is the major downstream effector for Insulin/IGF1 signaling [24] as is DAF-3/Co-Smad for the TGF-b pathway [25]. Mutations in daf-16 and daf-3 only partially suppress the Daf-c phenotype of daf- 25 (37.6% dauer larvae, n = 407 for daf-25(m362); daf-16(m26) and 60.0%, n = 167 for daf-25(m362); daf-3(mgDf90) at 25uC), indicat- ing that DAF-25 likely functions upstream of both pathways. Importantly, daf-10, which encodes an IFT protein (DAF-10/ IFT122) required for ciliogenesis [11], suppresses daf-25 (0% dauer larvae, n.200 for daf-25(m362); daf-6(e1387) compared to 97.5%, n = 281 for daf-25(m362) at 25uC), suggesting a function for DAF- 25 within sensory cilia. daf-6 mutants have closed amphid channels and cannot smell chemoattractants or form dauer larvae even though their cilia are present [5]. Interestingly, daf-6 mutations do not suppress the daf-25 Daf-c phenotype (97.4% dauer larvae, n = 312 for daf-25(m362); daf-6(e1377) at 25uC), indicating that DAF-25 acts downstream of DAF-6, and that environmental (ciliary) input is not required for the Daf-c phenotype. DAF-6/ PTCHD3 is expressed in the glial (sheath) cell that forms the amphid sensory channel, allowing contact of the sensory cilia to the environment through pores in the cuticle [26]. 8-bromo- cGMP rescues the dauer phenotype of daf-25 (0% dauer larva formation for daf-25(m362) on 8-bromo-cGMP, n = 72 compared to 32% dauer larva formation on the control, n = 65, both at 20uC), similar to that previously reported for daf-11 [12] indicating that DAF-25 functions upstream of the cGMP pathway in the cilia. Indeed the Daf-c phenotype of daf-25(m362) is very similar to that of daf-11(m84) at all temperatures tested (Table S1). The epistasis results are also similar to those for daf-11, indicating that both genes function at the same point in the genetic pathway— upstream of cilia formation and cGMP signaling in the cilia, and downstream of environmental input. daf-25 Mutants Exhibit Chemosensory Phenotypes Independent of Ciliary Ultrastructure Defects daf-25 mutants are temperature-sensitive Daf-c and defective in dauer recovery. They constitutively form virtually 100% dauer larvae at 25uC, which do not recover upon transfer to 15uC. The Daf-c phenotype is rescued by maternally contributed daf-25 as seen in the progeny of daf-25(m362) heterozygous hermaphrodites which form zero percent dauer larvae at 25uC (n.200). Moreover, daf-25 animals exhibit defective responses to various chemosensory stimuli as well as a moderate defect in response to osmotic stress (37 of 45 daf-25(m362) adults crossed the sucrose hyperosmotic boundary compared to 1 of 45 for N2, x2-p-value = ,0.00001, while 0 of 30 daf-25(m362) and N2 adults crossed a glycerol boundary). Adults are also defective in egg laying. Despite the fact that Daf-c genes in the IIS pathway (like daf-2 and age-1) extend adult lifespan [27], daf-25 mutants show no significant difference in lifespan from N2 (Figure S1). daf-25 mutants are defective in chemotaxis to at least four volatile odorants (Figure 1). Wild-type N2 adults were attracted to the compounds tested, the chemotaxis-defective mutant daf-11 was partially attracted, whereas the two daf-25 mutants tested were nearly unresponsive (Figure 1). DAF-11 and the cGMP pathway are known to regulate responses to the AWC neuron-mediated PLoS Genetics | www.plosgenetics.org 2 November 2010 | Volume 6 | Issue 11 | e1001199