DAF-25/Ankmy2 Functions in Ciliary cGMP Signaling (C) and daf-25(m362) (D). Also, no change in localization was seen for TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8/TTC8, OSM-5/ IFT88 and XBX-1/D2LIC in daf-25 mutants (Figure S6). In (E), DAF-11::GFP and XBX-1::tdTomato are co-expressed in the amphid cilia in daf-25(m362) mutants. XBX-1::tdTomato localizes to the basal body (indicated by arrowhead) and cilia while DAF-11::GFP localizes to the distal end of the dendrite (indicated by arrow). No overlap in protein localization is observed indicating that DAF-11::GFP shows very little, if any localization to the basal body and no expression in the cilia. XBX-1::tdTomato is expressed in all of the amphid cilia while DAF-11::GFP is expressed in a subset. doi:10.1371/journal.pgen.1001199.g005 The GFP reporter results suggest a potentially specific function for DAF-25 in cilia. This finding is consistent with the reported regulation of daf-25 by the ciliogenic DAF-19 RFX-type transcription factor [39]. Taken together, DAF-25 appears to be an adaptor protein required for the transport or tethering of the guanylyl cyclase DAF-11 within sensory cilia. Conservation of Function for DAF-25/Ankmy2 To ascertain if a functional association between DAF-25/ Ankmy2 and guanylyl cyclase is evolutionarily conserved, we used a pull-down experiment to test whether mouse Ankmy2 interacts with the retinal-specific guanylyl cyclase GC1, a mammalian homolog of DAF-11 present within ciliary photoreceptors. We amplified Ankmy2 cDNA from a mouse retinal cDNA preparation (gift from Simon Kaja), and constructed a cDNA clone with the rhodopsin 1D4 epitope to use for co-IP experiments with anti-1D4 monoclonal antibody [40]. We co-expressed both in HEK293 cells to test for GC1 co-immunoprecipitation with the 1D4 epitope- tagged Ankmy2 (HEK293 cells do not express rhodopsin). Pull- down of Ankmy2 co-precipitated GC1, but not another control protein (retinal membrane protein ABCA4; Figure 6). This indicates that the functional interaction between DAF-25/ Ankmy2 and guanylyl cyclase observed in ciliated sensory cells may be conserved between mouse and worm. Discussion In this study, we have identified in a genetic screen for Dauer formation mutants a novel MYND domain-containing ciliary protein, DAF-25, that is required for the proper localization of a guanylyl cyclase (DAF-11) to sensory cilia. Disruption of DAF-25 does not interfere with intraflagellar transport (IFT) or ciliary ultrastructure, but the protein accumulates in a che-11 retrograde IFT mutant. We therefore propose that DAF-25 is associated with IFT not as a ‘core’ protein but instead as an adaptor for transporting ciliary cargo. In our model, abrogation of DAF-25 would thereby not allow transport of DAF-11, which explains the improper localization of DAF-11 in daf-25 mutants at the very base of cilia and the similarity in phenotype between daf-11 and daf-25 mutants. The amino acid sequence and domain structure similarity between DAF-25 and Ankmy2 suggests an important function for the latter mammalian protein that may be similar to DAF-25 in C. elegans. We attempted to co-immunoprecipitate DAF-25 and DAF- 11 in C. elegans but were unable to satisfactorily remove a sufficient amount of background proteins to avoid confounding any identified interaction (data not shown). We also showed that the retinal guanylyl cyclase GC1 binds to Ankmy2, and we propose that the functional relationship between DAF-25 and DAF-11 is conserved between Ankmy2 and GC1 in ciliated photoreceptor cells. Indeed, Ankmy2 may be required for the transport of not only GC1 but perhaps other cilia-targeted guanylyl cyclases as well as other cilia-targeted proteins in mammals. Further studies will be required to experimentally confirm whether Ankmy2 is required for transport of GC1 to the rod outer segment, and to test if Ankmy2 lesions result in retinal disease or a ciliopathy syndrome that includes retinopathies. Mutations in ciliogenesis and cilia related genes cause human disease phenotypes including Bardet- Biedl syndrome, retinopathies, obesity, situs inversus and polycystic kidney disease, among others [41,42]. Interestingly, GC1 and the nuclear hormone receptor Nr2e3 shown to regulate Ankmy2 expression in mouse retina both harbor mutations in patients with retinal disease [43,44]. While this research was being conducted we became aware of another group that cloned and characterized chb-3 (Y48G1A.3/ daf-25/Ankmy2) as a suppressor of the che-2 body size phenotype [45]. Fujiwara et al., (in press) describe the cloning of chb-3/daf-25 and its essential role in GCY-12 cilia localization. They show that DAF-25 is required in a subset of sensory neurons to rescue the phenotypes they assayed (dauer formation and body size) using a tax-4 promoter. This indicates that DAF-25 function is required in the neurons where cGMP signaling takes place (TAX-4 is a subunit of cGMP-gated calcium channel). They also show expression of DAF-25 in the ASJ neurons (one pair of neurons where DAF-11 is expressed) is required for rescue of the dauer phenotype, also indicating a cell autonomous role for DAF-25. It is interesting that screens for the Daf-c and Chb (che-2 body size suppressor) phenotypes both resulted in the identification of daf- 25/chb-3 and separately identified its apparent ciliary cargos daf-11 and gcy-12, guanylyl cyclases that specifically work in dauer formation and body size, respectively. This indicates that DAF- 25/CHB-3/Ankmy2 may interact with cilia-targeted guanylyl cyclases in a general manner and that much of the phenotype of daf-25/chb-3 mutants reflects a global defect in cGMP signaling, potentially along with other unidentified cargo proteins. In conclusion, our findings uncover a novel ciliary protein that plays an important role in modulating the localization/function of cGMP signaling components, which are known to play a critical role in the function of ciliary photoreceptors [46]. DAF-25/ Ankmy2 may also play a role in the ciliary targeting of other as of yet identified proteins. As such, Ankmy2 could participate in phototransduction and be associated with retinopathies, and more generally, could be implicated in other ciliary diseases (ciliopa- thies). Methods Mapping, Epistasis, and Phenotyping daf-25 daf-25 mutations were created by treatment of N2 with 0.25 M EMS, or by mut-2 transposon mobility, and selection for constitutive dauer formation as previously described [22]. For 3- factor mapping, fog-1(e2121) unc-11(e47) was crossed with daf- 25(m362) and daf-25(m362) unc-35(e259) was crossed with dpy- 5(e61). Scoring the genotypes of the F2 progeny required the phenotyping of F3 progeny (due to the maternal effect of the daf-25 dauer phenotype). Pooled SNP mapping was completed as previously described [30] with some changes. In the Po generation, CB4856 males were crossed to daf-25;unc-11 double mutant hermaphrodites. The F1 males were crossed with CB4856 hermaphrodites. F2 hermaphrodites were selected by absence of Unc progeny. F3 hermaphrodites were placed one to a plate and were selected into wild type or mutant pools based on absence or presence of dauers in the F4. Wild type and mutant pools of F3 hermaphrodites were subject to SNP analysis as previously PLoS Genetics | www.plosgenetics.org 8 November 2010 | Volume 6 | Issue 11 | e1001199