B9 protein family conservation and schematic gene representations. (A) Homology with other organisms presented as % amino acid identity/% amino acid similarity. (B) Schematic representation of xbx-7, tza-1, and tza-2 genomic regions. Promoter X-box location, gene region encoding each B9 domain, gene region deleted in each mutant allele, and in the case of xbx-7, splice isoforms are shown. xbx-7(tm2705) and tza-1(tm2452) both contain internal in-frame deletions, whereas tza-2(ok2092) contains an internal frameshift deletion that results in a truncated protein. Exons are represented as black boxes. Diamond signifies site of xbx-7 alternative splicing.

DAF-19 regulation of xbx-7, tza-1, and tza-2 in C. elegans. Expression analysis of (A) xbx-7::YFP, (B) tza-1::DsRed2, and (C) tza-2::CFP in the head ciliated sensory neurons in (left panels) daf-19(m86) mutant and (right panels) daf-19(+) backgrounds. The expression of each transgene was absent in daf-19(m86) mutants while outcrossing to daf-19(+) background restored expression to wild-type levels. Arrowhead, position of the amphid neuron cell bodies from which the dendrites extend to the anterior of the worm. Cilia project from the (arrow) distal tips of the dendrites. For this and subsequent figures, anterior is toward the left.

Localization of XBX-7, TZA-1, and TZA-2 proteins to transition zones at the base of cilia in C. elegans. (A) An illustration depicting the anatomical positions of (left) amphid cilia bundles in the head and (right) phasmid cilia bundles in the tail. Cilia axonemes are represented by blue, transition zones by green, and the dendritic processes by red. (B–D) Transgenic lines were generated that expressed XBX-7::YFP, TZA-1::CFP, or TZA-2::CFP along with cilia/transition zone markers CHE-13::CFP or CHE-13::YFP, each under the control the endogenous promoter of the corresponding gene. (B) XBX-7::YFP localized to the transition zones at the base of cilia in the (top) amphid neurons of the head and (bottom) phasmid neurons of the tail but not along cilia axonemes. (C) TZA-1::YFP localized to the transition zones at the base of cilia in the (top) amphid neurons of the head and (bottom) phasmid neurons of the tail but not along cilia axonemes. In contrast to CHE-13::CFP localization, which was restricted to the cilia axonemes and transition zones, TZA-1::YFP was found at the transition zones and (top, brackets) extended slightly into the dendrites of the amphid neurons. In the (bottom) phasmid neurons, the (brackets) extension of TZA-1::YFP into the dendrites versus the restriction of CHE-13::CFP to the transition zones and cilia axonemes was more distinctive. (D) TZA-2::CFP localized to the transition zones at the base of cilia in the (top) amphid neurons of the head and (bottom) phasmid neurons of the tail but not along cilia axonemes. (E) Analysis of TZA-1::YFP and TZA-2::CFP colocalization at the base of cilia. In contrast to TZA-2::CFP localization, which was restricted to the transition zones, TZA-1::YFP was found at the transition zones and (brackets) extended into the dendrites of both the (top) amphid and (bottom) phasmid neurons. (F–I) Enlargement of phasmid cilia regions from B–E, respectively. (G) TZA-1::YFP and CHE-13::CFP and (I) TZA-1::YFP and TZA-2::CFP colocalized largely to the transition zones but not to (arrowheads) a small domain at the distal ends of the dendrites which contained TZA-1::YFP only.

Cilia morphology analysis of transition zone protein mutants. (A) Potential cilia defects were analyzed in xbx-7(tm2705), tza-1(tm2452), tza-2(ok2092), and nph-4(tm925) mutants by evaluating their ability of to absorb DiI into the ciliated sensory neurons. The ability of xbx-7(tm2705), tza-1(tm2452), tza-2(ok2092), and nph-4(tm925) mutants to absorb DiI was not affected compared with wild-type controls. However, in these mutant worms, the dye appeared to (arrowheads) concentrate in aggregates along the dendrites. This was (arrows) particularly evident in the nph-4(tm925) mutants and was not seen in the wild-type controls. N2 wild-type worms and daf-19(m86) mutants, which lack cilia, were used as positive and negative controls, respectively. (B) Cilia morphology was more directly analyzed in xbx-7(tm2705), tza-1(tm2452), tza-2(ok2092), and nph-4(tm925) mutants expressing the cilia marker CHE-13::YFP. (Top) head and (bottom) tail cilia morphology appeared overtly normal in these strains.

TZA-1 protein is required for proper localization of XBX-7 and TZA-2 to the transition zone. To analyze the effect of tza-1(tm2452), tza-2(ok2092), or nph-4(tm925) mutations on XBX-7::YFP localization, strains were generated by passing an extrachromosomal array encoding XBX-7::YFP from wild-type worms into the mutant backgrounds. Similarly, the TZA-1::YFP array was passed into xbx-7(tm2705), tza-2(ok2092), or nph-4(tm925) mutant background from wild-type, and the TZA-2::CFP array was passed into xbx-7(tm2705), tza-1(tm2452), or nph-4(tm925) mutant background from wild-type. (A) In contrast to (left) wild type, XBX-7::YFP was not detected at the transition zone in the (middle) tza-1(tm2452), or (right) tza-2(ok2092) mutants. (B) Compared with (left) wild type, TZA-1::YFP localization was unaltered in the background of (middle) xbx-7(tm2705) and (right) tza-2(ok2092) mutants. (C) TZA-2::CFP localization was not affected in (middle) xbx-7(tm2705) mutant background; however, it failed to localize correctly to the transition zone in (right) tza-1(tm2452) mutant background. (D) In the nph-4(tm925) mutant background, (left) XBX-7::YFP, (middle) TZA-1::YFP, and (right) TZA-2::CFP localization was not affected.

tza-1, tza-2, and nph-4 mutants exhibit defects in foraging behavior. Single worms were allowed to roam freely for 18 h on a bacteria lawn of 3.0-cm diameter, and area covered by the worm was quantified by overlaying the lawn with a grid and counting the number of squares of the grid that contained tracks. (A) Representative tracking patterns of (left) N2 wild-type, (middle) nph-4(tm925), and (right) tza-1;nph-4 worms. Wild-type worms roamed across the entire area of the bacteria lawns, whereas nph-4(tm925) mutants restricted movement to a smaller portion of the lawn. Exploratory behavior in xbx-7;nph-4, tza-1;nph-4, and nph-4;tza-2 double mutants was even more limited. (B) Graph of quantified tracking data for N2 wild type, xbx-7(tm2705), tza-1(tm2452), tza-2(ok2092), nph-4(tm925), xbx-7;nph-4, tza-1;nph-4, and nph-4;tza-2. Data are given as percent area units covered compared with N2 (set to 100%). Error bars, SEM. *p < 0.005; **p < 0.0001, compared with N2.

B9 gene;nph-4 double mutants exhibit dye-filling defects. Potential cilia abnormalities were analyzed in xbx-7;nph-4, tza-1;nph-4, and nph-4;tza-2 double mutants by evaluating their ability to absorb DiI into the ciliated sensory neurons. (A) xbx-7;nph-4 mutants displayed either (top) partial or (bottom) complete inability to dye-fill (left) amphid neurons and (right) phasmid neurons. (B) tza-1;nph-4 mutants displayed either (top) partial or (bottom) complete inability to dye-fill (left) amphid neurons and (right) phasmid neurons. (C) nph-4;tza-2 displayed either (top) partial or (bottom) complete inability to dye-fill (left) amphid neurons but were completely defective in dye-filling (right) phasmid neurons.

B9 gene;nph-4 double mutants exhibit cilia morphology and positioning defects. Cilia were analyzed in xbx-7;nph-4, tza-1;nph-4, and nph-4;tza-2 double mutant worms expressing CHE-13:: YFP as a cilia/transition zone marker. (A) A representative amphid cilia bundle in the head of an N2 wild-type worm. (B) A representative pair of phasmid cilia in the tail of an N2 wild-type worm. These cilia are positioned near the cuticle of the worm where they are exposed to the external environment. See Figure 3A for schematic representation of the N2 wild-type amphid and phasmid cilia bundles. (C) Amphid cilia bundle morphology was altered in xbx-7;nph-4 double mutant background. In many of the xbx-7;nph-4 double mutants, some of the cilia were (arrow) positioned posterior to the rest of the bundle. In other xbx-7;nph-4 double mutants, cilia failed to extend off the (arrowhead) transition zones at the distal ends of the dendrites. (D) Phasmid cilia in xbx-7;nph-4 double mutants appeared (top) almost normal in some mutant worms but in others they were (bottom) shortened and mispositioned relative to wild type. (E) Amphid cilia bundle morphology was (top and bottom) grossly abnormal in tza-1;nph-4 double mutants. The head region contained few cilia axonemes and lacked typical bundle arrangement. (F) tza-1;nph-4 double mutants exhibited stunted phasmid cilia that were observed (top) slightly or (bottom) severely mispositioned. (G and H) nph-4;tza-2 double mutants exhibited ciliary defects similar to those in tza-1;nph-4 double mutants, including (arrowhead) laterally oriented phasmid cilia in comparison to wild type.

B9 gene;nph-1 double mutants exhibit cilia morphology and positioning defects. Cilia were analyzed in nph-1;xbx-7, nph-1;tza-1, and nph-1;tza-2 double mutant worms expressing CHE-13::YFP as a cilia/transition zone marker. (A) A representative amphid cilia bundle in the head of an N2 wild-type worm. (B) A representative pair of phasmid cilia in the tail of an N2 wild-type worm. These cilia are positioned near the cuticle of the worm where they are exposed to the external environment. See Figure 3A for schematic representation of the N2 wild-type amphid and phasmid cilia bundles. (C) Amphid cilia bundle morphology was largely unaltered in nph-1;xbx-7 double mutant background. nph-1;xbx-7 double mutants occasionally exhibited cilia positioned (arrowhead) posterior to the rest of the bundle but otherwise resembled wild-type worms. (D) Phasmid cilia in nph-1;xbx-7 double mutants most often appeared (top) normal but in some cases were observed (bottom) shortened and mispositioned relative to wild type. (E) Amphid cilia bundle morphology was (top and bottom) grossly abnormal in nph-1;tza-1 double mutants. The head region contained (arrowhead) few cilia, consisted mostly of (arrow) transition zones lacking axonemes, and had no discernable amphid bundle arrangement. (F) nph-1;tza-1 double mutants exhibited stunted phasmid cilia that were observed (top) slightly or (bottom) severely mispositioned and often oriented laterally. (G) nph-1;tza-2 double mutants exhibited amphid ciliary defects more severe than nph-1;xbx-7 double mutants but less severe than nph-1;tza-1 double mutants. Although not perfectly arranged, (top and bottom) some bundling of multiple cilia was typically observed among (arrowhead) shortened and mispositioned cilia and (arrowhead) transition zones lacking axonemes. (H) In contrast to the relatively mild defects observed in the amphid bundles, the phenotype of phasmid cilia in nph-1;tza-2 double mutant worms mimicked the severity of defects seen in the phasmid cilia of nph-1;tza-1 double mutant worms.

The dendrites of ciliated sensory neurons in B9 gene;nph gene double mutants are malformed although the surrounding sheath cells are intact. (A–F) Transgenic B9 gene single mutants and B9 gene;nph-4 double mutants were generated that expressed the xbx-7::YFP transcriptional fusion to visualize the ciliated sensory neurons. (A, C, and E) Expression of xbx-7::YFP revealed that the (arrow) phasmid dendrites are properly extended from the (arrowhead) cell bodies in the background of single B9 gene mutations. (B, D, and F) The phasmid dendrites of B9 gene;nph-4 double mutant worms were shortened at variable lengths and sometimes (arrow) misdirected. (G and H) Morphological analysis of (left) amphid and (right) phasmid sheath cells in wild-type and tza-1;nph-4 double mutant worms. Transgenic worms were generated that coexpressed the tza-1::DsRed2 transcriptional fusion to visualize the ciliated sensory neurons and the f16f9.3::GFP transcriptional fusion to visualize the associated sheath cells. (G) In wild-type worms, the amphid and phasmid sheath cells fully surrounded the dendrites of the ciliated sensory neurons at (arrows) their distal tips where the cilia are formed. (H) Sheath cell morphology was unaffected in tza-1;nph-4 double mutants. The amphid sheath cells were properly extended to surround the distal tips of the associated neurons. Likewise, phasmid sheath cells in the tail were appeared normally extended while the phasmid neuron dendrites were (arrows) shortened and clearly dissociated from (arrowhead) the distal ends of the sheath cells.