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Figure S2.
Further characterization of ciliary phenotypes in Cmb mutants, related toFig. 2. (A) Appearance of Cmb mutant flies, as well as Cmb mutants rescued by the expression of a GFP-CMB transgene. Cmb mutants display abnormal wing posture, a phenotype associated with defective mechanosensation. (B) Fertility test performed on Cmb mutant males and females, Cmb mutants rescued by the expression of a GFP-CMB transgene or maintained over a balancer (Tm6), and Cmb mutants placed over a deficiency that covers the Cmb locus (Def 25, 26). Cmb mutant males but not females exhibit fully penetrant sterility, a defect rescued by the expression of the GFP transgene. Placing the mutant over a deficiency does not impact fertility, excluding potential nonallelic effects. Error bars are the mean ± SD. N = 3 single males, each crossed to four virgin females. (C) Schematic and immunofluorescence micrographs of scolopidia in chordotonal organ of the fly. SAS-4 and NompC were used to visualize basal body (green) and ciliary tip (magenta), respectively. Each scolopidium contains two ciliated nerve endings ensheathed by a glial cell, with the ciliary tips attached to the cuticle via a cap cell (Kernan, 2007). No gross ciliary morphological defects are observed in Cmb mutants. N = 63 control scolopidia, 63 Cmb mutant. A statistical test is t test with Welch’s correction. (D) DIC images of scolopidia. Cmb mutants display a larger distance between ciliary dilation and cap cell, indicative of ciliary positioning defects. Error bars are the mean ± SD. N = 32 control, 31 Cmb mutants. Student’s t test with Welch’s correction was used; ***P < 0.001. (E) Cross-sectional views of control and Cmb mutant scolopidia by transmission electron microscopy (TEM) from the distal tips (1) to the ciliary rootlets (6) below the basal body. Position was indicated by numbers in schematic on the left. Cmb mutants show minor structural defects, including broken axonemes and misplaced doublet microtubules (arrows). (F) Left: Analysis of flagellar movement of control and Cmb mutant sperm by high-speed video capture in dark-field microscopy. Sinusoidal motion can be seen in wild type. Arrowheads indicate the position of propagating peaks and troughs in image sequence. Cmb mutant sperm show severely compromised flagellar movement. Right: In contrast to controls, seminal vesicles of Cmb mutant flies are almost devoid of sperm, indicating defective movement of sperm to seminal vesicle. (G) Schematic and immunofluorescence images of Drosophila spermatogenesis. In Cmb mutants, the early stages of spermatogenesis appear superficially normal; however, in later stages, investment cones involved in individualizing sperm fail to form properly. Scale bars, 1 µm (C and D), 100 nm (E), 100 µm (F), 20 µm (G).

Further characterization of ciliary phenotypes in Cmb mutants, related to Fig. 2 . (A) Appearance of Cmb mutant flies, as well as Cmb mutants rescued by the expression of a GFP-CMB transgene. Cmb mutants display abnormal wing posture, a phenotype associated with defective mechanosensation. (B) Fertility test performed on Cmb mutant males and females, Cmb mutants rescued by the expression of a GFP-CMB transgene or maintained over a balancer (Tm6), and Cmb mutants placed over a deficiency that covers the Cmb locus (Def 25, 26). Cmb mutant males but not females exhibit fully penetrant sterility, a defect rescued by the expression of the GFP transgene. Placing the mutant over a deficiency does not impact fertility, excluding potential nonallelic effects. Error bars are the mean ± SD. N = 3 single males, each crossed to four virgin females. (C) Schematic and immunofluorescence micrographs of scolopidia in chordotonal organ of the fly. SAS-4 and NompC were used to visualize basal body (green) and ciliary tip (magenta), respectively. Each scolopidium contains two ciliated nerve endings ensheathed by a glial cell, with the ciliary tips attached to the cuticle via a cap cell (Kernan, 2007). No gross ciliary morphological defects are observed in Cmb mutants. N = 63 control scolopidia, 63 Cmb mutant. A statistical test is t test with Welch’s correction. (D) DIC images of scolopidia. Cmb mutants display a larger distance between ciliary dilation and cap cell, indicative of ciliary positioning defects. Error bars are the mean ± SD. N = 32 control, 31 Cmb mutants. Student’s t test with Welch’s correction was used; ***P < 0.001. (E) Cross-sectional views of control and Cmb mutant scolopidia by transmission electron microscopy (TEM) from the distal tips (1) to the ciliary rootlets (6) below the basal body. Position was indicated by numbers in schematic on the left. Cmb mutants show minor structural defects, including broken axonemes and misplaced doublet microtubules (arrows). (F) Left: Analysis of flagellar movement of control and Cmb mutant sperm by high-speed video capture in dark-field microscopy. Sinusoidal motion can be seen in wild type. Arrowheads indicate the position of propagating peaks and troughs in image sequence. Cmb mutant sperm show severely compromised flagellar movement. Right: In contrast to controls, seminal vesicles of Cmb mutant flies are almost devoid of sperm, indicating defective movement of sperm to seminal vesicle. (G) Schematic and immunofluorescence images of Drosophila spermatogenesis. In Cmb mutants, the early stages of spermatogenesis appear superficially normal; however, in later stages, investment cones involved in individualizing sperm fail to form properly. Scale bars, 1 µm (C and D), 100 nm (E), 100 µm (F), 20 µm (G).

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