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Figure 2.
Combover is required for ciliogenesis and proper cell division. (A) Schematic of ciliated tissues used for phenotypic analysis. Primary cilia are found in sensory bristles and chordotonal neurons, while motile cilia/flagella are found in testes (Jana et al., 2016). (B) Climbing assay used to assess defects in mechanosensation. Cmb mutant flies are severely uncoordinated, a phenotype rescued by the expression of CMB-GFP. PCP flies (Fy RNAi) show no detectable phenotype. Error bars are the mean ± SD. N > 10 flies per condition. A Kruskal–Wallis test with Dunn’s multiple comparisons test was performed; **P < 0.01, ****P < 0.0001. (C) Male fertility scored by crossing individual males with WT virgins and assessing the number of offspring. Cmb RNAi/mutant flies are fully male infertile. Error bars are the mean ± SD. N = 3 single males each crossed to four virgin females per condition. A Kruskal–Wallis test with Dunn’s multiple comparisons test was performed; *P < 0.05, **P < 0.01, ***P < 0.001. (D and E) TEM analysis of sperm axonemes in control and Cmb mutant testes. Cross-sectional views reveal missing axonemal doublets and fragmented axonemes (D), as well as overall lower number of cilia (number per cyst <64, E). Error bars are the mean ± SD. N = 38 cysts (control), 38 cysts (Cmb mutant). Student’s t test was used to assess statistical significance; *P < 0.05. (F) Embryonic viability test shows lethality in 50% of offspring of Cmb mutant females with heterozygous mutant males (viability could not be assessed for homozygous males due to their failure to mate and fertilize oocytes). Error bars are the mean ± SD. N = 2 virgin females crossed to one male per condition. Student’s t test was used to assess statistical significance; **P < 0.01. (G) Schematic of Drosophila syncytial-stage embryo showing synchronous nuclear divisions occurring close to the egg surface. (H) Live imaging of control and Cmb mutant early embryos (nuclear cycle 12) expressing the centriole marker ASL-GFP and H2A-RFP. Time shown in min:s. Cmb mutants show lagging chromosomes and increased frequency of nuclear fallout (orange dashed circle). Error bars are the mean ± SD. N = 5 control, 6 Cmb mutant embryos. Student’s t test was used to assess statistical significance; *P < 0.05. (I) Live imaging of control and Cmb mutant early embryos (nuclear cycle 12) expressing the centrosome marker RFP-CNN. Time shown in min:s. Cmb mutants show defects in centrosome separation subsequent to nuclear fallout (orange dashed circle) but not PCM recruitment. CNN centrosome intensity was measured at NEBD in nuclear cycle 12. Dots represent average intensity of all centrosomes in a single embryo. Error bars are the mean ± SD. N = 8 control, 6 Cmb mutant embryos. A Mann–Whitney test used to assess statistical significance. Scale bars, 100 nm (D), 500 nm (E), 10 µm (H and I). See also Fig. S2. NEBD, nuclear envelope breakdown; TEM, transmission electron microscopy.

Combover is required for ciliogenesis and proper cell division. (A) Schematic of ciliated tissues used for phenotypic analysis. Primary cilia are found in sensory bristles and chordotonal neurons, while motile cilia/flagella are found in testes (Jana et al., 2016). (B) Climbing assay used to assess defects in mechanosensation. Cmb mutant flies are severely uncoordinated, a phenotype rescued by the expression of CMB-GFP. PCP flies (Fy RNAi) show no detectable phenotype. Error bars are the mean ± SD. N > 10 flies per condition. A Kruskal–Wallis test with Dunn’s multiple comparisons test was performed; **P < 0.01, ****P < 0.0001. (C) Male fertility scored by crossing individual males with WT virgins and assessing the number of offspring. Cmb RNAi/mutant flies are fully male infertile. Error bars are the mean ± SD. N = 3 single males each crossed to four virgin females per condition. A Kruskal–Wallis test with Dunn’s multiple comparisons test was performed; *P < 0.05, **P < 0.01, ***P < 0.001. (D and E) TEM analysis of sperm axonemes in control and Cmb mutant testes. Cross-sectional views reveal missing axonemal doublets and fragmented axonemes (D), as well as overall lower number of cilia (number per cyst <64, E). Error bars are the mean ± SD. N = 38 cysts (control), 38 cysts (Cmb mutant). Student’s t test was used to assess statistical significance; *P < 0.05. (F) Embryonic viability test shows lethality in 50% of offspring of Cmb mutant females with heterozygous mutant males (viability could not be assessed for homozygous males due to their failure to mate and fertilize oocytes). Error bars are the mean ± SD. N = 2 virgin females crossed to one male per condition. Student’s t test was used to assess statistical significance; **P < 0.01. (G) Schematic of Drosophila syncytial-stage embryo showing synchronous nuclear divisions occurring close to the egg surface. (H) Live imaging of control and Cmb mutant early embryos (nuclear cycle 12) expressing the centriole marker ASL-GFP and H2A-RFP. Time shown in min:s. Cmb mutants show lagging chromosomes and increased frequency of nuclear fallout (orange dashed circle). Error bars are the mean ± SD. N = 5 control, 6 Cmb mutant embryos. Student’s t test was used to assess statistical significance; *P < 0.05. (I) Live imaging of control and Cmb mutant early embryos (nuclear cycle 12) expressing the centrosome marker RFP-CNN. Time shown in min:s. Cmb mutants show defects in centrosome separation subsequent to nuclear fallout (orange dashed circle) but not PCM recruitment. CNN centrosome intensity was measured at NEBD in nuclear cycle 12. Dots represent average intensity of all centrosomes in a single embryo. Error bars are the mean ± SD. N = 8 control, 6 Cmb mutant embryos. A Mann–Whitney test used to assess statistical significance. Scale bars, 100 nm (D), 500 nm (E), 10 µm (H and I). See also Fig. S2. NEBD, nuclear envelope breakdown; TEM, transmission electron microscopy.

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