XB-IMG-151062
Xenbase Image ID: 151062
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Figure 6. Wtip depletion affects posterior localization and growth of GRP cilia.(A) Efficiency and specificity of the Wtip knockdown. Embryos were injected with Wtip-FLAG RNA (1 ng), HA-RFP-Wtip RNA (HR-Wtip, 0.5 ng), control MO (CoMO, 30 ng), WtipMO1 (10 ng), or WtipMO2 (30 ng) as indicated. Embryo lysates obtained at stage 12 were immunoblotted with anti-FLAG and anti-HA antibody. Asterisks mark nonspecific signals from anti-HA antibody. α-tubulin (αtub) is a control for loading. (B–I) Effects of Wtip depletion (B–F) and rescue (G–I) on cilia position and length. Embryos were injected with CoMO (B), WtipMO1 (C), WtipMO2 (D) or WtipMO1 plus HA-RFP-Wtip RNA (75 pg) (G). GRP explants were prepared at stage 17 and stained with anti-acetylated α-tubulin (Ac-tub) antibody to visualize cilia. Membrane-associated mCherry marks the boundaries of cells injected with MOs. Anterior is to the top. (E,H) Percentage of cells with the indicated cilia position. The position of each cilium was assigned to the A, M or P location in each cell, as in Fig. 2. Significance was assessed by two-tailed t test comparing the frequencies of cilia positioned posteriorly (green). WtipMO1 to CoMO: p < 0.0001, WtipMO2 to CoMO: p = 0.0009. WtipMO1+ Wtip to WtipMO1: p = 0.35. (F,I) Bar graph showing the means +/− s.d. of cilia length in GRP cells depleted of Wtip. ****p < 0.0001, two-tailed t test. Representative images from three independent experiments are shown, and at least 7 explants were examined per group. The effects of WtipMO1 and WtipMO2 were visible in over 80% of the explants. Co-expression of HA-RFP-Wtip with WtipMO1 reduced the frequency of short cilia phenotype to 48% (n = 23). (E,F,H,I) Data are collected from six embryos in two independent experiments. (J,K) Embryos were injected with 100 pg of Histone-GFP RNA and CoMO (J,J’) or WtipMO1 (K,K’) (10 ng each). GRP explants from stage 17 embryos were double stained for GFP and γ-tubulin. Representative images from three independent experiments are shown, 6–10 explants were examined per group. The effect of WtipMO1 on γ-tubulin staining was visible in approximately 80% of the explants. Image published in: Chu CW et al. (2016) Copyright © 2016, Macmillan Publishers Limited. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license
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