Yasunaga T et al. (2011), Regulation of basal body and ciliary functions ...

XB-ART-43787

Mech Dev 2011 Jan 01;1287-10:376-86. doi: 10.1016/j.mod.2011.07.004.

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Regulation of basal body and ciliary functions by Diversin.

Yasunaga T , Itoh K , Sokol SY .

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The centrosome is essential for the formation of the cilia and has been implicated in cell polarization and signaling during early embryonic development. A number of Wnt pathway components were found to localize at the centrosome, but how this localization relates to their signaling functions is unclear. In this study, we assessed a role for Diversin, a putative Wnt pathway mediator, in developmental processes that involve cilia. We find that Diversin is specifically localized to the basal body compartment near the base of the cilium in Xenopus multi-ciliated skin cells. Overexpression of Diversin RNA disrupted basal body polarization in these cells, suggesting that tightly regulated control of Diversin levels is crucial for this process. In cells depleted of endogenous Diversin, basal body structure appeared abnormal and this was accompanied by disrupted polarity, shortened or absent cilia and defective ciliary flow. These results are consistent with the involvement of Diversin in processes that are related to the acquisition of cell polarity and require ciliary functions.

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Species referenced: Xenopus
Genes referenced: ankrd6 cetn4 mid1ip1 myc pnma2
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	Fig. 1. Diversin localizes to the basal body in multi-ciliated cells. Both ventral blastomeres of four-cell embryos were injected with the following mRNAs as indicated: Div-RFP (0.1–0.2 ng), Centrin2-RFP (0.4 ng), Centrin2-GFP (0.1 ng) or Mig12-GFP (0.1 ng). Injected embryos were fixed at stage 34 and cryosectioned for confocal imaging. (A) Experimental scheme. Plane of sectioning is indicated by a dashed line; arrow shows direction of viewing. D, Dorsal; V, Ventral. (B) Mig12-GFP and Centrin2-RFP label the striated rootlet and the basal body, respectively, as indicated in the cartoon on the right. Arrows represent basal body polarity. (C and D) Div-RFP localizes to the basal bodies labeled with Centrin2-GFP (C), but not to the striated rootlets marked by Mig12-GFP (D). Lower panels show boxed images at higher magnification, merged files are on the right (B–D). Representative confocal images of embryonic epidermis are shown. Scale bar, 2 μm.
	Fig. 2. Overexpressed Diversin disrupts basal body polarity. (A) Basal body polarity in the control RFP-expressing cell. (B) Basal body polarity was quantified by calculating circular standard deviations for individual multi-ciliated cells (see Section 4). Results of a representative experiment are shown as means ± SEM (n = 10 and 15 for RFP and Div-RFP, respectively). (C) Disrupted basal body polarity in Div-RFP-expressing cell. Div-RFP (0.3–0.5 ng) and control RFP (1 ng) RNAs were injected as described in Fig. 1. Merged files are on the right, lower panels are at higher magnification. Basal body polarity is represented by arrows.
	Fig. 3. Diversin is required for basal body polarity and ciliogenesis. (A) Specificity of DivMO. Embryos were injected with CoMO or DivMO, as indicated, together with XtDiv-GFP RNA (1 ng) and myc-γ tubulin RNA (0.1 ng). Western analysis of st. 11.5 embryo lysates shows specific inhibition of XtDiv-GFP by DivMO, but not CoMO. XtDiv-GFP was detected with anti-GFP, whereas myc-γ tubulin was detected with anti-Myc antibody. Anti-β-tubulin antibody was used to control loading. (B–D) MOs (20 ng) and RNAs encoding Centrin2-RFP (0.4 ng) and Mig12-GFP (0.1 ng) were injected into the animal-ventral region of four-cell embryos. (B) Basal body polarity was quantified in a representative experiment by calculating circular standard deviations (see Section 4). Results are shown as means ± SEM (n = 20 and 18 for CoMO and DivMO respectively). (C and D) The apical regions of multi-ciliated cells scored in (B) are shown as x–y (C and D) or x–z plane (C′ and D′) projections of serial optical sections (see also Fig. 1 legend). Arrows indicate basal body polarity. Top is apical in C′ and D′. (C and C′) CoMO does not affect basal body apical localization and polarity. (D and D′) DivMO disrupted basal body polarity and striated rootlet structure. Arrowheads point to defects in basal body apical docking.
	Fig. 4. Diversin is required for ciliary function in multi-ciliated cells. (A and B) Effects of Diversin MO (DivMO) on ciliogenesis. Morpholinos (20 ng each) and RNAs for Centrin2-RFP (0.4 ng) and Mig12-GFP (0.1 ng) were injected as described in Fig. 1, embryos were cultured until stage 34, fixed and stained for cilia with anti-acetylated tubulin antibody. (A) CoMO-injected cell. (B) DivMO-injected cell. Short cilia are marked by arrowheads at the base (red) and the tip (blue). (C) Basal body density is slightly reduced in multi-ciliated cells from embryos injected with DivMO. Number of basal bodies was counted in eight randomly picked multi-ciliated cells of CoMO or DivMO injected embryos. (D) DivMO-injected embryos contain shorter cilia. Cilia length was measured in multi-ciliated cells of CoMO- or DivMO-injected embryos shown in A and B. (E) Effects of DivMO on hovering movements. Morpholinos (20 ng) were injected into four ventral blastomeres of eight-cell embryos. Compared to CoMO, DivMO significantly decreased hovering movement velocity that was measured when control siblings reached stage 36 (see Section 4). Means ± standard deviations are shown, ∗p < 0.005.
	Fig. 5. Defective ciliogenesis in the gastrocoel roof plate of Diversin-depleted embryos. Morpholino oligonucleotides and GFP-CAAX RNA (150 pg) were coinjected into one dorsal blastomere of four-cell embryos. Gastrocoel roof plate (GRP) was dissected at stage 17 and stained with anti-GFP and anti-acetylated tubulin antibodies. Arrowheads (red channel only) indicate GRP cilia. The midline (M) is shown by a dashed line. Merged images are shown on the left. Div-MO, but not Co-MO, inhibited cilia formation in the GRP. (A) Control MO, 40 ng. (B) Diversin MO, DivMO, 40 ng. (C) Effects of DivMO on GRP cilia. The number of GRP cilia was scored in the area of 1900 mm2 (adjacent to the midline), which was randomly selected in morpholino-injected embryos. The area on the contra-lateral side was used as an additional control. DivMO drastically reduced cilia number.

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