Chu CW et al. (2016), Prickle3 synergizes with Wtip to regulate basal...

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Sci Rep 2016 Apr 11;6:24104. doi: 10.1038/srep24104.

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Prickle3 synergizes with Wtip to regulate basal body organization and cilia growth.

Chu CW , Ossipova O , Ioannou A , Sokol SY .

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PCP proteins maintain planar polarity in many epithelial tissues and have been implicated in cilia development in vertebrate embryos. In this study we examine Prickle3 (Pk3), a vertebrate homologue of Drosophila Prickle, in Xenopus gastrocoel roof plate (GRP). GRP is a tissue equivalent to the mouse node, in which cilia-generated flow promotes left-right patterning. We show that Pk3 is enriched at the basal body of GRP cells but is recruited by Vangl2 to anterior cell borders. Interference with Pk3 function disrupted the anterior polarization of endogenous Vangl2 and the posterior localization of cilia in GRP cells, demonstrating its role in PCP. Strikingly, in cells with reduced Pk3 activity, cilia growth was inhibited and γ-tubulin and Nedd1 no longer associated with the basal body, suggesting that Pk3 has a novel function in basal body organization. Mechanistically, this function of Pk3 may involve Wilms tumor protein 1-interacting protein (Wtip), which physically associates with and cooperates with Pk3 to regulate ciliogenesis. We propose that, in addition to cell polarity, PCP components control basal body organization and function.

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Species referenced: Xenopus laevis
Genes referenced: myc nedd1 pkm prickle1 prickle3 tub vangl2 wtip
???displayArticle.morpholinos??? prickle3 MO1 prickle3 MO2 wtip MO2 wtip MO3

Phenotypes: Xla Wt + prickle3 MO (fig.2.c, d) [+]

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	Figure 1. Localization of Pk3 and Vangl2 in the gastrocoel roof plate (GRP).(A) Schematic neurula embryo section illustrating the position of the GRP (yellow). The dorsal (D)- ventral (V) axis is indicated. NP, neural plate; N, notochord; S, somites. (B) Enrichment of GFP-Pk3 at the base of cilium in GRP cells (arrowhead). Embryos were injected with GFP-Pk3 RNA (0.25 ng). Protein localization is detected by GFP fluorescence in stage 15 GRP explants. Cilium is stained by anti-acetylated α-tubulin (Ac-tub) antibody. (C) En face immunostaining of endogenous Vangl2 in the GRP of stage 15 embryos. The anterior (A) - posterior (P) axis is indicated. Note the accumulation of Vangl2 at anterior cell boundaries. There is no staining in the absence of primary antibody (data not shown). (D–D”) GRP cells coexpressing GFP-Pk3 and HA-Vangl2 (150 pg of RNA each) at stage 15. Arrowheads mark anterior cell borders. (E) Scheme of Pk3 constructs. (F) Physical interaction between Pk3 and Vangl2 in transfected HEK293T cells. The indicated FLAG-Pk3 proteins were pulled down using anti-FLAG-agarose beads, and co-precipitation of HA-Vangl2 was examined. Protein levels are shown by immunoblotting with anti-FLAG and anti-HA antibodies.
	Figure 2. Pk3 is required for the posterior localization and growth of GRP cilia.(A) Efficiency of the Pk3 knockdown. Embryos were injected with Pk3-GFP RNA (2 ng) that contains MO target sites, control FLAG-GFP RNA (0.1 ng), control MO (CoMO, 80 ng), Pk3MO1 (15 ng), or Pk3MO2 (80 ng) as indicated. Embryo lysates obtained at stage 11 were immunoblotted with anti-GFP antibody. α-tubulin (αtub) is a control for loading. (B–I) Effects of Pk3 depletion (B–F) and rescue (G–I) on GRP cilia position and length. Embryos were injected at the 4–8 cell stage with CoMO (B), Pk3MO1 (C), Pk3MO2 (D) or Pk3MO1 plus GFP-Pk3 RNA (10 pg, G). GRP explants were prepared from stage 17 embryos and stained with anti-acetylated α-tubulin (Ac-tub) antibody to visualize cilia. Coinjection of membrane-associated mCherry RNA (Cherry, 100 pg) marks cell boundaries. (E,H) Percentage of cells with the indicated cilia position. Representative cells with the anterior (A), middle (M) or posterior (P) position of cilia are indicated in (C). Significance was assessed by two-tailed t test comparing the frequencies of cilia positioned posteriorly (green). (E) Pk3MO1 to CoMO: p = 0.0003, Pk3MO2 to CoMO: p < 0.0001. (H) Pk3MO1+Pk3 to Pk3MO1: p = 0.0002. (F,I) Cilia length in GRP cells depleted of Pk3, presented as means +/− s.d. ****p < 0.0001, two-tailed t test. Representative images from three to five experiments are shown, and at least 14 explants were examined per group in each experiment. The effects of Pk3MO1 and Pk3MO2 were evident in approximately 90% of the explants. Co-expression of GFP-Pk3 with Pk3MO1 reduced the frequency of short cilia phenotype to 36% (n = 58). (E,F,H,I) Data were collected from 6 to 10 explants per group in three independent experiments.
	Figure 3. Pk3 is required for the anterior polarization of Vangl2.(A,B) Immunostaining of Vangl2 (arrowheads) in stage 15 GRP cells from embryos injected with GFP RNA (0.2 ng, lineage tracer) and CoMO (15 ng, A,A’) or Pk3MO1 (15 ng, B,B’). (C,D) Embryos were injected with 2 ng of GFP-Pk3C RNA, and Vangl2 localization (C,C’) and cilia (marked by Ac-tub, D,D’) were visualized in GRP cells at stage 15 (C) and 17 (D) respectively. ZO-1 co-staining reveals cell boundaries. En face staining is shown, anterior is to the top. Representative images from three independent experiments are shown, with 6–10 explants per group.
	Figure 5. Pk3 and Wtip physically interact and colocalize at the basal body of GRP cells.(A) Protein interactions between Wtip and Pk3. HEK293T cells expressing Myc-Wtip and FLAG-Pk3 proteins were lysed and immunoprecipitated with anti-FLAG agarose beads. Protein levels were assessed after immunoblotting with anti-FLAG and anti-Myc antibodies. (B) Localization of Wtip at the base of the cilium in GRP cells. Embryos were injected with GFP-Wtip RNA (150 pg). Protein localization is revealed by epifluorescence and staining of acetylated α-tubulin (Ac-tub) in stage 15 GRP explants. (C,D) Colocalization of Pk3 and Wtip in the GRP cells. Embryos were injected with 250 pg of GFP-Pk3 RNA plus 100 pg (C) or 250 pg (D) of HA-RFP-Wtip RNA. Epifluorescence in stage 15 GRP explants is shown.
	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.
	Figure 7. Wtip functionally interacts with Pk3 to regulate GRP cilia growth and positioning.(A–D) Synergistic effects of Wtip and Pk3 depletion. Embryos injected with membrane-mCherry (Cherry) RNA and CoMO (5 ng, A), WtipMO1 (2.5 ng, B), Pk3MO1 (2.5 ng, C) or WtipMO1 plus Pk3MO1 (D) were stained with anti-acetylated α-tubulin (Ac-tub) antibody to visualize cilia. Membrane-mCherry marks the boundaries of the targeted cells. En face staining is shown, anterior is to the top. Representative images from three independent experiments are shown, with at least 14 explants examined per group in each experiment. While WtipMO1 and Pk3MO1 alone caused short cilia in 20% and none of the explants respectively, co-injection of both MOs increased the frequency of short cilia phenotype to 84% (n = 32). (E) 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. Significance was assessed by one-way ANOVA comparing frequencies of posteriorly positioned cilia (green) in the WtipMO1+Pk3MO1 group and the WtipMO1 group (p < 0.001) or the Pk3MO1 group (p < 0.001). (F) Bar graph showing the means +/− s.d. of cilia length in GRP cells injected with MOs. ***p < 0.001, one-way ANOVA. (E,F) Data are collected from six embryos in two independent experiments.
	Supplementary Figure 1. Pk3 lacking the C-terminal fragment does not affect GRP cilia. Embryos were injected with 2 ng of RNAs encoding GFP (A, A’) or GFP-Pk3δC (B, B’). Cilia were visualized by immunostaining of acetylated α-tubulin (Ac-tub) in GRP cells at stage 17. ZO-1 co-staining reveals cell boundaries. En face staining is shown, anterior is up.
	Supplementary Figure 2. Establishing basal body marker specificity in GRP cells. GRP explants of stage 17 embryos were co-immunostained for γ-tubulin and Centrin1 (A-A”) or Nedd1 (B-B”). Both Centrin1 and Nedd1 colocalize with γ-tubulin at the basal body in the GRP. En face staining is shown, anterior is up.
	Supplementary Figure 3. Expression of Wtip and Pk3 in Xenopus early embryos. GRP explants and whole embryos were subjected to wholemount in situ hybridization with Wtip or Pk3 anti-sense or control sense RNA probes as indicated. (A, B) Stage 10+ gastrulae. Wtip transcripts are weakly detected in the animal pole ectoderm when compared to the embryo hybridized with the corresponding sense probe. Animal pole is up. (C-E) Stage 18 neurulae. Wtip transcripts are detected in both neural and non-neural ectoderm. The neural folds and the neural crest area are strongly positive. (A-E) Anterior is to the left. (A, B, D) Lateral view, (C, E) dorsal view. (F, G) Both Pk3 and Wtip RNAs are present in the ventral side of GRP explants that were prepared prior to in situ hybridization from stage 17/18 embryos. For both anti-sense probes, staining appears stronger at the midline than in the adjacent endoderm. Ventral view is shown, anterior is up. (H-J) Transverse sections of the explants shown in (F, G). Wtip RNA is predominantly in the neural tissue, with lower levels in the GRP (arrow) and the mesoderm (I). Pk3 transcripts are present at the GRP midline (arrow), epidermis and the superficial layer of the neural plate (J). Notochord, N, is indicated by dashed outline.
	Supplementary Figure 4. Pk3 becomes polarized in the epidermis in the presence of Vangl2. Embryos were injected with 100 pg of pCS2-FLAG-GFP-Pk3 DNA (A, B) or 150 pg of GFP-Pk3 RNA plus 150 pg of HA-Vangl2 (VL2) RNA (C-F). Pk3 polarity was visualized by GFP fluorescence in the epidermis of fixed stage 30 embryos. In the absence of HA-Vangl2, fluorescence of injected GFP-Pk3 RNA is not visible at this stage (data not shown). No cortical Pk3 polarization is detectable in representative non-ciliated (A) or ciliated (B) cells, but Pk3 was associated with basal bodies (B). (C-F) Examples of Pk3 polarization in the presence of Vangl2 in ciliated (C, D) and non-ciliated (E, F) cells. Cell borders are marked by co-injected membrane-associated mCherry RNA (Cherry, 100 pg). Asterisks mark the cells showing Pk3 polarization. The anterior-posterior (A-P) and the dorsal-ventral (D-V) axes indicated in E also refer to other panels. Six embryos from two independent experiments were examined. Pk3 was enriched posteriorly in 62 % of mosaically-expressing cells and anteriorly in 38 % of the cells (n = 34).
	wtip (Wilms tumor 1 interacting protein) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior left.
	wtip (Wilms tumor 1 interacting protein)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17-18, transverse section, dorsal up.
	prickle3 ( prickle 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17-18, transverse section, dorsal up
	Figure 4. Pk3 depletion prevents γ-tubulin and Nedd1 recruitment to the basal body.Embryos were injected with 0.2 ng of RNA encoding GFP (A–D) or Histone-GFP (E,F) as a lineage tracer and CoMO (A,C,E) or Pk3MO1 (B,D,F) (15 ng each). GRP explants from stage 17 embryos were double stained for GFP and γ-tubulin (A,B), Nedd1 (C,D) or Centrin1 (E,F). En face staining is shown, anterior is at the top. Representative images from three to five independent experiments are shown, with 6–10 explants examined per group. The effects of Pk3MO1 on γ-tubulin and Nedd1 are detectable in the majority of cells depleted of Pk3 (n > 100).

References [+] :

Ambegaonkar, Coordination of planar cell polarity pathways through Spiny-legs. 2015, Pubmed