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Figure S5: Wdr5 is essential for apical expansion in MCCs. Related to Figure 4.
(A) Apical area of MCCs increases over development (from stage 21 to stage 28) in
uninjected controls but fails to increase in wdr5 morphants.
(B) Quantification of number of MCCs per unit area between controls and wdr5
morphants. n = number of embryos.
(C) Quantification of apical area over development (from stage 21 to stage 28) in
uninjected controls and wdr5 morphants. n = number of MCCs. |
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Figure 1. Wdr5 Regulates Ciliogenesis in MCCs Independently of H3K4MT
(A) X. tropicalis epidermal MCCs marked either by anti-acetylated α-tubulin (red) or membrane-RFP; actin is labeled with phalloidin and nuclei with Hoechst. Scanning electron microscope images of MCCs of Xenopus embryos (green stars mark MCCs). Uninjected control embryos are on the left, and embryos injected at one cell stage with wdr5 MO are on the right.
(B) Cilia-driven epidermal flow is visualized using red microbeads over the period of 6 s (see Video S1). Magenta arrowheads indicate bead displacement from starting point (dashed lines). Green arrowheads indicate no displacement. Green fluorescent protein traces the morpholino and RNA. Experimental conditions include wdr5 MO, wdr5 MO + human WT WDR5 RNA, wdr5 MO + human mutant S91K WDR5 RNA, and compared to uninjected controls.
(C) Quantification of maximum bead displacement (micron) in 6 s in uninjected controls, first wdr5 MO, second wdr5 MO, first wdr5 MO + WT-WDR5-3xGFP, first wdr5 MO + S91K-WDR5-3xGFP, and first wdr5 MO + K7Q-WDR5-3xGFP. n = number of embryos. ★★★ indicates statistical significance at p < 0.0005. Data is represented as mean ± SEM.
(D) X. tropicalis epidermal MCCs marked with anti-acetylated α-tubulin (green) for cilia and phalloidin (red) for actin. Percentage of embryos with wild-type-like cilia (magenta), partial rescue (green), or complete loss (cyan) of cilia under different conditions: uninjected controls, wdr5 MO, wdr5 MO + human WT-WDR5, and wdr5 MO + human S91K-WDR5. n = number of embryos. ★★★ indicates statistical significance at p < 0.0005. |
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Figure 7. Wdr5 Is Essential for Stabilization of F-Actin
(A) Schematic showing the experimental design to examine the rate of F-actin disassembly in MCCs of Xenopus embryos. To isolate the effect of WDR5 in disassembly, we exposed epidermal MCCs to Latrunculin A (LatA) treatment for 10 min. LatA specifically binds to G-actin in stoichiometric 1:1 ratio, preventing the F-actin polymerization. Thus, exposure to LatA prevents the assembly but not disassembly of F-actin, allowing us to evaluate the effect of WDR5 depletion on F-actin disassembly in MCCs. Based on earlier results that medial actin is necessary for apical expansion and our results that Wdr5 depletion leads to loss of medial F-actin enrichment, we specifically examined the medial F-actin intensity in controls and wdr5 morphants. Medial actin intensity was normalized to cortical actin to allow us to combine results from different experiments for statistical comparison.
(B) Quantification of normalized medial actin intensity in the MCCs of controls and the suboptimal dose of wdr5 morphants after exposure to DMSO (vehicle) or LatA (2 μM) for 10 min using two non-overlapping wdr5 MOs. Our results show a dramatic reduction in actin intensity in response to LatA in wdr5 morphants compared to DMSO only. n = number of MCCs. ★★ and ★★★★ indicate statistical significance at p < 0.005 and p < 0.00005. Data are represented as mean ± SEM.
(C) Immunofluorescence showing Xenopus epidermal MCCs labeled for F-actin (phalloidin) and cilia (anti-acetylated α-tubulin) in controls and wdr5 morphants after exposure to DMSO (vehicle) or LatA (2 uM) for 10 min. Arrowheads indicate actin enrichment in MCCs. The cartoon depicts the hypothesized rates of disassembly and the effect of LatA on medial actin enrichment in controls and wdr5 morphants.
(D) Schematic illustration of a model proposing the role of WDR5 in the formation of an MCC. WDR5 binds to basal bodies as basal bodies are synthesized deep in the cytoplasm. WDR5 then migrates apically, where F-actin organizes around WDR5. WDR5 interacts with F-actin to stabilize actin network essential for apical expansion and basal body distribution. In the final stages, WDR5 anchors basal bodies to the apical actin network to form functional MCCs. |
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Figure S1: Specificity of wdr5 MO and Wdr5 antibody. Related to Figure 1.
(A) Western Blot indicating Wdr5 levels in uninjected controls, wdr5 morphants, and
wdr5 morphants injected with human WDR5 RNA at stage 20. Numbers at the top are
normalized values for Wdr5 intensity based on loading control Gapdh.
(B) Western Blot indicating WDR5 levels in RPE cells using WDR5 antibody, WDR5
antibody mixed with blocking peptide in 1:1 and 1:2 volumetric ratio. A, B represent two
independent protein isolations.
(C) Western Blot indicating Wdr5 levels in uninjected controls, wdr5 morphants, and
wdr5 morphants injected with either human WT-WDR5-GFP RNA, human S91K-WDR5-
3xGFP RNA, or human K7Q-WDR5-3xGFP RNA. Numbers at the top are normalized
values for Wdr5 intensity based on loading control Gapdh.
(D-J) Percentage of embryos with present (magenta) or reduced (green) cilia driven
epidermal flow.
(D) Experimental conditions include uninjected controls and scrambled MO
(E) Experimental conditions include uninjected controls, wdr5 MO and wdr5 MO +
human WDR5-GFP.
(F) Experimental conditions include uninjected controls and second wdr5 MO.
(G) Experimental conditions include uninjected controls and wdr5 CRISPR.
(H) Experimental conditions include uninjected controls, wdr5 MO, wdr5 MO + human
WDR5 (K7Q)-3xGFP RNA, and human WDR5 (K7Q)-3xGFP RNA.
(I) Experimental conditions include uninjected controls, wdr5 MO, wdr5 MO + human
WT-WDR5 3xGFP RNA, and wdr5 MO + human WDR5 (26-334)-3xGFP RNA.
(J) Experimental conditions include uninjected controls, injected with wdr5 MO, wdr5 MO
+ human WT WDR5 RNA, and wdr5 MO + human mutant S91K WDR5 RNA.
n = number of embryos. ★★★ indicate statistical significance at P < 0.0005
(K) X. tropicalis epidermal MCCs in controls and CRIPSR injected embryos marked with
anti-acetylated α-tubulin (green) for cilia and phalloidin (magenta) for actin.
(L) Transcriptional profile with absolute number of transcripts per embryo of X. tropicalis
starting at single cell up to stage 28. |