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Fig. 6. katnal2is required for ciliogenesis. A-J) Multiciliated X. tropicalis embryonic epidermis following injection of katnal2 morpholino at two doses or CRISPR/Cas9 RNP, showing Centrin4-CFP (blue), Actin (phalloidin, green) and acetylated α-tubulin (Ac-α-tubulin, cilia, magenta). A) Control embryos (injected with Centrin4-CFP alone) show normal ciliogenesis. B-C) Embryos injected with 2–4 pmol of katnal2 morpholino show reduced number and length of cilia. D-E) Embryos injected with katnal2 CRISPR/Cas9 RNPs also show reduced number and length of cilia. F-J) High magnification view of each condition. Asterisks (⁎) mark cells that did not undergo katnal2 disruption (Centrin4-CFP negative). |
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Fig. S2. Validation of Katnal2 rabbit antibody. A-B) X. tropicalis embryonic epidermis stained for Katnal2 (green) and acetylated α-tubulin (Ac-α-tubulin, cilia, magenta) in the presence (A) or absence (B) of Katnal2 primary rabbit antibody. |
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Fig. S6. katnal2loss disrupts basal body distribution and actin network formation in theX. tropicalisembryonic epidermis. A) Control embryos showing actin (phalloidin, green), Centrin4-CFP (blue, basal bodies), and cilia (Ac-α-tubulin, magenta). B) Embryos injected with 4 pmol katnal2 morpholino show abnormal actin network formation (B), basal body distribution (B’), and cilia length and number (B”). C) Embryos injected with 6 pmol katnal2 morpholino show abnormal actin network formation (C), basal body distribution (C’), and cilia length and number (C”). |
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Fig. 2. Katnal2 localizes to epidermal cilia, basal bodies, and centrioles. A-B) Katnal2 rabbit antibody staining (green) of X. tropicalis multiciliated cells of the epidermis, co-stained with DAPI (nuclei, blue) and acetylated α-tubulin (Ac-α-tubulin; cilia, magenta). B) High-magnification image from the specimen shown in A. C) Katnal2 antibody staining (green) with Centrin4-CFP expression (magenta). D-H) KATNAL2-GFP (green) in the embryonic epidermis of X. laevis, co-stained for Centrin4-RFP (basal bodies/centrioles, magenta) and Ac-α-tubulin (cilia, blue). D-E) KATNAL2-GFP localizes to basal bodies in multiciliated cells. (E is close-up of D) F-H) KATNAL2-GFP localization in non-ciliated epidermal cells. G) KATNAL2-GFP signal in centrioles. H) KATNAL2-GFP signal in the midbody of dividing cells. |
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Fig. S5. katnal2loss-of-function phenotypes inX. laevis. A) Representative X. laevis embryos at stage 40 following katnal2 morpholino injection. Asterisk (⁎) indicates injected side. B) Representative X. laevis embryos at stage 44 following katnal2 morpholino injection. Asterisk (⁎) indicates injected side. C-E) Multiciliated embryonic epidermis following injection of katnal2 morpholino at two doses in X. laevis, showing Centrin4-CFP (blue), Actin (phalloidin, green) and acetylated α-tubulin (Ac-α-tubulin, cilia, magenta). C) Control embryos. D) Embryos injected with 2 pmol of katnal2 morpholino. E) Embryos injected with 4 pmol of katnal2 morpholino. Injected embryos show abnormal ciliogenesis. Asterisks (⁎) mark cells that were not targeted (Centrin4-CFP negative). |
