MCI can substitute for GMNC activity, but not vice versa, in MCC differentiation. (A) Pronephric duct of a 48 h post-fertilization (hpf) wild-type zebrafish embryo showing multiple cilia on MCCs (arrows). (B) Pronephric duct of a 48 hpf gmnc mutant zebrafish embryo showing severe lack of MCCs. Monocilia, which are not affected by the loss of Gmnc, are indicated (arrows). (C) Pronephric duct of a 48 hpf gmnc mutant embryo showing rescue of MCCs (arrows) on overexpression of mouse Mci (mMci) mRNA. (D) Quantification of the rescue of gmnc homozygous mutant zebrafish embryos with mMci mRNA. Seventy-two embryos overexpressing mouse MCI were genotyped. Seventeen were gmnc homozygotes, of which 14 showed MCC rescue in pronephric ducts (partial to full rescue in one or both ducts). Control represents an uninjected embryo. **P≤0.01. (E) Lentivirus-mediated overexpression of GFP in wild-type airway cell ALI culture does not affect MCC differentiation (E′-E‴ show individual channels). (F) Overexpression of GFP in Mci mutant airway cell ALI culture does not restore MCC differentiation (F′-F‴ show individual channels). (G) Overexpression of GMNC in wild-type airway cell ALI culture induces supernumerary MCC differentiation (G′-G‴ show individual channels). (H) Overexpression of GMNC in Mci mutant airway cell ALI culture does not rescue MCC differentiation (H′-H‴ show individual channels). (I) Overexpression of MCI in wild-type airway cell ALI culture induces supernumerary MCC differentiation (I′-I‴ show individual channels). (J) Overexpression of MCI in Mci mutant airway cell ALI culture rescues MCC differentiation (J′-J‴ show individual channels). In all preparations, cilia were stained with anti-acetylated tubulin antibodies (green in A-C; magenta in D-I) and nuclei with DAPI (blue). Overexpressed GFP, and GMNC and MCI were detected with anti-GFP and anti-HA antibodies (green in D-I). Lentivirus-mediated overexpression of GFP, MCI and GMNC in ALI cultures represents two independent biological replicates. Scale bars: 5 μm.
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