Villa M , Crotta S , Dingwell KS , Hirst EM , Gialitakis M , Ahlfors H , Smith JC , Stockinger B , Wack A .

MC_U117597139 Medical Research Council , MRC_MC_U117597139 Medical Research Council , MC_U117597140 Medical Research Council , Wellcome Trust

	Figure 1. Multiciliated cell generation is markedly reduced in AhR-deficient mTEC cultures.(a) Immunofluorescent staining of acetylated α-tubulin in AhR-sufficient and AhR-deficient mTEC cultures following 9 days of ALI. Arrowheads indicate the few multiciliated cells (acetylated α-tubulin+) developing from Ahr−/− cultures. Scale bars, 50 μm. (b) Number of acetylated α-tubulin+ cells per field in wt and Ahr−/− mTEC cultures after 9 days of ALI. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). n=5 random fields per group. (c) Quantification of acetylated α-tubulin fluorescence intensity from entire wells of AhR-sufficient and AhR-deficient mTEC cultures at day 9 of ALI. Fluorescence intensity of Ahr+/+ condition was set as 100%. n=3 wells per group. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data in a–c are from five independent experiments. (d) Scanning electron microscopy (SEM) images of AhR-sufficient and AhR-deficient mTEC cultures at 9 days of ALI. Dashed lines indicate the multiciliated cell perimeter. Scale bars, 1 μm. Data representative of two independent experiments. (e) Immunofluorescent staining of acetylated α-tubulin (cilia, violet) and γ-tubulin (centrioles/basal bodies, yellow) in AhR-sufficient and AhR-deficient mTEC cultures at 9 days of ALI. Scale bars, 10 μm. Data representative of three independent experiments.
	Figure 2. AhR deficiency affects both multiciliated cell commitment and cilia organization.(a) Immunofluorescent staining of acetylated α-tubulin (cilia, violet) and Foxj1 (yellow) in wt and Ahr−/− mTEC cultures at 9 days of ALI. Scale bars, 20 μm. Dashed boxes indicate insets (i,ii). Scale bars, 5 μm (for insets i and ii). Data representative of two independent experiments. (b) Immunofluorescent staining of acetylated α-tubulin (cilia, violet) and Foxj1 (yellow) in Ahr+/+ and Ahr−/− mTEC cultures following 9 days of ALI. Caption of the z projection of a single multiciliated cell showing distribution of the acetylated α-tubulin staining in the cytoplasm. Scale bars, 2 μm. Data representative of two independent experiments. (c) Number of Foxj1+ cells per field in AhR-sufficient and AhR-deficient mTEC cultures at day 9 of ALI. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). n=5 random fields per group, data from two independent experiments. (d) Fraction of Foxj1+ cells bearing a disorganized pattern of cilia (uneven distribution on the apical surface; intracytosolic staining of acetylated α-tubulin) in Ahr+/+ and Ahr−/− mTEC cultures after 9 days of ALI. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). n=5 random fields per group, data from two independent experiments.
	Figure 3. Deletion of Ahr leads to an aberrant cilia pattern in both mouse and Xenopus laevis embryos.(a) SEM images of tracheae from Ahr+/+ and Ahr−/− E18.5 embryos. Scale bars, 5 μm. (b) Relative fraction of multiciliated cells in E18.5 embryonic tracheae as in a, classified by their ciliar pattern (normal, sparse and patchy). No significant differences were observed in the number of multiciliated cells per field between the two genotypes. Mean±s.e.m.; two-way analysis of variance (variables: genotype and cilia pattern) with Sidak's multiple comparison test. Data from two independent litters (n=5–9). (c) Multiciliated cells containing Ahr1α/β morpholinos (Ahr1α/β MO—arrowheads) in Ahr1α/β MO-injected embryos develop very few cilia, and basal bodies fail to migrate and dock at the apical surface, compared either with cells that are uninjected (arrows) or with cells in embryos injected with control MO. Scale bars, 15 μm. Bottom panels show the accumulation of acetylated α-tubulin above the apical surface of the cell in control morphants, while in Ahr1α/β morphants, microtubules amass below the apical surface. Scale bars, 5 μm. (d) Quantification of integrated fluorescence intensity of acetylated α-tubulin above the apical surface of multiciliated cells, from the embryonic skin of X. laevis injected with Ahr1α/β or control MO. Mean; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (e) Fraction of multiciliated cells, from the embryonic skin of X. laevis injected with Ahr1α/β or control MO. Mean±maximum and minimum; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. Ctrl, control; MCCs, multiciliated cells.
	Figure 4. AhR controls Ccno expression and regulates the transcriptional programme required for ciliogenesis.(a,c) mRNA expression levels of the indicated genes in Ahr+/+ and Ahr−/− mTEC cultures at indicated days of ALI. Values are normalized to Hprt1. Mean±s.e.m.; two-way analysis of variance (variables: genotype and time). Data representative of at least three independent experiments. (b) Immunoblot and densitometric analysis of AhR protein expression as compared with β-actin, in AhR-sufficient and AhR-deficient mTEC cultures at days 0 and 8 of ALI. Data representative of three independent experiments. (d) Chromatin immunoprecipitation (ChIP) analysis of AhR interaction with the Ccno promoter in wt and Ahr−/− mTEC cultures at indicated days of ALI. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (e) Luciferase reporter assay was performed in AhR-sufficient and AhR-deficient mTEC cultures transfected at 5 days of ALI and analysed 36 h post transfection. Delta Ccno promoter lacks predicted AhR response elements. Mean±s.e.m.; Student's t-test. Data representative of two independent experiments. (f) Luciferase reporter assay was performed in NIH3T3 cells. Cells were co-transfected with a plasmid encoding AhR (pCMV-Ahr) or control vector (pCMV) and a luciferase-encoding reporter gene downstream of the Ccno promoter. Cells were analysed 36 h post transfection. Delta Ccno promoter lacks predicted AhR response elements. Empty indicates the control reporter vector. Mean±s.e.m. Student's t-test. Data representative of two independent experiments. (g) mRNA expression levels of Cdc20b (normalized to Hprt1) and mir449c (normalized to U6) in Ahr+/+ and Ahr−/− mTEC cultures at indicated days of ALI. Mean±s.e.m.; two-way analysis of variance (variables: genotype and time). Data are representative of three independent experiments. FC, fold change.
	Figure 5. AhR-mediated Ccno expression is prevented by Notch signalling and requires normoxic air exposure.(a) Quantification of acetylated α-tubulin fluorescence intensity from entire wells of AhR-sufficient mTEC cultures grown for 8 days in ALI, submersion or ALI under hypoxic condition. Fluorescence intensity of ALI condition was set as 100%. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). n=3 wells per group, data from three independent experiments. (b) mRNA expression levels of Ccno in Ahr+/+ mTEC cultures grown in ALI, submersion or ALI under hypoxic condition at indicated days of treatment. Values are normalized to Hprt1. Mean±s.e.m.; two-way analysis of variance (variables: treatment and time). Data representative of three independent experiments. (c) Chromatin immunoprecipitation (ChIP) analysis of AhR interaction with the Ccno promoter in Ahr+/+ mTEC cultures grown in ALI, submersion or ALI under hypoxic condition at indicated days of treatment. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (d) Quantification of acetylated α-tubulin fluorescence intensity from entire wells of AhR-sufficient and AhR-deficient mTEC cultures at indicated days of ALI in presence or absence of DAPT. Fluorescence intensity of Ahr+/+ mTEC culture at 3 days of ALI was set as 100%. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (e) mRNA expression levels of Ccno in Ahr+/+ and Ahr−/− mTEC cultures at indicated days of ALI in presence of DAPT or vehicle control. Values are normalized to Hprt1. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (f) ChIP analysis of AhR interaction with the Ccno promoter in AhR-sufficient mTEC cultures grown in ALI or submersion in the presence of DAPT or vehicle control. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of two independent experiments. Ctrl, control.
	Figure 6. Environmental cues differentially regulate AhR transcriptional activity and can interfere with each other.(a,b) mRNA expression levels of Ccno and Cyp1a1 in wt mTEC cultures treated with vehicle or FICZ (for 2 days before ALI onset) or grown in ALI for 4 days. Values are normalized to Hprt1. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (c) mRNA expression levels of Ccno in AhR-sufficient mTEC cultures at indicated days of ALI and treated with 3-methylcholanthrene (3MC) or vehicle starting 2 days before ALI onset. Values are normalized to Hprt1. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (d) Quantification of acetylated α-tubulin fluorescence intensity from entire wells of Ahr+/+ mTEC cultures at 8 days of ALI in the presence or absence of 3MC. Fluorescence intensity of vehicle-treated control (−) was set as 100%. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). Data representative of three independent experiments. (e) Immunofluorescent staining of acetylated α-tubulin (cilia, violet) and Foxj1 (yellow) in AhR-sufficient mTEC cultures at 8 days of ALI in the presence of 3MC or vehicle. Dashed boxes indicate insets (i,ii). Scale bars, 20 μm (5 μm for insets i and ii). (f) Number of Foxj1+ cells per field in AhR-sufficient mTEC cultures at 8 days of ALI in the presence or absence of 3MC. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). n=5 random fields per group. (g) Fraction of Foxj1+ cells bearing a disorganized pattern of cilia (uneven distribution on the apical surface; intracytosolic staining of acetylated α-tubulin) in Ahr+/+ mTEC cultures at 8 days of ALI in the presence or absence of 3MC. Mean±s.e.m.; Student's t-test (unpaired, two-tailed). n=5 random fields per group. Data in e–g are representative of two independent experiments.

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