Journal: PLoS Genetics

Article Title: Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

doi: 10.1371/journal.pgen.1005627

Figure Lengend Snippet: (A) Limb bud sections (left) from E11.5 embryos were stained for acetylated tubulin (Tub Ac , green) to mark the ciliary axonemes, γ-tubulin (red) to mark the basal bodies and DAPI (blue) to mark nuclei. Scale bar, 5 μm. Transmission electron microscopy (TEM, right) for each genotype. Scale bar, 200 nm. (B) Quantification of limb bud cilia in control, Tctn1 -/- Nphp4 n/+ , and Tctn1 -/- Nphp4 n/n mutants from TEM fields of view. Error bars represent the standard error of the mean. Statistical significance according to unpaired Student’s t -tests (* p <0.05; ** p <0.001). (C) Fibroblasts derived from E13.5 limb buds of the indicated genotypes stained for Tub Ac (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). White arrowheads indicate cilia in Tctn1 -/- Nphp4 n/+ and Tctn1 -/- Nphp4 n/n mutants. Scale bar, 10 μm. (D) Quantification of proportion of ciliated cultured limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s t -tests (* p <0.05; ** p <0.0001). (E) Immunostaining of limb bud fibroblasts for Nphp1 (red), Tub Ac (green), and γ-tubulin (cyan). Scale bar, 1 μm.

Article Snippet: Antibodies used were: acetylated tubulin (clone 6-11B-1; Sigma), γ-tubulin (C-20; Santa Cruz), Arl13b (17711-1-AP; Proteintech), Nphp1 (gift from Dr. Greg Pazour), Smoothened (ab38686; Abcam) and Gpr161 (13398-1-AP; Proteintech).

Techniques: Staining, Transmission Assay, Electron Microscopy, Control, Derivative Assay, Cell Culture, Standard Deviation, Immunostaining

Journal: PLoS Genetics

Article Title: Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

doi: 10.1371/journal.pgen.1005627

Figure Lengend Snippet: (A) Lateral views of wild type, Tctn1 -/- , Bbs1 -/- and Tctn1 -/- Bbs1 -/- embryos at E14.5. Exencephaly is apparent in the Tctn1 -/- Bbs1 -/- double mutant. Alcian blue staining of the corresponding genotype’s forelimb (top) and hindlimb (bottom) are included, with asterisks indicating extra digits. Scale bars, 1 mm. (B-D) Number of digits in the (B) forelimbs and (C) hindlimbs, and (D) the incidence of exencephaly for the indicated genotypes. Numbers of animals analyzed for polydactyly and exencephaly are shown in and Tables. (E) Limb bud fibroblasts of the indicated genotypes immunostained for Tub Ac (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). Scale bar, 10 μm. (F) Quantification of proportion of ciliated limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s t -tests (* p <0.0001). (G) Limb bud fibroblasts immunostained for Nphp1 (red), Tub Ac (green), and γ-tubulin (blue). Scale bar, 1 μm.

Article Snippet: Antibodies used were: acetylated tubulin (clone 6-11B-1; Sigma), γ-tubulin (C-20; Santa Cruz), Arl13b (17711-1-AP; Proteintech), Nphp1 (gift from Dr. Greg Pazour), Smoothened (ab38686; Abcam) and Gpr161 (13398-1-AP; Proteintech).

Techniques: Mutagenesis, Staining, Standard Deviation

Journal: Science Advances

Article Title: Primary cilium remodeling mediates a cell signaling switch in differentiating neurons

doi: 10.1126/sciadv.abb0601

Figure Lengend Snippet: ( A ) Immunostaining to detect endogenous IFT88 (red), Arl13b (blue), and Tuj-1 (green). Cyan dashed lines demarcate differentiating cells, and boxed regions are enlarged in right-hand panels followed by a cartoon of representative IFT88 localization (BB, basal body). Primary cilium is highlighted by white dashed lines. Top right panels show primary cilium in cell body of Tuj1 + cell extending axon in the basal region of the spinal cord. Bottom right panel shows quantification of percentage of cells with detectable IFT88 in ciliary tip (present) at different stages of apical process retraction and axonogenesis. Scale bars, 5 and 0.7 μm (enlarged region). ( B ) Immunofluorescence and Airyscan enhanced-resolution microscopy of misexpressed PACT-TagRFP (red) and endogenous IFT88 (white). ( C ) Airyscan 3D reconstruction of primary cilia (red) at different stages of apical process retraction (stages are summarized in the diagram on the left; movies S4 to S7). Cyan dashed lines demarcate the tip of apical process, red dashed lines demarcate the centrosome, and white dashed lines demarcate the remodeled cilium. Scale bar, 1 μm. ( D ) Time-lapse sequence of differentiating neuron misexpressing IFT88-mNeonGreen undergoing apical process retraction (movie S8) and axonogenesis (movie S9). Cyan dashed lines outline differentiating cell. Enlarged regions indicate representative still images from time-lapse sequences. Blue arrows indicate IFT88 in reassembled primary cilium. Scale bars, 5 μm (process retraction), 10 μm (axonogenesis), and 0.7 μm (enlarged regions).

Article Snippet: Primary antibodies were used at the following dilutions: Tuj1 (80120, BioLegend) 1:1000, Arl13b (17711-1-AP, Proteintech), Smo (20787-1-AP, Proteintech) 1:50, IFT88 (13967-1-AP, Proteintech) 1:200, and GPR161(13398-1-AP, Proteintech).

Techniques: Immunostaining, Immunofluorescence, Microscopy, Sequencing

Journal: PLoS Genetics

Article Title: Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

doi: 10.1371/journal.pgen.1005627

Figure Lengend Snippet: (A) Limb bud sections (left) from E11.5 embryos were stained for acetylated tubulin (Tub Ac , green) to mark the ciliary axonemes, γ-tubulin (red) to mark the basal bodies and DAPI (blue) to mark nuclei. Scale bar, 5 μm. Transmission electron microscopy (TEM, right) for each genotype. Scale bar, 200 nm. (B) Quantification of limb bud cilia in control, Tctn1 -/- Nphp4 n/+ , and Tctn1 -/- Nphp4 n/n mutants from TEM fields of view. Error bars represent the standard error of the mean. Statistical significance according to unpaired Student’s t -tests (* p <0.05; ** p <0.001). (C) Fibroblasts derived from E13.5 limb buds of the indicated genotypes stained for Tub Ac (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). White arrowheads indicate cilia in Tctn1 -/- Nphp4 n/+ and Tctn1 -/- Nphp4 n/n mutants. Scale bar, 10 μm. (D) Quantification of proportion of ciliated cultured limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s t -tests (* p <0.05; ** p <0.0001). (E) Immunostaining of limb bud fibroblasts for Nphp1 (red), Tub Ac (green), and γ-tubulin (cyan). Scale bar, 1 μm.

Article Snippet: Antibodies used were: acetylated tubulin (clone 6-11B-1; Sigma), γ-tubulin (C-20; Santa Cruz), Arl13b (17711-1-AP; Proteintech), Nphp1 (gift from Dr. Greg Pazour), Smoothened (ab38686; Abcam) and Gpr161 (13398-1-AP; Proteintech).

Techniques: Staining, Transmission Assay, Electron Microscopy, Control, Derivative Assay, Cell Culture, Standard Deviation, Immunostaining

Journal: PLoS Genetics

Article Title: Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

doi: 10.1371/journal.pgen.1005627

Figure Lengend Snippet: (A and B). mRNA levels of Gli1 and Ptch1 normalized to β-actin in forelimb bud cells treated with DMSO or SAG. Error bars represent standard deviations. Statistical significance according to unpaired Student’s t -tests (* p <0.05). (C) Limb bud fibroblasts treated with DMSO or SAG, then immunostained for Smo (red), Tub Ac (blue), and γ-tubulin (green). (D) Quantitation of Smo ciliary intensity in DMSO or SAG treated limb bud fibroblasts. (E) Limb bud fibroblasts treated with DMSO or SAG, then immunostained for Gpr161 (red), Tub Ac (blue), and γ-tubulin (green). (F) Quantitation of Gpr161 ciliary intensity in DMSO or SAG treated limb bud fibroblasts. Error bars represent standard error of the mean. Statistical significance according to unpaired Student’s t -tests (* p <0.05). Scale bars, 2 μm.

Article Snippet: Antibodies used were: acetylated tubulin (clone 6-11B-1; Sigma), γ-tubulin (C-20; Santa Cruz), Arl13b (17711-1-AP; Proteintech), Nphp1 (gift from Dr. Greg Pazour), Smoothened (ab38686; Abcam) and Gpr161 (13398-1-AP; Proteintech).

Techniques: Quantitation Assay

Journal: PLoS Genetics

Article Title: Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

doi: 10.1371/journal.pgen.1005627

Figure Lengend Snippet: (A) Lateral views of wild type, Tctn1 -/- , Bbs1 -/- and Tctn1 -/- Bbs1 -/- embryos at E14.5. Exencephaly is apparent in the Tctn1 -/- Bbs1 -/- double mutant. Alcian blue staining of the corresponding genotype’s forelimb (top) and hindlimb (bottom) are included, with asterisks indicating extra digits. Scale bars, 1 mm. (B-D) Number of digits in the (B) forelimbs and (C) hindlimbs, and (D) the incidence of exencephaly for the indicated genotypes. Numbers of animals analyzed for polydactyly and exencephaly are shown in and Tables. (E) Limb bud fibroblasts of the indicated genotypes immunostained for Tub Ac (green), Arl13b (red), γ-tubulin (cyan), and DAPI (blue). Scale bar, 10 μm. (F) Quantification of proportion of ciliated limb bud fibroblasts. Error bars represent the standard deviation. Statistical significance according to unpaired Student’s t -tests (* p <0.0001). (G) Limb bud fibroblasts immunostained for Nphp1 (red), Tub Ac (green), and γ-tubulin (blue). Scale bar, 1 μm.

Article Snippet: Antibodies used were: acetylated tubulin (clone 6-11B-1; Sigma), γ-tubulin (C-20; Santa Cruz), Arl13b (17711-1-AP; Proteintech), Nphp1 (gift from Dr. Greg Pazour), Smoothened (ab38686; Abcam) and Gpr161 (13398-1-AP; Proteintech).

Techniques: Mutagenesis, Staining, Standard Deviation

Journal: PLoS Genetics

Article Title: Conserved Genetic Interactions between Ciliopathy Complexes Cooperatively Support Ciliogenesis and Ciliary Signaling

doi: 10.1371/journal.pgen.1005627

Figure Lengend Snippet: (A and B) mRNA levels of Gli1 and Ptch1 normalized to β-actin in forelimb bud cells treated with DMSO or SAG. Error bars represent the standard deviations. Statistical significance according to unpaired Student’s t -tests (* p <0.05). (C) Limb bud fibroblasts treated with DMSO or SAG, then immunostained for Smo (red), Tub Ac (blue), and γ-tubulin (green). (D) Quantitation of Smo ciliary intensity in DMSO or SAG treated limb bud fibroblasts. * p <0.05 in unpaired Student’s t -tests. (E) Limb bud fibroblasts treated with DMSO or SAG, then immunostained for Gpr161 (red), Tub Ac (blue), and γ-tubulin (green). (F) Quantitation of Gpr161 ciliary intensity in DMSO or SAG treated limb bud fibroblasts. * p <0.01 and **p<0.001 in unpaired Student’s t -tests with respect to Tctn1 +/- Bbs1 +/- mutant fibroblasts treated with DMSO. Error bars represent standard error of the mean. Scale bars, 2 μm.

Article Snippet: Antibodies used were: acetylated tubulin (clone 6-11B-1; Sigma), γ-tubulin (C-20; Santa Cruz), Arl13b (17711-1-AP; Proteintech), Nphp1 (gift from Dr. Greg Pazour), Smoothened (ab38686; Abcam) and Gpr161 (13398-1-AP; Proteintech).

Techniques: Quantitation Assay, Mutagenesis