Journal: Journal of Developmental Biology

Article Title: Loss of Planar Cell Polarity Effector Fuzzy Causes Renal Hypoplasia by Disrupting Several Signaling Pathways

doi: 10.3390/jdb10010001

Figure Lengend Snippet: Gdnf/c-Ret signaling in E14.5. Fuzzy−/− embryonic kidneys. ( A ) In situ hybridization with antisense c - Ret , Gdnf and Wnt11 cDNA probes on wildtype and Fuzzy−/− sections; scale bars = 100 µM; sections from 3 embryos per genotype were analyzed. ( B ) Heatmap of 39 Ret pathway genes from the RNAseq analysis of wildtype and Fuzzy−/− kidneys, 3 kidneys per genotype were used. ( C ) Principal component analysis of the Ret pathway genes; each sample per genotype is designated as 1, 2 or 3; this numeration corresponds to the order of samples in the heatmap. ( D ) Wildtype versus Fuzzy−/− fold change in the expression of selected Ret pathway genes analyzed by RNAseq and quantitative PCR. ( E ) Validation of selected Ret pathway genes by quantitative PCR. Standard errors of mean (SEM) are shown. 3 biological samples per genotype, 3 technical replicates per sample were used; the experiments were repeated twice. * p < 0.05.

Article Snippet: The slides were hybridized at 65 °C in the hybridization buffer (7.5 mM Tris-HCl, 1 mM Tris-base, 5 mM NaH 2 PO 4 ·2H 2 O, 5 mM Na 2 HPO 4 , 5 mM EDTA, pH 8.0, 1x Denhardt’s solution, 50% deionized Formamide, 10% dextran sulfate, 1mg/ml yeast tRNA) with added DIG-labeled RNA probes (1:40,000 dilution) in a hybridization oven (Robbins Scientific ® -1000, Sunnyvale, CA, USA) overnight in a sealed humidified chamber.

Techniques: In Situ Hybridization, Expressing, Real-time Polymerase Chain Reaction, Biomarker Discovery

Journal: Journal of Developmental Biology

Article Title: Loss of Planar Cell Polarity Effector Fuzzy Causes Renal Hypoplasia by Disrupting Several Signaling Pathways

doi: 10.3390/jdb10010001

Figure Lengend Snippet: Ciliogenesis and Shh signaling in E14.5 Fuzzy−/− kidneys. ( A ) Confocal images of wildtype and Fuzzy−/− kidney sections immunostained with anti-Arl13b antibody (red) to detect primary cilium and anti- γ -tubulin antibody (light blue) to detect basal body; DBA (green) stains UB cells and DAPI (blue) stains nuclei. Intermittent white lines designate UB structures in which analyses of the ciliary length and numbers were carried out; scale bars = 25 µM. The inserts are the magnified areas within the UB tips designated by the squares. The cilia were counted only if both the Arl13b and γ-tubulin staining were detected for the same cilium; representative cilia/basal bodies are depicted by white arrows; insert scale bars = 8 µM. ( B ) Statistical analysis of ciliated cells in wildtype ( n = 16 tissue fields) and Fuzzy−/− ( n = 10 tissue fields). ( C ) Measurements of ciliary length in UB cells of wildtype (total n = 281 cilia) and Fuzzy−/− (total n = 67 cilia) were made using sections from 4 embryos per genotype. ( D ) In situ hybridization with antisense Ptch1 ( Patched1 ) and Gli1 probes on wildtype and Fuzzy−/− sections; scale bars = 100 µM. 4 WT and 3 Fuzzy−/− embryos were analyzed. **** p < 0.0001.

Article Snippet: The slides were hybridized at 65 °C in the hybridization buffer (7.5 mM Tris-HCl, 1 mM Tris-base, 5 mM NaH 2 PO 4 ·2H 2 O, 5 mM Na 2 HPO 4 , 5 mM EDTA, pH 8.0, 1x Denhardt’s solution, 50% deionized Formamide, 10% dextran sulfate, 1mg/ml yeast tRNA) with added DIG-labeled RNA probes (1:40,000 dilution) in a hybridization oven (Robbins Scientific ® -1000, Sunnyvale, CA, USA) overnight in a sealed humidified chamber.

Techniques: Staining, In Situ Hybridization