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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 17, 2020 |
| Title |
Hedgehog signaling pathway regulates gene expression profiling of epididymal principal cells through the primary cilium |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Background. Primary cilia (PC) are solitary antennae present at the cell surface. These non-motile cilia play an important role in organ development and tissue homeostasis through the transduction of the Hedgehog (Hh) signaling pathway. We recently revealed the presence of PC in the epithelium of the developing epididymis, an organ of the male reproductive system whose dysfunction triggers male infertility. Acknowledging that systemic blockade of the Hh pathway trigger epididymal dysfunctions in vivo, our main goals were 1) to portray the epididymal Hh environment, 2) to determine the direct responsiveness of epididymal epithelial cells to Hh, and 3) to define the contribution of PC to the transduction of this pathway. Results. The Hh ligands Indian and Sonic hedgehog (Ihh and Shh) were respectively located in principal and clear cells of the mouse epididymis by immunofluorescent staining. The propensity of epididymal principal cells to respond to Hh signaling was assessed on immortalized epididymal DC2 cells by western-blot, confocal imaging and 3D-reconstruction. Our results indicate that epididymal principal cells secrete Ihh and expose PC that co-localize with the conventional acetylated tubulin/Arl13b ciliary markers, as well as with GLI3 Hh signaling factor. Gene expression microarray profiling indicated that the expression of 43 and 248 genes was respectively and significantly modified following pharmacological treatment of DC2 cells with the Hh agonist SAG (250 nM) or the Hh antagonist cyclopamine (20 µM) compared with the control. Among Hh target genes identified, 6.7 % presented perfect matches for GLI-transcription factor consensus sequences, and the majority belonged to interferon-dependent immune response and lipocalin 2 pathways. Finally, the contribution of epididymal PC to the transduction of canonical Hh pathway was validated by ciliobrevinD treatment, which induced a significant decrease of PC length and the expressional reduction of Hh signalling targets. Conclusions. All together our data indicate that PC from epithelial principal cells regulate gene expression profile through a possible autocrine Hh signaling. This provides new hypotheses regarding the potential contribution of PC and Hh signaling in intercellular cross-talk and immunological regulation of the epididymis.
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| Overall design |
Immortalized DC2 cells were treated for 24 hrs with 250 nM of Smoothened agonist (SAG, n=3, SAG 1, SAG 2 and SAG 3), 20 uM of Cyclopamine (Cyclo, n=3, Cyclo 1, Cyclo 2 and Cyclo 3) and control medium condition (Ctrl, n=3, Ctrl 1, Ctrl 2 and Ctrl 3). Total RNA extraction was performed with the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol, and potential genomic DNA contamination was eliminated with the RNase-free DNase set (Qiagen). Total RNA was eluted in RNAse-free water and its concentration was quantified with a NanoDrop 1000 microvolume spectrophotometer (Thermo Scientific). Ribonucleic acid quality was assessed with the Eukaryote Total RNA Pico Series II kit (Agilent) on a 2100 Bioanalyzer by the transcriptomic core facility of the CHUQ. The minimal accepted number for RNA integrity was 7. DNA microarray analyses were carried out with Affymetrix Mouse Clariom S arrays (Thermofisher) according to the Affymetrix standard protocol. Briefly, total RNA (100 ng per sample) was labeled using the GeneChip® WT Plus Reagent Kit protocol and hybridized to the arrays as described by the manufacturer (Affymetrix, Thermofisher). The cRNA hybridization cocktail was incubated overnight at 45°C while rotating in a hybridization oven. After 16 h of hybridization, the cocktail was removed and the arrays were washed and stained in an Affymetrix GeneChip fluidics station 450, according to the Affymetrix-recommended protocol. The arrays were scanned using the Affymetrix GCS 3000 7G and the Gene-Chip Command Console Software (AGCC) (Affymetrix, Thermofisher) to produce the probe cell intensity data (CEL). The image data were analyzed by using the Affymetrix Expression Console Software to perform the quality control, the background subtraction and the normalization of probe set intensities with the method of Robust Multiarray Analysis (RMA). Microarray analyses were performed by the CHU de Québec Research Center (CHUL) Gene Expression Platform, Quebec, Canada.
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| Contributor(s) |
Belleanée C, Girardet L, Bernet A, Soule D, Beauparlant CJ, Droit A, Cyr D |
| Citation(s) |
32283570 |
| Submission date |
Jan 16, 2020 |
| Last update date |
Apr 19, 2020 |
| Contact name |
Ezequiel L Calvo |
| E-mail(s) |
cezequiel@yahoo.com
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| Organization name |
CRCHUL
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| Department |
Molecular Endocrinilogy
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| Lab |
Microarrays
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| Street address |
2705 Boul. Laurier
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| City |
Quebec |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 4G2 |
| Country |
Canada |
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| Platforms (1) |
| GPL23038 |
[Clariom_S_Mouse] Affymetrix Clariom S Assay, Mouse (Includes Pico Assay) |
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| Samples (9)
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| Relations |
| BioProject |
PRJNA601778 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE143829_RAW.tar |
10.6 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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