Computational protocol: Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development

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Protocol publication

[…] Slides were imaged using a Leica DM6000 slide microscope and either 10x, 20x, or 40x objectives. Confocal microscopy was performed using a Leica SP5 DM and either 20x or 40x objectives. The quantification of Merkel cells per mm of skin was performed as described []. Briefly, the length of each section was measured and the number of positively stained cells was counted. Typical section lengths were between 7–14 mm. We counted a large number of Merkel cells in the controls conditions (>300 Krt8(+) cells) and then counted the number of Merkel cells in a similar length of skin for the each mutant line. Typically, at least 100 mm of skin were counted for each condition. The quantification of Merkel cell clusters was done using Leica LAS AF software; tile scan images of the dorsal skin where acquired and the number of clusters of ≥3 Krt8(+) cells per mm2 was quantified for different areas of the dorsal skin of at least 3 different individuals. Merkel cells in glabrous paw skins were quantified using Leica LAS AF software; 10x tile scan images of sections of the paws where acquired. The number of Merkel cells was quantified on the zoomed-in images and the length of the Krt14(+) epidermis was measured on the large-scale tiled images, using the Leica LAS AF software and ImageJ software. PH3(+) cells were quantified using a fluorescence microscope. The length of each section was measured and the number of positively stained cells was counted. Whenever it was ambiguous whether a PH3(+) cells was in the epidermis, an image with Krt14 counter staining was acquired. For the Shh overexpression experiment, PH3(+) cells where quantified using Leica LAS AF software; the number of PH3(+) cells were quantified on images and the area of Krt14(+) epidermis was measured using ImageJ software. BrdU(+) cells where quantified using Leica LAS AF software; nuclear DAPI staining was used to quantify the total number of cells and the % of BrdU(+) cells was calculated. Gli1(+) cells were quantified in the same manner. Fluorescence intensity was calculated from at least three raw, single-channel greyscale images per condition using Leica LAS AF software. Staining in different skin cell populations was compared to background levels of fluorescence, which were measured as non-nuclear areas of the skin and areas outside of the skin in the same image. [...] RNA samples were amplified and labeled using the Low Input Quick Amp Labeling Kit (Agilent Technologies, USA) according to manufacturer's instructions. In brief, 100ng of total RNA from each sample was used to prepare Cyanine-3 (Cy3)-labeled cRNA for hybridization. The Universal Mouse Reference RNA (Agilent Technologies) was Cyanine-5 (Cy5)-labeled and used as an internal control, with dye swaps between samples and reference. The RNA Spike-In kit (Agilent Technologies) was used as an external control to monitor the microarray workflow and accuracy.For microarray hybridization, 300ng of labeled sample was fragmented and hybridized against 300ng of labeled Universal Mouse Reference RNA on SurePrint G3 Mouse GE 8X60K microarrays (Agilent) for 17 hours at 65°C in a rotating hybridization oven (Agilent). Following hybridization, microarrays were washed with GE wash buffer 1 (Agilent) for 1 min at room temperature and with GE wash buffer 2 (Agilent) for 1 min at 37°C. Microarrays were scanned using a SureScan Microarray Scanner (Agilent Technologies). The scanned images were analyzed with Agilent Feature Extraction v12.0.1.1 and GeneSpring v13.0 software (Agilent Technologies). Statistical analysis was carried out using an unpaired t-test and genes with a p-value <0.05 and an absolute fold change ≥2 were considered significantly differentially expressed.For the construction of the heatmaps, Log2 normalized expression values generated by the GeneSpring software were used for control and EED cKO samples. Fold changes were then generated between the control and EED cKO average expression levels to demonstrate differences in the expression values between the two conditions. Known genes of signaling pathways involved in hair follicle morphogenesis and skin development (Wnt, Shh, FGF, BMP, and Notch signaling) [], as well as Merkel cell-specific genes were mined from the KEGG database. Expression values for these genes were selected from the data and used for the construction of the heatmaps. Individual heatmaps were built with the R package ggplot2.The microarray data for this publication were deposited in NCBI’s Gene Expression Omnibus [] and can be viewed using GEO Series accession number GSE83244 ( […]

Pipeline specifications

Software tools ImageJ, Agilent Feature Extraction, GeneSpring GX, Ggplot2
Databases GEO
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens