CD1 mice from Charles River labs were used for all experiments. Notch Reporter transgenics46
were obtained from Jackson laboratories (strain Tg(Cp-EGFP)25Gaia/J, stock #005854) and outbred over multiple generations to the CD1 background, where they were maintained as homozygotes. Homozygous Lox-stop-Lox-RosaNICD-IRES-GFP
male breeder mice47
were obtained from Jackson laboratories (strain Gt(ROSA)26Sortm1(Notch1)Dam/J, stock #008159) and crossed to CD1 females when used for lentiviral injections. RBPJfl/fl
were bred as homozygotes for lentiviral injections, or crossed to K14-Cre49
females for analyses of the conditional mutant phenotype. BrdU (50 µg/g) was injected intraperitoneally to pregnant females 4–6h before sacrificing by administration of CO2
. All animals were maintained in an AAALAC-approved animal facility and procedures were performed using IACUC-approved protocols.
Constructs and RNAi
All shRNAs except shDctn1-1721
were obtained from The Broad Institute’s Mission TRC-1 mouse library, and were present in the pLKO.1 lentiviral backbone, which harbours a puromycin-resistance cassette. shRNA sequences were cloned from the library vectors into our modified pLKO H2B-mRFP1, H2B-YFP, or H2B-CFP vectors.19
The lentiviral Notch reporter was generated by cloning a KpnI-XbaI fragment containing the 4 CBF1 binding elements, SV40 minimal promoter, and EGFP from Addgene clone 1770546
into pLKO shScramble
. For expression of EYFP-mInsc, mRFP1-Numb, and mRFP1-LGN (FL and ΔC), the pLKO backbone was modified to delete the puromycin-resistance gene following the PGK promoter, and replace it with a custom multiple cloning site (pLKO PGK MCS). This facilitated subsequent cloning and allowed the use of a single lentivirus that could both express a cDNA of interest and an shRNA. The 72kD Numb isoform was cloned from mouse cDNA by PCR and fused to mRFP1, while EYFP-mInsc was reported previously.7
LGNΔC was produced according to the published mutant,21
which lacks exons 13, 14, and the coding region of the last exon 15. This truncates the protein at aa474, deleting the GoLoco motifs that mediate LGN’s interaction with Gαi/Gαo. It was also empirically found to delete the epitope for our LGN antibody (see below), which was raised to the C-terminus.
Viruses were produced as described19
. The following shRNAs were used: shLGN-1617
(Sigma SHC002). Detailed maps and constructs are available upon request. Full hairpin sequences (minus AgeI and EcoRI cloning sites) are listed below:
Primary mouse keratinocytes were maintained in E medium with 15% FBS and 50 µM CaCl2 (low Ca2+ medium). For viral infections, keratinocytes were plated in 6-well dishes at 100,000 cells per well and incubated with lentivirus in the presence of polybrene (100 µg/mL). After 2d, we positively selected infected cells with puromycin (1–2 µg/mL) for 4–7 d, and processed them for mRNA and protein analyses. Calcium shift assays were performed as follows. Keratinocytes were seeded at a saturating density (200,000 cells/24-well) onto coverslips coated with collagen and fibronectin. Cells were switched to high Ca2+ (1.5 mM) medium 16–24 h later, and grown for the indicated period of time (24–72 h). Cells were fixed with 4% paraformaldehyde for 10 min at RT. Immunostaining was performed using the same protocol as for slides (below). As differentiation is sensitive to cell density, nuclei/field were quantifed using Metamorph and only images with between 1800–2000 nuclei/10× field were quantified for K10 expression.
Antibodies, Immunohistochemistry and Imaging
Antisera against LGN were raised in guinea pigs using the C-terminus (aa 376–572) of LGN fused to GST, and were affinity purified using HiTrap NHS columns conjugated to purified immunogen protein (Pierce). Embryos were either embedded whole (<E16.5) or skinned and flat-mounted on paper towels. Both infected and littermate controls were embedded together in a single block to control for potential variability in immunostaining conditions. Embryos and skin were embedded unfixed in OCT (Tissue Tek), except for Notch reporter sections, which were prefixed for 1h in 4% paraformaldehyde in order to preserve the cytoplasmic GFP signal. To detect the reporter, antibodies against GFP were used, coupled to either fluorescent secondary antibodies or POD-conjugated secondaries which were amplified using the TSA Plus fluorescein or Cy5 system (Perkin Elmer). For BrdU immunostaining, slides were treated with 1N HCl for 1 h at 37°C before adding the anti-BrdU antibody.
Frozen sections were cut at a thickness of 8–10 µm on a Leica cryostat and mounted on SuperFrost Plus slides (Fisher). Slides were air-dried for 30 minutes, then fixed for 10 minutes with 4% paraformaldehyde, rinsed with PBS, then blocked for 1 h in gelatin block (5% NDS, 1% BSA, 2% gelatin, 0.2% triton-X in PBS) or BSA/NDS block (3% BSA, 5% NDS, 0.2% triton-X in PBS) before incubating in primary antibody diluted in block at 4°C overnight. After washing with PBS, secondary antibodies, conjugated to Alexa-488 (Molecular Probes), Cy3, RRX, DyLight 549, or Cy5 (Jackson Laboratories), were added for 1–2 h at RT. Slides were washed, counterstained with DAPI (0.5 µg/mL) and mounted in ProLong Gold (Invitrogen). Imaging was performed on a Zeiss Axioplan 2 using 10×/0.45 air, 20×/0.8 air, 63×/1.4 oil, or 100×/1.4 oil Plan-Apochromat objectives and the following Chroma filter sets: 49003 ET YFP (YFP), 49008 ET TR C94094 (mRFP1), 49004 ET dsR C94093 (Cy3, DyLight 549), 41008 Cy5 (Cy5), 41001 FITC (AlexaFluor 488/GFP); or a Zeiss LSM 510 Meta scanning confocal microscope with 40×/1.2 air or 63×/1.4 oil objective.
The following primary antibodies were used: Mouse IgM anti-NuMA (BD Biosciences, 1:200), Rb anti-pericentrin (Covance, 1:500), Rb anti-Gαi3 977 (gift of T. Gettys, 1:400), Rb anti-RFP (MBL, 1:4000), Rb mAb anti-survivin (Cell Signaling, 1:400), GP anti-K5 (Fuchs lab, 1:200), Rt mAb anti-Ecad (Fuchs lab, 1:500), Rb anti-K10 (Covance, 1:1000), Rb anti-K14 (Fuchs lab, 1:500), Rt anti-CD104/β4 integrin (BD Pharmingen), Rb anti-loricrin (Fuchs lab, 1:1000), Rb anti-involucrin (Covance, 1:1000), Rb anti-filaggrin (Covance, 1:1000), Rb anti-Hes1 (Fuchs lab, 1:500), Chicken anti-GFP (Abcam, 1:5000), Rb anti-GFP (Invitrogen, 1:5000), Hamster mAb anti-Notch3 (Biolegend, 1:400), Rb anti-Notch3/NICD3 (Abcam ab23426, 1:400), Mouse mAb anti-acetylated tubulin (Sigma, 6-11B-1, 1:400), Rb anti-γtubulin (Abcam, 1:500), Mouse mAB anti-βtubulin (Sigma, TUB 2.1, 1:500), Rb anti-AGS3 pep32 (gift of S. Lanier, 1:500), Rb anti-AGS3 pep22 (S. Lanier, 1:200), Rb anti-aPKC (Santa Cruz, N-17, 1:200), Rb anti-Par3 (Upstate/Millipore, 1:500), Rt anti-HA (Roche, 1:200), Rt anti-BrdU (Abcam, 1:200), Rb anti-RFP-HRPDirecT (MBL, 1:2000), Goat anti-Dctn1 (Abcam ab11806, 1:500).
mRNA was isolated using Absolutely RNA miniprep or microprep kits (Stratagene), and was quantified using a Nanodrop spectrophotometer. cDNA was synthesized from 2–500 ng of total RNA using either Superscript III with oligo-dT primers or Superscript VILO with random-primers (Invitrogen). Real-time qPCR was performed on a LightCycler 480 (Roche), and relative quantification performed using Roche software, with data normalized relative to cyclophilin
) and Hprt1
(using the geometric mean of the Cp values from both reference genes). To confirm the functionality of the primer sets used, multiple primer pairs were designed and tested for each gene; efficiencies of primer pairs were determined empirically (>1.8); specificity confirmed by the absence of product in samples prepared without reverse transcriptase (−RT controls), and product sizes calculated by melting curve analysis and confirmed by gel electrophoresis. The following primer sequences were used:
|Forward primers||Reverse primers|
Gel electrophoresis was performed using 4–12% NuPAGE Bis-Tris gradient gels (Invitrogen), transferred overnight at 100 mA to nitrocellulose membranes. Membranes were blocked for 1h in Odyssey blocking buffer (LI-COR), then incubated with primary antibodies in Odyssey block + 0.1% Tween-20 overnight at 4°C with gentle agitation. Membranes were rinsed several times in PBT (PBS + 0.1% Tween-20) before incubating in secondary antibodies diluted in Odyssey block for 30 minutes at RT in the dark. Membranes were washed in PBT, then in PBS before imaging on a LiCor infrared scanner. Quantification of band intensities was performed using Odyssey 3.0 software. Primary antibodies used were: GP anti-LGN (Fuchs lab, 1:2000), Rb anti-LGN (S. Bahria, 1:2000), Mouse IgG anti-βactin (Sigma, 1:5000), Rb anti-Hprt1 (Abcam, 1:2000). Secondary antibodies were conjugated to IRDye680 or IRDye800CW (LiCor and Rockland), and were used at 1:15000.
Embryos from K14H2B-GFP+/+ male × CD1 female matings, injected with lentivirus at E9.5, were collected at either E15.5 or E18.5 and processed as follows. For E15.5 embryos, back and head skin were dissected, and digested in 0.25% collagenase (Sigma) in HBSS for 1 h at 37°C with intermittent trituration and shaking. Epidermis was separated from dermal fibroblasts by filtering through a 70 µm filter and collecting the retaining epidermis. For E18.5 embryos, back and head skin were dissected and treated with dispase for 1 h at 37°C with shaking. The epidermis was peeled away from the underlying dermis using fine forceps. Isolated collagenase- or dispase-treated epidermis was then treated with 0.25% Trypsin-EDTA (Invitrogen) for 15 mins at RT with shaking. Keratinocytes were isolated by filtering through a 70µm cell strainer, retaining the flow-through cell suspension. PBS + 1% FBS (treated with BioRad Chelex to remove calcium) was added to inactivate trypsin, and cells were collected by centrifugation for 5 minutes at 300 × g. Cell pellets were resuspended in PBS + 1% FBS and stained with CD49f/α6 integrin-Alexa647 (AbD Serotec) diluted 1:50 for 30 minutes on ice. DAPI (20 ng/mL) was used for live/dead exclusion. FACS isolations were performed on a BD FacsAria 2 equipped with 355, 405, 488, 561, and 640nm lasers. Cells were gated as a6hi (basal) and a6low (suprabasal), and sorted for GFP+RFP+ (transduced, knockdown) and GFP+RFP− (internal control) populations. GFP+RFP− littermates were also sorted as controls. Sorted cells were validated by post-sort analysis on a BD LSR II; and RFP, GFP and α6 integrin expression assessed by RT-qPCR on RNA isolated from sorted populations. Sorted cells were divided for protein, RNA, and cell cycle analyses.
For cell cycle analyses, ~200,000 cells were resuspended in 150 µL of PBS, then fixed by drop-wise addition of 4 volumes of ice-cold 100% EtOH while vortexing. Cells were fixed for 15 minutes on ice, and stored at 4°C. After fixation, cells were centrifuged for 5 mins at 1000 × g, resuspended in PBS, and centrifuged again. Cells were resuspended in propidium iodide solution (10 µg/mL) with RNAse A (250 µg/mL), and stained for 15 minutes at 37°C in the dark. Cell cycle analysis was performed on a BD LSR II, and data processed and graphed using FlowJo 8.8.4.
Histology and electron microscopy analysis
Skin samples were fixed in 2% glutaraldehyde, 4% PFA, and 2 mM CaCl2 in 0.05 M sodium cacodylate buffer, pH 7.2, at RT for >1 h, postfixed in 1% osmium tetroxide and processed for Epon embedding; semi-thin sections (1 µm) were stained with toluidine blue and examined by light microscopy. For transmission electron microscopy, ultrathin sections (60–70 nm) were counterstained with uranyl acetate and lead citrate. EM images were taken with a transmission electron microscope (Tecnai G2–12; FEI) equipped with a digital camera (Model XR60; Advanced Microscopy Techniques, Corp.).
Dye exclusion assays were performed essentially as described.28
Essentially, unfixed embryos are immersed in a low pH X-gal substrate solution (100 µM NaPO4
, 1.3 mM MgCl2
, 3 mM K3
, 3 mM K4
, 1mg/mL X-gal, 0.01% sodium deoxycholate, 0.2% NP-40, pH 4.5) at 30–37°C for several hours to overnight until colour develops. Tails were snipped to serve as a positive control for staining. The principle of the assay is that at low pH, skin contains abundant β-galactosidase activity, so when the epidermis has incomplete barrier function, X-gal is cleaved and the blue precipitate is deposited.
Measurements, Quantification, Graphing, and Statistics
Spindle orientation was determined by measuring the angle between the centrosomal axis and the basement membrane in late prophase and metaphase cells, when two centrosomes were observed at opposite sides of the cell (in early prophase, the centrosomal pair is localized apically). LGN orientation was determined by measuring the angle defined by a line transecting the middle of the LGN crescent through the cell center, relative to the basement membrane. Stages of mitosis were defined as follows: early prophase cells had condensed chromatin lacking a clearly-defined pair of centrosomes; late prophase cells had a pair of centrosomes positioned at opposing poles; metaphase cells resembled late prophase cells but displayed aligned chromosomes characteristic of the metaphase plate. All cells were positive for phospho-histone H3 and LGN.
Axis of division was determined in anaphase/telophase cells, as it became obvious from analyses of metaphase spindle orientation that cells at this stage were dynamic, and spindle orientation was not necessarily predictive of the ultimate plane of division. Because phospho-histone H3 staining is weak or undetectable at this stage of the cell cycle, we utilized a novel marker to identify anaphase/telophase cells. Survivin/Birc5 is a component of the chromosomal passenger complex (CPC), together with INCENP/Aurora B kinase and Borealin/Dasra B. At prometaphase/metaphase, the CPC localizes to the inner centromeres, but at anaphase it translocates to the central spindle, and then finally to the midbody during cytokinesis. We therefore found this antibody to be an effective marker for anaphase/telophase cells, as survivin was present at the midzone between two daughter nuclei, allowing us to distinguish definitively between mitotic nuclei from a single cell and closely juxtaposed nuclei from neighbouring cells (this was confirmed secondarily by using the cell membrane marker E-cadherin). Angle of division was determined by measuring the angle defined by the plane transecting two daughter nuclei relative to the plane of the basement membrane.
Backskin thickness was quantified by taking >40 measurements/embryo of RFP+ regions from 5 random 20× fields arrayed from anterior to posterior. Epidermal thickness was measured as the distance from the basement membrane (labelled with β4 integrin) to the skin surface. Measurements of individual embryos are displayed as box and whisker plots (), with the dimensions of the box encompassing the 25–75% percentile, the horizontal bar representing the mean, and the error bars representing the minimum and maximum values. These values were normalized to the mean thickness of uninfected embryos from the same litter in order to control for subtle differences in gestational age between litters. Spinous/granular layer thickness in analyses of RBPJ mutants and NICD rescue experiments was calculated using Metamorph. A common threshold intensity was set for K10 fluorescent intensity, creating a binary image, whose area was calculated, and divided by the length of the section to determine average thickness. 10–40 sections of head and anterior backskin were quantified for each genotype, from >3 embryos. Data presented are the mean ± SEM.
Data were analyzed and statistics performed (unpaired two-tailed student’s t-tests or Chi-square tests) in Prism 5 (GraphPad). For determination of axis of cell division, the number of cells analyzed (n) is indicated in the radial histograms, and included cells from 3 or more embryos of the same age. Radial histograms of angle of division were plotted in Origin 8.1 (OriginLab) from raw data binned into 10° increments. All other graphs were prepared in Prism.