We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.
Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.
Traditionally embryonic skin has been cultured by dissecting from the mouse embryo and mounting on a polycarbonate Nuclepore membrane. The membrane is then floated on culture medium, thus maintaining an air-liquid interface across the surface of the developing tissue3,4. This technique has been used to assay melanoblast behavior by fixing the tissue and assessing melanoblast distribution using β-galactosidase as a marker7. We have developed a method that allows live-imaging of fluorescently labeled melanoblasts in ex vivo skin culture6. Here we describe the method in detail from dissection, to setup, to live confocal imaging and include some recent improvements.
Melanoblasts are the embryonic precursors of melanocytes, the cells that produce pigment in hair and skin. Melanoblasts arise in the neural crest adjacent to the neural tube at around embryonic day 9 (E9) in the developing mouse embryo. Subsequently they migrate along a dorsolateral pathway between the ectoderm and the developing somites. At E12.5 they move from the dermis to the epidermis where they proliferate and continue their migration. The primary hair follicle pattern begins to form in the epidermis at E14.5 and by E15.5 melanoblasts are localizing to these follicles. For a review of melanoblast/melanocyte development see Thomas & Erickson (2008)1. In order to label the melanoblast population we have combined Tyr::CreB animals that express Cre-recombinase driven by the mouse tyrosinase promoter8 with R26YFPR animals that express yellow fluorescent protein (YFP) conditionally from the ROSA26 locus9.
We describe a method to culture embryonic skin and capture images using an inverted confocal microscope. It is adapted form the original method described in Mort et al. (2010)6. The present method allows imaging in a 6-well format and removes the reliance on Nuclepore membranes and on matrigel to support the culture. Instead using a small block of 1% agarose to stabilize the embryonic skin. Removing the reliance on matrigel is important especially in situations where the response of the embryonic skin to soluble growth factors is the focus of study. Our original method has already been used to gain new insights into melanoblast development10-13 and we anticipate that the improvements we describe here will make it more powerful as an experimental technique especially where multiple parallel cultures are a requirement.
All animal work was performed in accordance with institutional guidelines under license by the UK Home Office (project license number PPL 60/3785 and 60/4424).
1. Preparation
2. Procedure
3. Live Imaging
Figure 2 shows representative results from a time lapse experiment using embryonic skin from Tyr::CreB x R26YFPR embryos at E14.5. 6 embryonic skin cultures were imaged by confocal microscopy every 2 min for 18 hr. The freeware image analysis software package ImageJ was used to analyze the behavior of the YFP-labeled melanoblasts in the 6 movies. The melanoblasts were automatically tracked using the wrMTrck plugin developed by Jesper Søndergaard Pedersen (http://www.phage.dk/plugins/wrmtrck.html) the tracking results are displayed in Figure 2. Movies 1-3 are representative of the experiment and correspond to panels A and G. They show the Transmitted light, YFP and composite views respectively. The migrating melanoblast population is extraordinarily active. Cells move almost constantly and only pause when they are about to divide by mitosis. It is also possible to observe the development of the primary hair follicle pattern over the culture period in the transmitted light channel (Figures 3A-3X and Movie 1).
Figure 1. Equipment and setup. A: The live imaging chamber base consists of a clear perspex housing with identical outside dimensions to a 6-well culture dish. It contains six holes in which the 6 individual chamber assemblies can slot. B: Each insert consists of 4 components; an imaging clip, an imaging insert, a lummox membrane, and an O-ring. The clip and the insert are lathed from black acetyl plastic, we found this to have the lowest autofluorescence of the plastics we tested. The O-ring is made from PVC and is used to clamp the lummox membrane onto the bottom of the insert forming the air liquid interface (ALI). The imaging clip (black acetyl) has several holes to allow circulation of culture medium. C: The central shaft of the clip is filled with 1% agarose before use to provide a firm flat surface on which to mount the skin. The skin is attached to the clip with suture thread before being pushed into the insert, sandwiching the skin between the agarose and lummox membrane. The insert is then filled with culture medium. D: To dissect and manipulate the embryonic skin a single tooth brush bristle is mounted into a slit in the flat end of a bamboo kebab skewer. A pair of hair sticks can be used in combination with fine forceps to dissect embryonic skin and mount in the imaging chamber. E: Photographs of the components detailed in A-C. Please click here to view a larger version of this figure.
Figure 2. Representative results of 6 movies recorded in parallel. A-F: Tracking results from 6 movies recorded in parallel to demonstrate the capabilities of the system. Each movie was recorded at 2-min time points for a total of 18 hr (see Movies 1-3 for the corresponding movie of panels A and G). The positions of the cells in frame one of the movie are shown and the tracking results are superimposed on top. G-L: Summary of the tracking results plotted as zeroed tracking data.
Figure 3. Representative images from time-lapse movies of hair follicle development and of a melanoblast undergoing mitosis. A-X: Selected, cropped frames from Movie 1. Primary hair follicles become visible over the period of skin culture from 0-18 hr. An LUT was applied to the images. A'-X': Selected, cropped frames from Movie 2. Representative images of melanoblasts migrating in the developing epidermis and an individual cell (see arrow in A') undergoing mitosis (arrow in T'). Please click here to view a larger version of this figure.
Movie 1: Representative result of the transmitted light channel from an 18-hr time-lapse experiment. The primary hair-follicle pattern, although not evident at the start of the movie, becomes more prominent as the culture progresses.
Movie 2: Representative result of the YFP channel from an 18-hr time-lapse experiment. The melanoblast population is a highly dynamic and migratory population. Rare stationary cells are usually undergoing mitosis (see Figure 3).
Movie 3: Representative result of the composite channel from an 18-hr time-lapse experiment. The melanoblast population can be seen to migrate within the developing epidermis and interact with the developing hair follicles.
We describe a method to culture embryonic skin that is particularity amenable to live-cell imaging on inverted confocal microscopes. The method includes recent improvements that allow 6 cultures to be imaged in parallel and removes the reliance on matrigel and Nuclepore membranes of the original method6. The crucial technical difference from similar techniques is the use of a gas-permeable lummox membrane to establish an air liquid interface and also to act as a coverslip. We have successfully maintained the cultures with continuous confocal imaging for up to 48 hr. The high resolution and excellent signal to noise ratio of the confocal images allows automated cell tracking to analyze melanoblast behavior in the resulting time-lapse movies.
Using this technique it is possible to investigate the dynamics of melanoblast dispersal and study their interaction with the epidermis and the developing hair follicles. The critical steps in the protocol are to carry out a careful dissection that does not damage the developing epidermis and to mount the skin samples dermal side against the agarose without kinks or stretches. The procedure described in this paper works best between E13.5 and E15.5. Before E13.5 the skin is very fragile and after E15.5 it is relatively thick and hard to penetrate by confocal microscopy. The capabilities of the microscope system used are also very important. We typically use a Nikon A1R confocal microscope equipped with Nikon's proprietary perfect focus system (PFS). Using PFS greatly reduces the number of bad movies caused by stage drift, similar systems are available for other platforms.
One disadvantage of this technique is the requirement for specialized culture equipment. However the design is simple and the chamber could be easily assembled by any small technical workshop. In summary we present a simple method that should be of broad use for studies of embryonic skin development and melanoblast behavior. It is anticipated that the technique could also be modified for other scenarios where an air-liquid interface is a requirement for successful culture.
The authors have nothing to disclose.
The work was supported by core funding from the Medical Research Council and from Medical Research Scotland, grant award 436_FRG_L_1006. We are grateful to Craig Nicol for preparing technical drawings. We are grateful to Matthew Pearson and Paul Perry for their imaging support.
DMEM high glucose | Biochrom AG | F0475 | without phenol red |
Penicillin | Sigma | P3032 | |
Streptomycin | Sigma | S9137 | |
Fetal calf serum | Hyclone | SV30160.03 | |
Glutamax | Gibco | 35050-038 | |
Ethanol | Generic | ||
Live imaging chamber | Custom made | ||
Lummox dishes | Sarstedt | 94.6077.410 | |
6-well plate | Greiner Bio-One | 657-160 | |
Single edged razor blade | Fisher Scientific | 1244-3170 | |
Agarose | Biogene | 300-300 | |
Fine pastette | Generic | ||
PBS | Generic | ||
Kebab skewers | Waitrose | Bamboo BBQ skewers 30cm | |
Toothbrush | Generic | ||
Petri dishes | Greiner Bio-One | 633185 | |
Suture thread | Look | SP115 | Black silk suture thread |