Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Summary

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.

Abstract

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 follicles^1,2. 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 culture^3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue^3,6. 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.

Introduction

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 tissue^3,4. 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 imag....

Protocol

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

1. Label the melanoblasts in the developing skin by combining an appropriate Cre-recombinase expressing mouse line with a fluorescent reporter mouse line. Tyr::CreA, Tyr::CreB⁸ and Wnt1::Cre¹⁴ have been used successfully as Cre-expressing lines and R26YFPR⁹ as a reporter line.
2. Prepare the culture medium in a laminar flow hood; supplement Dulbecco's Modified Eagle Medium (DMEM) w....

Results

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 (

Discussion

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 c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 30 cm
Toothbrush	Generic
Petri dishes	Greiner Bio-One	633185
Suture thread	Look	SP115	Black silk suture thread