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.

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
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	<li>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 R26YFPR...

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	<li>Thomas, A. J., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+making+of+a+melanocyte:+the+specification+of+melanoblasts+from+the+neural+crest.">The making of a melanocyte: the specification of melanoblasts from the neural crest.</a> Pigment Cell &amp; Melanoma Research. 21 (6), 598-610 (2008).</li><li>Mayer, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+migratory+pathway+of+neural+crest+cells+into+the+skin+of+mouse+embryos.">The migratory pathway of neural crest cells into the skin of mouse embryos.</a> Developmental Biology. 34 (1), 39-46 (1973).</li><li>Kashiwagi, M., Huh, N., Turksen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+Culture+of+Developing+Mouse+Skin+and+Its+Application+for+Molecular+Mechanistic+Studies+of+Morphogenesis.">Organ Culture of Developing Mouse Skin and Its Application for Molecular Mechanistic Studies of Morphogenesis.</a> Epidermal Cells: Methods and Protocols. 289, 39-45 (2005).</li><li>Kashiwagi, M., Kuroki, T., Huh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+hair+follicle+formation+by+epidermal+growth+factor+in+an+organ+culture+of+developing+mouse+skin.">Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin.</a> Developmental Biology. 189 (1), 22-32 (1997).</li><li>Van Der Wel, L. I., Wei, L., Prens, E. P., Laman, J. D., Companjen, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+ex+vivo+skin+organ+culture+system+for+functional+studies.">A modified ex vivo skin organ culture system for functional studies.</a> Archives of Dermatological Research. 293 (4), 184-190 (2001).</li><li>Mort, R. L., Hay, L., Jackson, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+live+imaging+of+melanoblast+migration+in+embryonic+mouse+skin.">Ex vivo live imaging of melanoblast migration in embryonic mouse skin.</a> Pigment Cell &amp; Melanoma Research. 23 (2), 299-301 (2010).</li><li>Jordan, S. A. A., Jackson, I. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGF+(KIT+ligand)+is+a+chemokinetic+factor+for+melanoblast+migration+into+hair+follicles.">MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles.</a> Developmental Biology. 225 (2), 424-436 (2000).</li><li>Delmas, V., Martinozzi, S., Bourgeois, Y., Holzenberger, M., Larue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre-mediated+recombination+in+the+skin+melanocyte+lineage.">Cre-mediated recombination in the skin melanocyte lineage.</a> Genesis. 36 (2), 73-80 (2003).</li><li>Srinivas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1 (1), (2001).</li><li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+Drives+Melanoblast+Organization+during+Mouse+Development+by+Orchestrating+Pseudopod.">Rac1 Drives Melanoblast Organization during Mouse Development by Orchestrating Pseudopod.</a> Driven Motility and Cell-Cycle Progression. 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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...

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 &#946;-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 &#38; 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&#160;et al.&#160;(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.

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	<tbody>
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			<td height="21" style="height:21px;width:216px;">DMEM high glucose</td>
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			<td style="width:119px;">F0475</td>
			<td style="width:167px;">without phenol red</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:132px;">Sigma</td>
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			<td style="width:132px;">Custom made</td>
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			<td style="width:132px;">Sarstedt</td>
			<td style="width:119px;">94.6077.410</td>
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			<td style="width:132px;">Generic</td>
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			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:132px;">Generic</td>
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			<td height="21" style="height:21px;width:216px;">Kebab skewers</td>
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			<td>Bamboo BBQ skewers 30cm</td>
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			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">633185</td>
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ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration

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&#160;cultures in parallel.

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&#160;min&#160;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 S&#248;ndergaard Pedersen (<a href="http://www.phage...

Watch this Scientific Journal Video about Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration at JoVE.com

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

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.

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

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

ex-vivo-culture-mouse-embryonic-skin-live-imaging-melanoblast

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Biology

12.5K visualizações.  University of Edinburgh.  Descrevemos a dissecção e ex vivo cultura de rato pele embrionária. O sistema mantém uma cultura de interface ar-líquido através da superfície do tecido e permite imagens em um microscópio invertido. Melanoblastos, um componente da pele em desenvolvimento, são marcadas por fluorescência, permitindo o seu comportamento a ser observada por meio de microscopia confocal. 