Mort Richard L, Keighren Margaret, Hay Leonard, Jackson Ian J
MRC Human Genetics Unit, MRC IGMM, Western General Hospital, University of Edinburgh.
MRC Human Genetics Unit, MRC IGMM, Western General Hospital, University of Edinburgh;
J Vis Exp. 2014 May 19(87):51352. doi: 10.3791/51352.
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 culture(6). 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.
成黑素细胞是源自神经嵴的黑素细胞前体；这些细胞负责在皮肤和毛发中产生色素。成黑素细胞穿过胚胎的表皮，随后在发育中的毛囊中定植（1,2）。神经嵴细胞迁移在体外得到了广泛研究，但体内方法仍未得到很好的发展，尤其是在哺乳动物系统中。一种替代方法是使用离体器官型培养（3 - 6）。培养小鼠胚胎皮肤需要在组织表面维持气液界面（ALI）（3,6）。由于缺乏一种不仅能维持这种气液界面，还能使培养物倒置从而与短工作距离物镜和大多数共聚焦显微镜兼容的良好方法，小鼠胚胎皮肤的高分辨率实时成像受到了阻碍。本文描述了一种方法的近期改进，该方法使用透气膜来克服这些问题，并允许对离体培养的胚胎皮肤进行高分辨率共聚焦成像（6）。通过使用表达成黑素细胞特异性Cre重组酶的小鼠品系与R26YFPR报告基因品系相结合，我们能够对这些皮肤培养物中的成黑素细胞群体进行荧光标记。该技术允许对成黑素细胞进行实时成像，并观察它们的行为以及与它们发育所在组织的相互作用。文中包含了代表性结果，以展示同时对6个培养物进行实时成像的能力。
