Format

Send to

Choose Destination
Dev Growth Differ. 2015 Apr;57(3):232-41. doi: 10.1111/dgd.12201. Epub 2015 Mar 4.

In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos.

Author information

1
Department of Zoology, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto, 606-8502, Japan; Graduate School of Biological Sciences, Nara Institute of Science and Technology, NARA, Takayama, Ikoma, 630-0192, Japan.

Abstract

During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin-containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross-talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus-mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication-Competent Avian sarcoma-leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back-transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)(+) Neo(r+) -electroporated melanocytes from Hypeco nera were selectively grown in G418-supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS-mOrange, mOrange(+) keratinocytes and transplanted EGFP(+) melanocytes were frequently juxtaposed each other. High-resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.

KEYWORDS:

G418; dendrite; in ovo electroporation; melanin; retrovirus

PMID:
25739909
DOI:
10.1111/dgd.12201
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center