Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants.
The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.
Unité de Génétique et Biologie du Développement, Institut Curie, PSL Research University, CNRS UMR 3215, INSERM U934; catherine.corbel@curie.fr.
Catherine
C
Corbel
Unité de Génétique et Biologie du Développement, Institut Curie, PSL Research University, CNRS UMR 3215, INSERM U934.
Edith
E
Heard
Development. 2013 Feb;140(4):861-72
23362347
J Embryol Exp Morphol. 1981 Apr;62:217-27
6895086
Placenta. 2008 Mar;29 Suppl A:S4-9
18083226
Curr Opin Cell Biol. 2009 Jun;21(3):359-66
19477626
Semin Cell Dev Biol. 2000 Apr;11(2):105-13
10873707
J Vis Exp. 2007 Feb 25;(2):160
18830421
Methods Enzymol. 2004;376:405-19
14975321
Methods Mol Biol. 2008;463:297-308
18951174
Mol Reprod Dev. 1993 Apr;34(4):357-68
8471259
Int Rev Cytol. 1996;165:53-119
8900957
Science. 2004 Jan 30;303(5658):644-9
14671313
Nat Methods. 2012 Jul;9(7):671-5
22930834
Methods Mol Biol. 2013;1042:13-31
23979997
Nat Methods. 2005 Sep;2(9):663-5
16118636
Nature. 2005 Nov 17;438(7066):369-73
16227973
Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21301-6
23236188
Placenta. 2003 Feb-Mar;24(2-3):123-30
12596737
Nature. 2010 Oct 21;467(7318):977-81
20962847
J Histochem Cytochem. 2002 Sep;50(9):1219-27
12185200
Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5198-203
19273861
Reproduction. 2004 Jul;128(1):53-71
15232064