High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization

Abstract

Mammalian preimplantation embryos provide an excellent opportunity to study temporal and spatial gene expression in whole mount in situ hybridization (WISH). However, large-scale studies are made difficult by the size of the embryos (∼60 μm diameter) and their fragility. We have developed a chamber system that allows parallel processing of embryos without the aid of a microscope. We first selected 91 candidate genes that were transcription factors highly expressed in blastocysts, and more highly expressed in embryonic (ES) than in trophoblast (TS) stem cells. We then used the WISH to identify 48 genes expressed predominantly in the ICM and to follow several of these genes in all seven preimplantation stages. The ICM-predominant expressions of these genes suggest their involvement in the pluripotency of embryonic cells. This system provides a useful tool to a systematic genome-scale analysis of preimplantation embryos.

An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells

“…The expression of this gene was not detected in either ICM or TE in blastocysts by whole mount in situ hybridization (Yoshikawa et al, 2006). It has been shown that REST binds to the promoter region of Pou5f1 in ES cells (Boyer et al, 2005;Loh et al, 2006) and its expression is suppressed when Pou5f1 was repressed in mouse ES cells .…”

“…The expression of this gene was not detected in either ICM or TE in blastocysts by whole mount in situ hybridization (Yoshikawa et al, 2006). It has been shown that REST binds to the promoter region of Nanog was originally isolated as a gene whose overexpression maintains the undifferentiated state of ES cells in the absence of LIF and has been used as a marker for pluripotency (Chambers et al, 2003;Mitsui et al, 2003).…”

“…Previously we reported the development of a protocol for a high-throughput whole mount in situ hybridization of preimplantation mouse embryos and the expression patterns of 98 genes in mouse blastocysts (Yoshikawa et al, 2006). We adapted this protocol to mouse ES cell culture (see the detailed protocol in Supplement).…”

“…In this case, genes with absolute expression levels estimated at ≥1 copy/cell in ES cells and < 1 copy/cell in TS cells were considered "ES-enriched". Genes predominantly expressed in the inner cell mass (ICM) of mouse blastocysts were previously identified using whole-mount in situ hybridization (Yoshikawa et al, 2006) and 61 of these genes were included in the candidate list. Finally, unpublished gene lists from experiments performed in our laboratory were added, as well as individual genes of interest under study.…”

Expression profiling of the mouse early embryo: Reflections and perspectives

“…Although large-scale ISH/WISH methods have been applied to mouse intestine (Komiya et al, 1997), E9.5 embryos (Neidhardt et al, 2000;Gitton et al, 2002), and E9.5 and E10.5 embryos (Reymond et al, 2002), it has been difficult to apply this method to early embryos, because of their small size (ϳ80-m diameter in the case of preimplantation embryos) and fragility. This problem has been overcome recently by a chamber system that uses both transwell inserts for parallel processing and capillary action for gentle buffer exchanges (Yoshikawa et al, 2006).…”