Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions

doi:10.1016/0168-9525(93)90162-B

Trends in Genetics

Volume 9, Issue 5, May 1993, Pages 162-167

https://doi.org/10.1016/0168-9525(93)90162-B Get rights and content

Abstract

Non-isotopic whole-mount in situ hybridization of mRNA is a novel technique that has greatly facilitated the precise three-dimensional localization of transcripts from genes whose expression is important during development. This methodology has recently been applied to the study of the mouse embryo and offers particular advantages over conventional procedures.

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Citation Excerpt :
Genital ridges were dissected from fetal mice, and fixed in 4% paraformaldehyde (PFA) overnight (o/n) at 4 °C. WISH with chromogen NBT/BCIP staining was carried out as described elsewhere (Rosen and Beddington, 1993; Schulz et al., 2007; Wei et al., 2011). Antisense and sense probes were synthesized using Digoxigenin RNA labeling mix (Roche) according to the manufacturer's instructions.
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Tamoxifen, as well as most endocrine-disrupting chemicals, affects the reproductive system and sexual development, but little is known about its disruption of the molecular pathways regulating mammalian sex determination. In fetal mice, the expression levels and pattern of key genes involved in controlling sexually dimorphic balance were analyzed both in vivo and in vitro by using whole-mount in situ hybridization and quantitative-PCR. Developmental tamoxifen exposure induced abnormal up-regulation of the testis differentiation marker Pdfgra in Leydig cells and of Sox9 and Fgf9 in Sertoli cells in XX gonad. Immunohistochemistry analysis confirmed the over-expression of SOX9 protein. Accordingly, the ovary development marker Foxl2 was depressed at both the mRNA and protein levels. The increase in testosterone and the reduction in 17β-estradiol and progesterone were observed by using the in vitro assay with organotypic cultures. Taken together, results indicated that tamoxifen induced the ectopic expression of well-established sex-specific genes during the critical developmental period, thus resulting in abnormal testicular development in the XX gonad of mammals. This study facilitates a better understanding of the molecular mechanisms of antiestrogens and possibly of compounds that interrupt estrogen signaling by other modes of action, and the association with the pathogenesis of human sexual developmental disorders.

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Trends in Genetics

Technical focusWhole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions

Abstract

EMBO J.

Cell

Nature

Dev. Biol.

Chromosoma

Cell

Cell

Dev. Biol.

Development

Methods Cell Biol.

Nature

Development

Cell

Curr. Opin. Cell Biol.

Technical focus
Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions