Elsevier

Gene Expression Patterns

Volume 12, Issues 1–2, January–February 2012, Pages 85-93
Gene Expression Patterns

Identification of a conserved cis-acting region driving expression of mouse Eomesodermin to the primitive streak, node, and definitive endoderm

https://doi.org/10.1016/j.gep.2011.06.003Get rights and content

Abstract

The cis-acting elements that regulate Eomes transcription during embryonic development are largely unknown. Here we identify a conserved cis-acting region (EoIV) located ∼20 kb upstream of the Eomes coding region that faithfully drives reporter expression to sites of Eomes expression during gastrulation. Transgenic EoIV-hsp68-GFP expression was evident in the epiblast of early-streak stage mouse embryos at the site where the primitive streak is initiated. At the mid- and late-streak stages, EoIV-hsp68-GFP expression was found in the streak, node region and definitive endoderm with a particular intensive GFP expression in the node region. At the early head fold stage, GFP was expressed in the node region and the surrounding endoderm. In contrast to earlier reports of Eomes mRNA expression, we confirmed Eomes protein expression in the node of early head fold embryos by immunohistochemistry. In vitro, EoIV-hsp68-GFP expression was activated ES cells differentiating into primitive streak-like progeny in response to Bmp and activin treatment.

Highlights

► The Eomes gene contains a conserved region (EoIV) at −20 kb. ► EoIV-hsp68-GFP is expressed at sites of Eomes expression during gastrulation. ► EoIV-hsp68-GFP is expressed in primitive streak-like ES cell progeny in vitro.

Section snippets

Expression of Eomes in mouse embryos

To gain a better understanding of Eomes expression in gastrulating mouse embryos we used whole-mount immunohistochemistry (IHC) and high resolution confocal scanning to map the expression of Eomes from pre-streak (E6.0) to early head fold (E7.75–8.0). At E6.0, prior to streak formation, we detected Eomes in the ExE and VE (Fig. 1A–D). As the streak formed at approximately E6.25, we found Eomes in the posterior epiblast, at the site of streak initiation (Fig. 1E–H) while expression was

Discussion

Developmentally regulated genes often harbor complex cis-regulatory landscapes and two cis-regulatory regions driving Eomes expression to specific embryonic tissues have already been identified (Chandler et al., 2009, Lettice et al., 2003, Mao et al., 2008, Pernaute et al., 2010, Ribich et al., 2006, Wunderle et al., 1998). Here we identify a third cis-acting region that drives Eomes expression to the primitive streak, node and emerging DE. Eomes is necessary for DE formation in the mouse

IHC

For immunohistochemistry embryos were incubated in 0.5% TNB blocking reagent (PerkinElmer, Waltham, Massachusetts, USA) with 0.1% Triton X-100-100 (Sigma Chemicals Co, St. Louis, USA) for 2 h at RT to promote antibody penetration and block unspecific antigen binding. Following embryos were incubated with a mix of two primary antibodies diluted in 0.5% TNB and 0.1% Triton X-100 over night at 4 °C. The antibodies used in this study were Polyclonal Rabbit-anti-Tbr2 (Eomes) 1:5000 (Chemicon

Acknowledgments

We are grateful to Doug Melton and Roland Stein for sending us Rosa26 ES cells and the hsp68 promoter, respectively. We thank Ragna Jørgensen and Anita Sharma Friismose for excellent technical assistance, Søren Refsgaard Lindskog for help with sequence and data analysis, Jan Nygaard Jensen, Jacob Hecksher-Sørensen, Jacob Hald, Jonas Ahnfelt-Rønne, Maria Winzi and Mark Kalisz for fruitful discussions. This work was made possible by funding from DACSDOC and NIH (DK 072495).

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