The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo.

Reciprocal inductive interactions between the embryonic and extraembryonic tissues establish the anterior-posterior (AP) axis of the early mouse embryo. The anterior visceral endoderm (AVE) signaling center emerges at the distal tip of the embryo at embryonic day 5.5 and translocates to the prospective anterior side of the embryo. The process of AVE induction and migration are poorly understood. Here we demonstrate that the T-box gene Eomesodermin (Eomes) plays an essential role in AVE recruitment, in part by directly activating the homeobox transcription factor Lhx1. Thus, Eomes function in the visceral endoderm (VE) initiates an instructive transcriptional program controlling AP identity.

Transthyretin mouse transgenes direct RFP expression or Cre-mediated recombination throughout the visceral endoderm.

Transthyretin (Ttr) is a thyroid hormone transport protein secreted by cells of the visceral yolk sac and fetal liver in developing embryos, and by hepatocytes and the choroid plexus epithelium of the brain in adult mice. Spatiotemporal localization of Ttr mRNA during embryogenesis suggested that Ttr regulatory elements might drive transgene expression throughout the visceral endoderm of early mouse embryos. We use Ttr cis-regulatory elements to generate Ttr::RFP and Ttr::Cre strains of mice, driving red fluorescent protein (RFP) and a nuclear-localized Cre recombinase, respectively. Visualization of RFP fluorescence in Ttr::RFP transgenics confirms reporter localization throughout the visceral endoderm in early embryos and in the visceral yolk sac and fetal liver of later stage embryos. Using both GFP-based and LacZ-based Cre reporter strains, we demonstrate that in Ttr::Cre transgenics, Cre-mediated recombination occurs throughout the visceral endoderm. The Ttr::Cre strain can therefore be used as a tool for genetic modifications within the visceral endoderm lineage.

Ex utero culture and live imaging of mouse embryos.

Mouse genetic approaches when combined with live imaging tools have the potential to revolutionize our current understanding of mammalian biology. The availability and improvement of a wide variety of fluorescent proteins have provided indispensable tools to visualize cells in living organisms. It is now possible to generate genetically modified mouse strains expressing fluorescent proteins in a tissue-specific manner. These reporter-expressing strains make it possible to image dynamic cell behaviors in the context of a living embryo. Since mouse embryos develop within the uterus, live imaging experiments require culture conditions that closely mimic those in vivo. Over the past few decades, significant advances have been made in developing conditions for culturing both pre- and postimplantation stage embryos. In this chapter, we will discuss methods for ex utero culture of preimplantation and postimplantation stage mouse embryos. In particular, we will describe protocols for collecting embryos at various stages, setting up culture conditions for imaging and using laser scanning confocal microscopy to visualize live processes in mouse embryos expressing fluorescent reporters.

The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages.

The cell movements underlying the morphogenesis of the embryonic endoderm, the tissue that will give rise to the respiratory and digestive tracts, are complex and not well understood. Using live imaging combined with genetic labeling, we investigated the cell behaviors and fate of the visceral endoderm during gut endoderm formation in the mouse gastrula. Contrary to the prevailing view, our data reveal no mass displacement of visceral endoderm to extraembryonic regions concomitant with the emergence of epiblast-derived definitive endoderm. Instead, we observed dispersal of the visceral endoderm epithelium and extensive mixing between cells of visceral endoderm and epiblast origin. Visceral endoderm cells remained associated with the epiblast and were incorporated into the early gut tube. Our findings suggest that the segregation of extraembryonic and embryonic tissues within the mammalian embryo is not as strict as believed and that a lineage previously defined as exclusively extraembryonic contributes cells to the embryo.

Eomes::GFP-a tool for live imaging cells of the trophoblast, primitive streak, and telencephalon in the mouse embryo.

Expression of T-box family member Eomesodermin (Tbr2) is spatiotemporally restricted in the mouse embryo; initially expressed in extraembryonic lineages in the sequential progression from the trophectoderm of the blastocyst, its derivatives the extraembryonic ectoderm, and thereafter the chorion, in addition to the visceral endoderm and primitive streak at gastrula stages, and the telencephalon at later stages. We describe the spatiotemporal expression of GFP in embryos of a Tg(Eomes::GFP) BAC transgenic strain, and have compared it with the localization of endogenous Eomes transcripts and protein. Our analysis reveals the following: (1) robust easily visualized reporter expression in live hemizygous transgenic embryos, (2) increased levels of expression in live homozygous transgenic embryos that are compatible with embryo viability, and (3) a close correlation between endogenous Eomes and GFP reporter expression in BAC transgenic embryos. These features establish the Tg(Eomes::GFP) BAC transgenic strain as a novel reagent for both live imaging and the isolation of Eomes expressing cells from specific locations within the embryo.

Tg(Afp-GFP) expression marks primitive and definitive endoderm lineages during mouse development.

Alpha-fetoprotein (Afp) is the most abundant serum protein in the developing embryo. It is secreted by the visceral endoderm, its derivative yolk sac endoderm, fetal liver hepatocytes, and the developing gut epithelium. The abundance of this protein suggested that Afp gene regulatory elements might serve to effectively drive reporter gene expression in developing endodermal tissues. To this end, we generated transgenic mouse lines Tg(Afp-GFP) using an Afp promoter/enhancer to drive expression of green fluorescent protein (GFP). Bright GFP fluorescence allowed the visualization, in real time, of visceral endoderm, yolk sac endoderm, fetal liver hepatocytes, and the epithelium of the gut and pancreas. Comparison of the localization of green fluorescence with that of endogenous Afp transcripts and protein indicated that the regulatory elements used to generate these mouse lines directed transgene expression in what appeared to be all Afp-expressing cells of the embryo, but only in a subset of fetal liver cells. The bright GFP signal permitted flow cytometric analysis of fetal liver hepatocytes. These mice represent a valuable resource for live imaging as well as identification, quantitation, and isolation of cells from the primitive and definitive endoderm lineages of the developing mouse embryo.