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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Genesis. Author manuscript; available in PMC Jun 9, 2010.
Published in final edited form as:
PMCID: PMC2882854
NIHMSID: NIHMS208881
Eomes::GFP—A Tool for Live Imaging Cells of the Trophoblast, Primitive Streak, and Telencephalon in the Mouse Embryo
Gloria S. Kwon1,2 and Anna-Katerina Hadjantonakis1*
1Developmental Biology Program, Sloan-Kettering Institute, New York, New York
2Neuroscience Program, Weill Graduate School of Medical Sciences of Cornell University, New York, New York
*Correspondence to: Anna-Katerina Hadjantonakis, Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021. hadj/at/mskcc.org
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.
Keywords: mouse embryo, T-box, Eomesodermin, Tbr2, green fluorescent protein, live imaging, blastocyst, trophectoderm, extraembryonic ectoderm, primitive streak, telencephalon, limb, digit condensation, stomach, pancreas
Eomesodermin (Eomes or Tbr2) a member of the T-box family of transcriptional regulators was originally identified for its ability to specify mesoderm in Xenopus (Ryan et al., 1996). Mouse Eomes mutants have uncovered its role in the regulation of trophoblast lineage specification and mesoderm migration during embryonic development and the development of cell-mediated immunity in the adult (Pearce et al., 2003; Russ et al., 2000). The tightly regulated spatiotemporal expression of Eomes (Bulfone et al., 1999; Ciruna and Rossant, 1999; Hancock et al., 1999) and the paucity of available transgenic mouse strains expressing GFP in a lineage-specific fashion in early mouse embryos led us to determine if an Eomes::GFP BAC transgenic mimicked endogenous gene expression, and could therefore be used as a reporter strain for live imaging (Lee et al., 2001; Zhang et al., 2004, 2005).
The Tg(Eomes::GFP) BAC transgene was obtained from a library generated by the GENSAT consortium (Gong et al., 2003). The BAC covers ~225 kb of mouse genomic DNA, containing sequences spanning ~186 kb upstream and ~18 kb downstream of the Eomes locus. Enhanced green fluorescent protein (EGFP) and a pA sequence were inserted directly upstream of the Eomes coding region preserving gene structure while providing a readout of promoter activity (Fig. 1a). Tg(Eomes::GFP) BAC transgenic animals were generated through the GENSAT consortium (http://www.gensat.org/index.html). Hemizygous and homozygous transgenics were viable and fertile and indistinguishable from non-transgenic littermates. Hemizygous Tg(Eomes:GFP)/+ embryos were used for all data presented. Homozygotes exhibited identical localization of GFP, but with increased levels of fluorescence.
FIG. 1
FIG. 1
Schematic representation of Tg(Eomes::GFP) BAC transgene and onset of GFP expression in live peri-implantation mouse embryos. (a) Map of the GENSAT Tg(Eomes::GFP) BAC transgene spanning 186 kb upstream and 18 kb downstream of the mouse Eomes locus. Gray (more ...)
Previous studies have documented Eomes gene expression and protein localization in mouse oocytes and preimplantation embryos (McConnell et al., 2005). Since expression assayed by wholemount in situ hybridization or lacZ staining in a targeted allele initiates in the trophectoderm of the blastocyst, we imaged live Tg(Eomes::GFP) embryos from compacting morula to late blastocyst stages. We were unable to detect GFP expression in early blastocysts (~E3.5; Fig. 1b,c); however, weak expression within the trophectoderm could be seen to initiate in later hatched blastocysts (~E4.0; Fig. 1d–h). By peri-implantation (~E4.5) embryos exhibited an increased level of GFP fluorescence restricted to the trophectoderm (Fig. 1i–m). The localization of GFP expression in Tg(Eomes::GFP) embryos mirrored endogenous Eomes, albeit somewhat temporally delayed (Hancock et al., 1999; Strumpf et al., 2005). Any lag in GFP expression could in part be due to the fact that the routine detection of endogenous Eomes transcripts or protein utilizes an amplification step, whereas in the Tg(Eomes::GFP) transgenics the visualization of GFP is a direct and linear readout of promoter activity in living embryos, and thus sufficient levels of detectable protein must be present.
By the pre-streak stage, robust fluorescence was detected exclusively in the extraembryonic ectoderm, a derivative of the polar trophectoderm (Fig. 2a,b). Subsequently, by the early-streak stage GFP expression was maintained in extraembryonic regions, but also had initiated within the primitive streak concomitant with its formation (Fig. 2d,e). Visualization of endogenous Eomes transcripts by in situ hybridization of the same embryos live imaged for GFP established the correlation between endogenous Eomes expression and reporter expression at these stages (Fig. 2c,f). In addition, GFP fluorescence in the extraembryonic ectoderm and primitive streak closely correlated with the distribution of GFP transcripts (Fig. 2m–p). These observations imply that the cis-acting regulatory elements necessary for Eomes activation within the trophoblast, extraembryonic ectoderm, and primitive streak are present within the BAC. Previous work on the dissection of the Xenopus Eomes promoter led to the identification of an activin responsive element (ARE) and two FAST2 binding sites necessary for mesodermal expression that were positioned upstream of the transcriptional start site (Ryan et al., 2000). Database searches revealed that both ARE and FAST2 sites were present within the region located immediately upstream of the first exon of the mouse Eomes locus and were contained within the BAC insert (data not shown).
FIG. 2
FIG. 2
GFP expression in the extraembryonic ectoderm and early primitive streak of live gastrula stage embryos. (a,b,d,e) Pre-streak stage (~E5.75) to early streak stages (~E6.5). (c,f) In situ hybridization of the same embryos to detect Eomes. (g,h) Late-streak (more ...)
Knock-in strains with primitive streak-specific fluorescent protein expression have been reported (Hart et al., 2002; Huber et al., 2004). In these cases a GFP reporter was introduced into an endogenous locus by gene targeting in embryonic stem cells. However, since knock-in alleles perturb wild type gene function, these strains are not favored as neutral indicators for analyzing mutants due to the possibility of genetic interactions and epistasis effects. The Tg(Eomes::GFP) strain therefore provides a solution, since it is the first transgenic strain reported to exhibit robust GFP expression within the early primitive streak of the mouse embryo concominant with the onset of gastrulation.
By the late-streak stage (~E7.5) when Eomes was localized to the chorion and primitive streak (Fig. 2i), GFP expression was downregulated within the extraembryonic ectoderm, while expression within the epiblast was observed but at reduced intensity, encompassing an expanded domain including nascent mesoderm cells emanating from the primitive streak, likely resulting from the perdurance of the fluorescent protein in cells emanating from the primitive streak. (Fig. 2g,h). By early headfold stages (E7.75) when Eomes was maintained in the chorion, but downregulated in the primitive streak (Fig. 2l), GFP fluorescence was undetectable in all regions of the embryo (Fig. 2j,k). These observations reveal that GFP fluorescence was extinguished prior to downregulation of endogenous Eomes, suggesting the following: (1) Distinct cis-acting regulatory elements are necessary for maintenance of Eomes expression. Such cis-regulatory elements might reside at, or co-operate with, sites distant to the Eomes locus not contained within the BAC, alternatively the introduction of EGFP into the Eomes locus may have perturbed the spacing and thus function of such elements. (2) Perdurance of GFP does not extend over more than a period of a few hours. (3) The Tg(Eomes::GFP) strain can be used to visualize, and thus discriminate, early vs. later primitive streak.
Live imaging of midgestation embryos revealed fluorescence in two locations: the central nervous system (CNS) and developing limbs which correlated with previously reported endogenous Eomes and persisted until perinatal stages when it was downregulated (Bulfone et al., 1999; Ciruna and Rossant, 1999; Hancock et al., 1999; Russ et al., 2000).
In the CNS, fluorescence was first observed at E10.0 within the telencephalon (Fig. 3a), and became more robust by E12.5–E14.5 (Fig. 3b,c), then diminished by P0 (Fig. 3d). We investigated the colocalization of GFP and endogenous Eomes protein (Fig. 3e–h) and determined that GFP was expressed in a larger population of cells. High magnification views of sectioned brains at E14.5 revealed that GFP was detectable in a subpopulation of cells expressing and colocalized with Eomes protein predominantly within the subventricular zone (SVZ), but GFP expressing cells were also present in the intermediate zone (IZ) to marginal zone (MZ) (Fig. 3i–l). This absence of GFP expression in Eomes +ve cells of the SVZ is likely to be analogous to the delay in onset of detectable fluorescence observed in blastocyst stage embryos (discussed previously). We also noted that GFP fluorescence extended to more differentiated regions of the neocortex where Eomes protein was not present, likely due to perdurance of the fluorescent protein.
FIG. 3
FIG. 3
GFP expression within the telencephalon of Tg(Eomes::GFP) embryos at midgestation and early postnatal stages. (a) Onset of GFP fluorescence in the central nervous system (CNS) at E10.0. (b,c) Wholemount brightfield and GFP overlays depicting GFP fluorescence (more ...)
We next determined colocalization of the GFP reporter with molecular markers representing the different cell types present in the telecephalon, including both distinct classes of neural progenitors and postmitotic cells. Pax6 marks a population of radial glial progenitor cells (Gotz et al., 1998) fated for glutamatergic differentiation (Schuurmans et al., 2004). Pax6 antibody staining revealed no overlap between Pax6 expression in the VZ and GFP expression in the SVZ and MZ, suggesting that the transgene was not prematurely activated (Fig. 3m–p). However, staining with T-brain1 (Tbr1) antibody revealed a subpopulation of cells co-expressing GFP and Tbr1 (Fig. 3q–t). Since Eomes (Tbr2) and Tbr1 are expressed in mutually exclusive populations of cells (Englund et al., 2005), this result suggests that GFP fluorescence extended into regions containing postmitotic neurons and that this could result from perdurance of the fluorescent protein or downregulation of the reporter. Furthermore, staining with the M-phase marker phosphohistone-H3 revealed colocalization with a subpopulation of GFP expressing cells representing a non-surface dividing intermediate progenitor cell population (Fig. 3u–x).
To establish if the expanded domain of GFP fluorescence observed in Tg(Eomes::GFP) brains was due to perdurance of GFP protein, we processed sequential sections to determine and compare the localization of GFP protein, GFP transcripts, and Eomes transcripts. GFP transcripts were more restricted than GFP protein (Fig. 4a,b,d–e), and were localized to an identical domain as Eomes transcripts, residing predominantly within the subventricular and intermediate zones at E14.5 (Fig. 4c), and the hippocampus at P0 (Fig. 4f). Thus perdurance of the fluorescent protein results in the expanded domain of GFP fluorescence, such that the GFP reporter acts as a lineage tracer for postmitotic neurons that have recently extinguished Eomes having migrated into the cortical plate (cp).
FIG. 4
FIG. 4
The expanded domain of GFP fluorescence within the telencephalon of Tg(Eomes::GFP) embryos is due to perdurance of GFP protein. (a) Brightfield and GFP overlay of vibratome sections of E14.5 brains illustrating GFP localization. (b,c) GFP expression by (more ...)
Within the developing limb, a focus of GFP fluorescence was observed at the base of digit IV (Fig. 3b–c, Fig. 4i). Sections through the forelimb at E12.5 revealed that GFP was localized in a ring of cells around the condensations associated with the fourth digit (Fig. 4j–n). In addition, Eomes transcripts within the forelimb at this stage were also restricted to the same ring of cells at the base of the fourth digit, and thus closely correlated with transgene expression (Fig. 4g–h).
At midgestation through fetal stages additional sites of readily detectable GFP expression included discrete populations of cells within the stomach, pancreas, and placenta (Fig. 5). Within the E14.5 placenta, a small population of fluorescent cells were observed within the parietal yolk sac (Fig. 5a,b). These cells were present in the endodermal layer of the parietal yolk sac, as at this stage no GFP expression was observed in trophoblast giant cells, which are derivatives of the trophectoderm. This population persisted until birth.
FIG. 5
FIG. 5
Additional sites of GFP fluorescence in Tg(Eomes::GFP) embryos at midgestation to fetal stages of development. (a,b) A population of fluorescent cells present in the placenta of E14.5 embryos. (c–e) Rendered confocal images depicting nuclear counterstain (more ...)
Transgene expression was also observed in the pylorus of the stomach (Fig. 5h–k), with increased magnification revealing the elongated cell morphology of GFP-positive cells (Fig. 5l). E-cadherin antibody staining revealed that these GFP positive cells represented a subpopulation of cells within the mucosa (Fig. 5s–u). GFP fluorescence was also observed within the developing pancreas (Fig. 5m–r) at E14.5. Thus, to better define the identity of these cells, an antibody against pancreatic duodenal homeobox-1 (Pdx1) was used to stain sections through the pancreas. The staining revealed that there was no colocalization of Pdx1 and GFP (Fig. 5v–x). Since Pdx1 is present in endocrine progenitor cells at E14.5, and is later involved in β-cell maturation, the GFP positive cells observed within the pancreas are unlikely to represent the endocrine cells of the islets of Langerhans, including the alpha-(glucagon-secreting) and beta-(insulin-secreting) cells, but more likely to represent a subpopulation of pancreatic exocrine cells.
In summary we have demonstrated that GFP expression in the GENSAT Tg(Eomes::GFP) BAC transgenic strain closely correlates with endogenous mouse Eomes expression during embryonic development. This strain therefore represents a potentially useful tool for live imaging cell behavior in the trophoblast and its derivative extraembryonic ectoderm, the early primitive streak at gastrulation, as well as cell populations within the telencephalon during neurogenesis in addition to populations of cells within the limb, stomach, and pancreas during later fetal stages (Hadjantonakis et al., 2003). Furthermore lineage specific GFP expression also permits the isolation of Eomes expressing cells from specific locations within the embryo (Hadjantonakis and Nagy, 2000). The Eomes BAC represents a reagent that can be retrofitted to replace the native EGFP cassette with spectral variant or subcellularly localized fluorescent proteins for higher resolution live imaging, or site specific recombinase variants for lineage-specific gene modification (Branda and Dymecki, 2004; Hadjantonakis and Papaioannou, 2004; Rhee et al., 2006).
Embryo Collection
Preimplantation embryos were recovered in M2 medium and cultured in KSOM medium (Chemicon Specialty Media, Temecula, CA) in an organ culture dish (BD Falcon, cat no. 353037) or, for live imaging, under mineral oil in a MatTek glass bottom dish (cat. no. P35G-1.5-14C) at 37°C, 5% CO2. Postimplantation embryos and organs were dissected in modified PB-1 (Papaioannou and West, 1981; Whittingham and Wales, 1969) medium containing 10% fetal bovine serum (FBS). Live embryos were counterstained with 7.5 mM DRAQ5 (DAKO, Carpinteria, CA).
Vibrating Microtome Sectioning and Counterstaining
Embryos were fixed in 4% PFA/PBS for 2–12 h after dissection, washed in PBS, and embedded in 4% low-melt agarose, 5% sucrose in PBS. Blocks were cut out of embedding molds, trimmed using a razor blade, and then mounted onto a vibrating microtome chuck (Leica VT1000S) using superglue. Sections were cut at a thickness of 25–200 μm. Embryos were counterstained with Hoechst (cat no. H3570, Invitrogen) and AlexaFluor633 phalloidin (cat no. A22284, Invitrogen).
In Situ Hybridization and Immunochemistry
In situ hybridizations were performed according to standard protocols (Nagy et al., 2003). For immunohistochemistry, brains were cut into 70 μm-thick sections, blocked for 1 h at RT in PBSMT, and then incubated with one of the following antibodies diluted in PBSMT at 4° overnight: anti-Tbr2 (1:200; cat. no. AB9618, Chemicon International), anti-Tbr1 (1:500; Hsueh et al., 2000), anti-Pax6 (1:200; Developmental Studies Hybridoma Bank), anti-pHH3 (1:500; cat. no. 06-570, Upstate Biotechnology), anti-E-cadherin (1:200; cat. no. U-3254, Sigma), or anti-Pdx1 (1:200; cat. no. ab47267, Abcam). The next day, the sections were washed with PBSMT and then incubated with Vectastain blocking solution (Vector Labs, Burlingame, CA) for 1 h at RT, followed by incubation with a biotinylated secondary antibody at 4° overnight. The sections were then washed with PBSMT and rinsed in PBT before being incubated with anti-rabbit Streptavidin-AlexaFluor546 (Molecular Probes, Eugene, OR) for 1 h at RT in the dark. Finally, sections were washed in PBT before being imaged.
Image Acquisition
Laser scanning confocal data were acquired using a Zeiss LSM510 META laser scanning confocal mounted on a Zeiss Axiovert 200M microscope. Whole embryos were kept in PBS in MatTek glass bottom dishes during imaging. Widefield images were acquired using a Zeiss Axiocam MRc or MRm camera mounted on a Leica MZ16FA microscope.
ACKNOWLEDGMENTS
We thank Shiaoching Gong and Mark Tomishima for advice on GENSAT BAC construction; Terry Capellini for discussions on limb expression; Stewart Anderson for antibodies and advice on transgene expression in the telencephalon; Sonja Nowotschin for comments on the manuscript; Ginny Papaioannou for in situ probes, assistance, and insightful discussions pertaining transgene expression and comments on the manuscript.
Contract grant sponsor: NIH, Contract grant number: RO1-HD052115
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