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• W2109293927 abstract "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. 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. In the mouse, gastrulation is the event that results in the formation of the three germ layers from the pluripotent epiblast and leads to the elaboration of the embryonic axes. Prior to the initiation of gastrulation, the mouse embryo comprises a bilaminar cup-shaped structure consisting of visceral endoderm and the epiblast, which will give rise to the fetus and extraembryonic mesoderm. The visceral endoderm is involved in nutrient uptake and transport and also plays a critical role in the morphogenesis and patterning of the epiblast (Hogan and Zaret, 2002Hogan B.L.M. Zaret K.S. Development of the endoderm and its tissue derivatives.in: Rossant J. Tam P.L. Mouse Development. Academic Press, San Diego, CA2002: 301-330Crossref Google Scholar, Srinivas, 2006Srinivas S. The anterior visceral endoderm-turning heads.Genesis. 2006; 44: 565-572Crossref PubMed Scopus (33) Google Scholar). There is increasing evidence for extensive cellular heterogeneity, dynamic cell behaviors, and intrinsic cell polarity within the visceral endoderm (Mesnard et al., 2006Mesnard D. Guzman-Ayala M. Constam D.B. Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning.Development. 2006; 133: 2497-2505Crossref PubMed Scopus (176) Google Scholar, Rivera-Perez et al., 2003Rivera-Perez J.A. Mager J. Magnuson T. Dynamic morphogenetic events characterize the mouse visceral endoderm.Dev. Biol. 2003; 261: 470-487Crossref PubMed Scopus (94) Google Scholar, Rivera-Perez and Magnuson, 2005Rivera-Perez J.A. Magnuson T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3.Dev. Biol. 2005; 288: 363-371Crossref PubMed Scopus (179) Google Scholar, Rodriguez et al., 2005Rodriguez T.A. Srinivas S. Clements M.P. Smith J.C. Beddington R.S. Induction and migration of the anterior visceral endoderm is regulated by the extra-embryonic ectoderm.Development. 2005; 132: 2513-2520Crossref PubMed Scopus (108) Google Scholar, Thomas and Beddington, 1996Thomas P. Beddington R. Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo.Curr. Biol. 1996; 6: 1487-1496Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). The visceral endoderm is set aside as a lineage in the late blastocyst, in contrast to the definitive endoderm which arises from the epiblast at gastrulation. It is thought that the visceral endoderm is fated to form the endoderm of the extraembryonic visceral yolk sac, whereas the definitive endoderm exclusively represents the embryonic gut endoderm, giving rise to the epithelial lining of the respiratory and digestive tracts and their associated organs including the lungs, liver, and pancreas (Hogan and Zaret, 2002Hogan B.L.M. Zaret K.S. Development of the endoderm and its tissue derivatives.in: Rossant J. Tam P.L. Mouse Development. Academic Press, San Diego, CA2002: 301-330Crossref Google Scholar). At gastrulation, cells that will form the mesoderm and definitive endoderm are recruited from the epiblast and ingress through the primitive streak, a morphologically distinct structure which marks the posterior end of the embryo. Mesoderm emanates from the primitive streak as two bilateral wings of cells that spread anteriorly as they circumnavigate the space between two epithelia: the visceral endoderm on the outer surface of the embryo and the inner epiblast (Kinder et al., 1999Kinder S.J. Tsang T.E. Quinlan G.A. Hadjantonakis A.K. Nagy A. Tam P.P. The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo.Development. 1999; 126: 4691-4701PubMed Google Scholar, Lawson et al., 1991Lawson K.A. Meneses J.J. Pedersen R.A. Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.Development. 1991; 113: 891-911PubMed Google Scholar, Parameswaran and Tam, 1995Parameswaran M. Tam P.P. Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation.Dev. Genet. 1995; 17: 16-28Crossref PubMed Scopus (118) Google Scholar, Tam and Beddington, 1987Tam P.P. Beddington R.S. The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis.Development. 1987; 99: 109-126PubMed Google Scholar). In contrast to mesoderm, the cell movements underlying the morphogenesis of the definitive endoderm are not well understood (Lewis and Tam, 2006Lewis S.L. Tam P.P. Definitive endoderm of the mouse embryo: formation, cell fates, and morphogenetic function.Dev. Dyn. 2006; 235: 2315-2329Crossref PubMed Scopus (117) Google Scholar). Fate-mapping experiments performed by single-cell labeling, embryo painting, and cell transplantation have revealed that the definitive endoderm is derived from cells that occupy a distal position within the prestreak (PS) to midstreak (MS) epiblast (Lawson et al., 1986Lawson K.A. Meneses J.J. Pedersen R.A. Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer.Dev. Biol. 1986; 115: 325-339Crossref PubMed Scopus (166) Google Scholar, Lawson and Pedersen, 1987Lawson K.A. Pedersen R.A. Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse.Development. 1987; 101: 627-652PubMed Google Scholar, Tam and Beddington, 1987Tam P.P. Beddington R.S. The formation of mesodermal tissues in the mouse embryo during gastrulation and early organogenesis.Development. 1987; 99: 109-126PubMed Google Scholar, Tam and Beddington, 1992Tam P.P. Beddington R.S. Establishment and organization of germ layers in the gastrulating mouse embryo.Ciba Found. Symp. 1992; 165: 27-41PubMed Google Scholar). Studies on prestreak to early streak (PS-ES) stage embryos suggested that the visceral endoderm is displaced proximally by cells that derive from the anterior primitive streak (Lawson et al., 1986Lawson K.A. Meneses J.J. Pedersen R.A. Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer.Dev. Biol. 1986; 115: 325-339Crossref PubMed Scopus (166) Google Scholar). By the early to late bud (EB-LB) stage, visceral endoderm cells in proximal regions contributed to the yolk sac and yolk sac-embryo junction (Lawson et al., 1986Lawson K.A. Meneses J.J. Pedersen R.A. Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer.Dev. Biol. 1986; 115: 325-339Crossref PubMed Scopus (166) Google Scholar, Tam et al., 2004Tam P.P. Khoo P.L. Wong N. Tsang T.E. Behringer R.R. Regionalization of cell fates and cell movement in the endoderm of the mouse gastrula and the impact of loss of Lhx1(Lim1) function.Dev. Biol. 2004; 274: 171-187Crossref PubMed Scopus (53) Google Scholar). Further support for the proximal-ward movement of the visceral endoderm comes from the analysis of panvisceral endoderm markers, which initially mark the cells overlying both embryonic and extraembryonic regions at PS-ES stages and are subsequently restricted to the visceral layer of the yolk sac at the no bud to early headfold (OB-EHF) stages (Downs and Davies, 1993Downs K.M. Davies T. Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope.Development. 1993; 118: 1255-1266PubMed Google Scholar, Duncan et al., 1994Duncan S.A. Manova K. Chen W.S. Hoodless P. Weinstein D.C. Bachvarova R.F. Darnell Jr., J.E. Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst.Proc. Natl. Acad. Sci. USA. 1994; 91: 7598-7602Crossref PubMed Scopus (301) Google Scholar, Tam et al., 2004Tam P.P. Khoo P.L. Wong N. Tsang T.E. Behringer R.R. Regionalization of cell fates and cell movement in the endoderm of the mouse gastrula and the impact of loss of Lhx1(Lim1) function.Dev. Biol. 2004; 274: 171-187Crossref PubMed Scopus (53) Google Scholar). The prevailing model suggests that definitive endoderm cells reach the outer surface of the embryo by inserting into and establishing a congruent epithelium with the preexisting visceral endoderm cell layer at the distal tip of the embryo. The proposed unidirectional (distal to proximal) displacement of the layer of cells at the surface of the embryo involves the active expansion of the definitive endoderm that pushes the adjacent epithelium of the visceral endoderm proximally toward extraembryonic regions (Hogan and Zaret, 2002Hogan B.L.M. Zaret K.S. Development of the endoderm and its tissue derivatives.in: Rossant J. Tam P.L. Mouse Development. Academic Press, San Diego, CA2002: 301-330Crossref Google Scholar). By the late streak (LS) stage, the endodermal layer overlying the epiblast is believed to be exclusively populated by the definitive endoderm, except for a region overlying the posterior primitive streak (Lawson et al., 1986Lawson K.A. Meneses J.J. Pedersen R.A. Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer.Dev. Biol. 1986; 115: 325-339Crossref PubMed Scopus (166) Google Scholar, Lawson and Pedersen, 1987Lawson K.A. Pedersen R.A. Cell fate, morphogenetic movement and population kinetics of embryonic endoderm at the time of germ layer formation in the mouse.Development. 1987; 101: 627-652PubMed Google Scholar, Tam and Beddington, 1992Tam P.P. Beddington R.S. Establishment and organization of germ layers in the gastrulating mouse embryo.Ciba Found. Symp. 1992; 165: 27-41PubMed Google Scholar). Soon after its formation, the gut endoderm folds and is internalized to form the gut tube (Hogan and Zaret, 2002Hogan B.L.M. Zaret K.S. Development of the endoderm and its tissue derivatives.in: Rossant J. Tam P.L. Mouse Development. Academic Press, San Diego, CA2002: 301-330Crossref Google Scholar, Tremblay and Zaret, 2005Tremblay K.D. Zaret K.S. Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues.Dev. Biol. 2005; 280: 87-99Crossref PubMed Scopus (212) Google Scholar) from which the respiratory and digestive tracts and associated visceral organs will develop (Wells and Melton, 1999Wells J.M. Melton D.A. Vertebrate endoderm development.Annu. Rev. Cell Dev. Biol. 1999; 15: 393-410Crossref PubMed Scopus (408) Google Scholar). Because no previous studies have examined the morphogenetic events underlying this process with a high degree of spatial or temporal resolution, little is known of the precise sequence of events and the cell behaviors involved. Anticipating that the events surrounding endoderm formation would be rapid and dynamic, we adopted a live imaging approach to investigate gut endoderm morphogenesis (Hadjantonakis et al., 2003Hadjantonakis A.K. Dickinson M.E. Fraser S.E. Papaioannou V.E. Technicolour transgenics: imaging tools for functional genomics in the mouse.Nat. Rev. Genet. 2003; 4: 613-625Crossref PubMed Scopus (131) Google Scholar). Our strategy involved genetic labeling of the entire visceral endoderm lineage for visualization of cells at high resolution in individual embryos and at multiple time points. To label the entire visceral endoderm, we used the Afp::GFP strain of mice (Kwon et al., 2006Kwon G.S. Fraser S.T. Eakin G.S. Mangano M. Isern J. Sahr K.E. Hadjantonakis A.K. Baron M.H. Tg(Afp-GFP) expression marks primitive and definitive endoderm lineages during mouse development.Dev. Dyn. 2006; 235: 2549-2558Crossref PubMed Scopus (58) Google Scholar), in which the enhanced fluorescent protein variant, EGFP, was placed downstream of cis-regulatory elements from the mouse Alpha-fetoprotein (Afp) locus (Andrews et al., 1982Andrews G.K. Dziadek M. Tamaoki T. Expression and methylation of the mouse alpha-fetoprotein gene in embryonic, adult, and neoplastic tissues.J. Biol. Chem. 1982; 257: 5148-5153Abstract Full Text PDF PubMed Google Scholar, Krumlauf et al., 1985Krumlauf R. Hammer R.E. Tilghman S.M. Brinster R.L. Developmental regulation of alpha-fetoprotein genes in transgenic mice.Mol. Cell. Biol. 1985; 5: 1639-1648Crossref PubMed Google Scholar, Kwon et al., 2006Kwon G.S. Fraser S.T. Eakin G.S. Mangano M. Isern J. Sahr K.E. Hadjantonakis A.K. Baron M.H. Tg(Afp-GFP) expression marks primitive and definitive endoderm lineages during mouse development.Dev. Dyn. 2006; 235: 2549-2558Crossref PubMed Scopus (58) Google Scholar, Tilghman and Belayew, 1982Tilghman S.M. Belayew A. Transcriptional control of the murine albumin/alpha-fetoprotein locus during development.Proc. Natl. Acad. Sci. USA. 1982; 79: 5254-5257Crossref PubMed Scopus (282) Google Scholar). Our results reveal a complex series of events that result in extensive mixing between the visceral endoderm and epiblast lineages during the morphogenesis of gut endoderm. At MS-LS stages, the visceral endoderm epithelium overlying the epiblast is dispersed concomitant with the intercalation of cells originating in the epiblast. Visceral endoderm cells are not displaced to extraembryonic regions. Instead, they remain in association with the epiblast and constitute a subpopulation of cells contributing to the gut tube. These dispersed visceral endoderm derived-cells are organized around distinct morphological signaling centers of the embryo, including the primitive streak, node, and midline. Taken together, our experiments provide the first glimpse of the live cell behaviors during gut endoderm morphogenesis in the mouse. Furthermore, they uncover a lineage relationship between the visceral endoderm of the early postimplantation mouse embryo and the endoderm-derived tissues of the fetus and adult. To visualize visceral endoderm cell dynamics and cell fate during endoderm morphogenesis, we used the Afp::GFP strain of mice (Kwon et al., 2006Kwon G.S. Fraser S.T. Eakin G.S. Mangano M. Isern J. Sahr K.E. Hadjantonakis A.K. Baron M.H. Tg(Afp-GFP) expression marks primitive and definitive endoderm lineages during mouse development.Dev. Dyn. 2006; 235: 2549-2558Crossref PubMed Scopus (58) Google Scholar). Live imaging of PS through MS stage Afp::GFP embryos confirmed continuous GFP expression exclusively in the surface cell layer, the location of the visceral endoderm (see Movie S1 available online). Three-dimensional (3D) rendering of xyz data confirmed Afp-GFP+ localization correlated with Afp mRNA localization in stage-matched embryos (Figures 1A and 1B; Figure S1A and Movie S2). Live imaging of embryos from LS through EHF stages (E7.0–E7.5), the time when definitive endoderm emerges (Lin et al., 1994Lin T.P. Labosky P.A. Grabel L.B. Kozak C.A. Pitman J.L. Kleeman J. MacLeod C.L. The Pem homeobox gene is X-linked and exclusively expressed in extraembryonic tissues during early murine development.Dev. Biol. 1994; 166: 170-179Crossref PubMed Scopus (98) Google Scholar, Tam and Beddington, 1992Tam P.P. Beddington R.S. Establishment and organization of germ layers in the gastrulating mouse embryo.Ciba Found. Symp. 1992; 165: 27-41PubMed Google Scholar), revealed a region of continuous Afp-GFP+ cells positioned proximally overlying the extraembryonic ectoderm and proximal epiblast (yellow arrowheads, Figures 1C and 1D) and a distinct distally positioned, dispersed population of Afp-GFP+ cells overlying the distal two-thirds of the epiblast (blue arrowheads, Figures 1C and 1D; Movie S3). The dispersed Afp-GFP+ population represented scattered single cells with irregular morphology (Figure 1E). Posterior views, however, showed a coherent sheet of Afp-GFP+ cells overlying the primitive streak (Figure 1F), likely corresponding to the posterior visceral endoderm (PVE) or its descendents (Rivera-Perez and Magnuson, 2005Rivera-Perez J.A. Magnuson T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3.Dev. Biol. 2005; 288: 363-371Crossref PubMed Scopus (179) Google Scholar). These observations contrasted with the prevailing model in which the visceral endoderm is sustained as a contiguous epithelium that is displaced to extraembryonic regions by emergent definitive endoderm. 3D reconstructions of LS-EHF imaging data confirmed that scattered Afp-GFP+ cells persisted on the surface layer of the embryo. By the late headfold (LHF) to early somite (ESom) stages (E7.75–8.25), GFP-negative cells predominated in the surface layer of the embryo. However, individual Afp-GFP+ cells were still present scattered over the distal region of the embryo as visualized in lateral views (Figure 1G; Movie S4) and were more abundant in posterior and distal locations. Posteriorly, Afp-GFP+ cells formed a contiguous sheet of cells overlying the primitive streak (Movie S5), and distally they were organized around the node and anterior midline (Figure 1H; Movie S6). To validate that the scattered population of cells overlying the epiblast was of visceral endoderm origin, we generated additional transgenic strains using cis-regulatory elements from another endoderm-specific gene, Transthyretin (Ttr). Cis-regulatory elements from the Ttr locus had previously been shown to drive transgene expression within the yolk sac endoderm and fetal liver (Costa et al., 1990Costa R.H. Van Dyke T.A. Yan C. Kuo F. Darnell Jr., J.E. Similarities in transthyretin gene expression and differences in transcription factors: liver and yolk sac compared to choroid plexus.Proc. Natl. Acad. Sci. USA. 1990; 87: 6589-6593Crossref PubMed Scopus (58) Google Scholar). Ttr mRNA expression assayed by in situ hybridization had also been shown to localize to the entire visceral endoderm at PS stages (Mesnard et al., 2006Mesnard D. Guzman-Ayala M. Constam D.B. Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning.Development. 2006; 133: 2497-2505Crossref PubMed Scopus (176) Google Scholar). Sequence comparisons of the Afp and Ttr cis-regulatory elements failed to reveal any large regions of homology, so these represented independent reporters of the visceral endoderm lineage. We generated Ttr::RFP strains in which the 3 kb upstream region and first exon of Ttr was used to drive expression of a monomeric red fluorescent protein (RFP) transgene (Campbell et al., 2002Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. A monomeric red fluorescent protein.Proc. Natl. Acad. Sci. USA. 2002; 99: 7877-7882Crossref PubMed Scopus (1917) Google Scholar). High-resolution imaging of Ttr::RFP embryos demonstrated a correlation of fluorescence with endogenous Ttr expression and revealed a scattered population of Ttr-RFP+ cells overlying the epiblast in LS-EB stage embryos (Figure 2A). This cell population resembled the population of Afp-GFP+ cells observed in Afp::GFP embryos. To determine whether the same cell population was labeled in the two transgenic strains, we generated double transgenic Afp::GFP ; Ttr::RFP embryos. Dispersed double fluorescent (Afp-GFP+; Ttr-RFP+) cells (yellow) predominated over the distal two-thirds of the epiblast, confirming that the same cell population is labeled in both transgenic strains (Figure 2B). Our findings suggested that either visceral endoderm cells became dispersed but remained overlying the epiblast or that the Afp::GFP transgene was activated within a subpopulation of epiblast-derived cells that occupied the outermost layer of the embryo overlying the epiblast. Support for the prevailing model of endoderm formation comes from the analysis of molecular markers (such as Afp and Ttr mRNAs), which are documented as either encompassing the entire epiblast (PS-MS stages) or being fully displaced (LS-EB stages). Our live imaging observations were in disagreement with these data. We therefore compared the localization of Afp-GFP+ cells to GFP mRNA in Afp::GFP embryos. We live imaged consecutively staged PS-EHF embryos (∼E5.75–E7.5), then processed them for whole-mount GFP mRNA in situ hybridization (Figures 2C–2F). This allowed us to determine whether GFP fluorescence was due to perdurance of the fluorescent protein in cells that were derived from the visceral endoderm. It would also reveal whether the Afp cis-regulatory elements active within visceral endoderm cells were not responsive to downregulation or if the Afp::GFP transgene was being activated in epiblast-derived cells in the outermost layer of the embryo. At PS-ES stages (E5.75–6.25), GFP protein localization tightly correlated with GFP mRNA in Afp::GFP embryos, being widespread throughout the visceral endoderm (Figures 2C and 2D). However, by the LS-EB stages, when Afp-GFP+ cells changed to a contiguous sheet overlying the extraembryonic ectoderm and proximal epiblast, and a scattered population of cells overlying the distal two-thirds of the epiblast, GFP mRNA was detected only in the visceral endoderm overlying extraembryonic regions and proximal epiblast (Figures 2E and 2F). This indicates that GFP protein perdures and functions as a short-term lineage tracer in Afp::GFP embryos and that scattered Afp-GFP+ cells overlying the epiblast originate within the visceral endoderm. To confirm that the scattered population of cells on the embryo surface was not due to heterogeneity in transgene expression, whereby neighboring visceral endoderm-derived cells exhibit concerted downregulation of cis-regulatory elements, we generated an additional transgenic strain where Cre recombinase was placed downstream of the Ttr-regulatory elements. Identical results were obtained when the Ttr::Cre strain was crossed to either GFP-based or LacZ-based Cre reporter strains. At PS stages, the entire visceral endoderm was labeled with the Cre reporter in agreement with observations made with the Afp::GFP and Ttr::RFP transgenes (Figure 2G). By the EB stage, a dispersed population of Cre reporter-expressing cells remained overlying the epiblast in Ttr::Cre embryos (Figure 2H). These data confirm that concerted downregulation of the Afp::GFP and Ttr::RFP transgenes did not occur, that the scattered population of cells overlying the epiblast is visceral endoderm derived, and that neighboring cells are of a different origin. When investigating Afp and Ttr mRNA expression (Law and Dugaiczyk, 1981Law S.W. Dugaiczyk A. Homology between the primary structure of alpha-fetoprotein, deduced from a complete cDNA sequence, and serum albumin.Nature. 1981; 291: 201-205Crossref PubMed Scopus (96) Google Scholar, Yamamura et al., 1985Yamamura K. Miki T. Suzuki N. Ebihara T. Kawai K. Kumahara Y. Honjo T. Introduction of mouse C epsilon genes into Cos-7 cells and fertilized mouse eggs.J. Biochem. (Tokyo). 1985; 97: 333-339PubMed Google Scholar) in whole-mount in situ preparations of LS-EB stage embryos, the time during which the visceral endoderm became dispersed, a small percentage of embryos exhibited speckled staining overlying the epiblast (black arrowheads, Figure S1A) in all batches of embryos examined. Although this type of coloration might commonly be overlooked and attributed to nonspecific background staining, it may instead reveal a rapid downregulation of marker expression. To determine whether scattered Afp-GFP+ cells overlying the epiblast expressed markers of the visceral endoderm, we analyzed the mRNA expression and protein distribution of Hnf4α, a visceral endoderm marker in PS-EB stage (E5.75–E7.5) embryos (Chen et al., 1994Chen W.S. Manova K. Weinstein D.C. Duncan S.A. Plump A.S. Prezioso V.R. Bachvarova R.F. Darnell Jr., J.E. Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos.Genes Dev. 1994; 8: 2466-2477Crossref PubMed Scopus (465) Google Scholar, Duncan et al., 1994Duncan S.A. Manova K. Chen W.S. Hoodless P. Weinstein D.C. Bachvarova R.F. Darnell Jr., J.E. Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo: HNF-4 is a marker for primary endoderm in the implanting blastocyst.Proc. Natl. Acad. Sci. USA. 1994; 91: 7598-7602Crossref PubMed Scopus (301) Google Scholar, Taraviras et al., 1994Taraviras S. Monaghan A.P. Schutz G. Kelsey G. Characterization of the mouse HNF-4 gene and its expression during mouse embryogenesis.Mech. Dev. 1994; 48: 67-79Crossref PubMed Scopus (141) Google Scholar). Low-magnification whole-mount imaging revealed that both Hnf4α mRNA and protein, which were localized throughout the visceral endoderm overlying both the epiblast and extraembryonic ectoderm at PS-ES stages (E5.75–E6.0), were downregulated in regions overlying the epiblast by the MS-LS stage (E6.5–7.0) and were localized proximally by the EB stage (E7.25–7.5) (Figures 2I, 2M, and 2Q; Figure S1). We confirmed that Hnf4α protein colocalized with GFP and was restricted to the surface layer of the embryo (Figure S1). High-magnification covisualization and quantification of Hnf4α protein and GFP in Afp::GFP embryos revealed that at the PS stage, all Afp-GFP+ cells exhibited nuclear-localized Hnf4α staining at equal levels (Figures 2J–2L). By the MS stage, even though Hnf4α protein was detected in all cells, those more distally positioned, overlying the epiblast, exhibited reduced levels of protein (white arrowheads, Figures 2N–2P). By the EB stage, Hnf4α protein was predominantly localized proximally overlying the extraembryonic ectoderm (Figures 2R–2T). Close inspection, however, revealed that some scattered Afp-GFP+ cells overlying the proximal epiblast expressed Hnf4α (white arrowheads, Figures 2R–2T) but that more distally located Afp-GFP+ cells lacked detectable Hnf4α expression (orange arrowheads, Figures 2S and 2T). These data indicate that distally positioned visceral endoderm-derived cells overlying the epiblast downregulate visceral endoderm markers. To confirm the visceral endoderm origin of cells observed overlying the epiblast in the Afp and Ttr transgenic strains, we needed to exclude the possibility that cells derived from the epiblast had activated transgene expression. To do this, we used three different approaches. First, we used an epigenetic approach to label cells of the epiblast by analyzing Xm/XpGFP female embryos carrying a paternally inherited copy of a widely expressed X-linked GFP transgene (Hadjantonakis et al., 1998bHadjantonakis A.K. Gertsenstein M. Ikawa M. Okabe M. Nagy A. Non-invasive sexing of preimplantation stage mammalian embryos.Nat. Genet. 1998; 19: 220-222Crossref PubMed Scopus (114) Google Scholar). In such embryos, the XpGFP transgene is imprinted, and thus silenced, in cells of the visceral endoderm and extraembryonic ectoderm, while cells of the epiblast are subject to random X-inactivation resulting in mosaic GFP expression (Hadjantonakis et al., 2001Hadjantonakis A.K. Cox L.L. Tam P.P. Nagy A. An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta.Genesis. 2001; 29: 133-140Crossref PubMed Scopus (93) Google Scholar). Imaging of double transgenic Xm/XpGFP; Ttr::RFP OB-EHF stage embryos revealed individual GFP+ or RFP+ cells. However, no double fluorescent cells were observed in over 200 cells counted (Figures 3A–3H). This indicates that X-GFP+ and Ttr-RFP+ cells in the outer layer of the embryo overlying the epiblast have distinct origins. (A–H) 3D reconstructions of laser scanning confocal z stacks taken through an LB stage XmXpGFP; Ttr::RFPTg/+ embryo. (E–H) High-magnification views of boxed region. RFP+ cells, all of which are located on the surface of the embryo (white arrowheads); GFP+ cells located on the surface of the embryo (orange arrowheads); GFP+ cells that are not superficially located (blue arrowheads)." @default.
• W2109293927 created "2016-06-24" @default.
• W2109293927 creator A5014695527 @default.
• W2109293927 creator A5015625581 @default.
• W2109293927 creator A5024702853 @default.
• W2109293927 date "2008-10-01" @default.
• W2109293927 modified "2023-10-12" @default.
• W2109293927 title "The Endoderm of the Mouse Embryo Arises by Dynamic Widespread Intercalation of Embryonic and Extraembryonic Lineages" @default.
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