Investigation of cell lineage and differentiation in the extraembryonic endoderm of the mouse embryo

Development ◽  
1982 ◽  
Vol 68 (1) ◽  
pp. 175-198
Author(s):  
R. L. Gardner
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Early Embryo ◽  
Visceral Endoderm ◽  
Primitive Endoderm ◽  
Developmental Potential ◽  
Extraembryonic Endoderm ◽  
Parietal Endoderm ◽  
Endodermal Cells

The technique of injecting genetically labelled cells into blastocysts was used in an attempt to determine whether the parietal and visceral endoderm originate from the same or different cell populations in the early embryo. When the developmental potential of 5th day primitive ectoderm and primitive endoderm cells was compared thus, only the latter were found to colonize the extraembryonic endoderm. Furthermore, single primitive endoderm cells yielded unequivocal colonization of both the parietal and the visceral endoderm in a proportion of chimaeras. However, in the majority of primitive endodermal chimaeras, donor cells were detected in the parietal endoderm only, cases of exclusively visceral colonization being rare. Visceral endoderm cells from 6th and 7th day post-implantation embryos also exhibited a striking tendency to contribute exclusively to the parietal endoderm following blastocyst injection. The above findings lend no support to a recent proposal that parietal and visceral endoderm are derived from different populations of inner cell mass cells. Rather, they suggest that the two extraembryonic endoderm layers originate from a common pool of primitive endoderm cells whose direction of differentiation depends on their interactions with non-endodermal cells.

scholarly journals The origin of mouse extraembryonic endoderm stem cell lines

2021 ◽  
Author(s):  
Jiangwei Lin
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Cell Lineage ◽  
Preimplantation Embryos ◽  
Visceral Endoderm ◽  
Primitive Endoderm ◽  
Cellular Origin ◽  
Extraembryonic Endoderm ◽  
Parietal Endoderm ◽  
Stem Cell Lines

Mouse extraembryonic endoderm stem (XEN) cell lines can be derived from preimplantation embryos (pre-XEN) and postimplantation embryos (post-XEN). XEN cells share a gene expression profile and cell lineage potential with primitive endoderm (PrE) blastocysts. However, the cellular origin of XEN cells in embryos remains unclear. Here, we report that post-XEN cell lines are derived both from the extraembryonic endoderm and epiblasts of postimplantation embryos and that pre-XEN cell lines are derived both from PrE and epiblasts of blastocysts. Our strategy consisted of deriving post-XEN cells from clumps of epiblasts, parietal endoderm (PE) and visceral endoderm (VE) and deriving pre-XEN cell lines from single PrE and single epiblasts of blastocysts. Thus, XEN cell lines in the mouse embryo originate not only from PrE and PrE-derived lineages but also from epiblast and epiblast-derived lineages of blastocysts and postimplantation embryos.

An in situ cell marker for clonal analysis of development of the extraembryonic endoderm in the mouse*

Development ◽  
1984 ◽  
Vol 80 (1) ◽  
pp. 251-288
Author(s):  
R. L. Gardner
Keyword(s):  
Enzyme Activity ◽  
Malic Enzyme ◽  
Cell Mass ◽  
Differential Staining ◽  
Wild Type ◽  
Primitive Endoderm ◽  
Extraembryonic Endoderm ◽  
Blue Tetrazolium ◽  
Malic Enzyme Activity ◽  
Parietal Endoderm

Conditions were found for staining whole mid-gestation capsular parietal endoderms and visceral yolk sacs for malic enzyme activity that gave excellent discrimination between wildtype (Mod-1+/Mod-1+) cells and mutant (Mod-ln/Mod-1n) cells that lack the cytoplasmic form of the enzyme. Reciprocal blastocyst injection experiments were undertaken in which single primitive endoderm cells of one genotype were transplanted into embryos of the other genotype. In addition, Mod-1+/Mod-1+ early inner cell mass (ICM) cells were injected into Mod-1n/Mod-1n blastocysts, either in groups of two or three singletons or as daughter cell pairs. A substantial proportion of the resulting conceptuses showed mosaic histochemical staining in the parietal endoderm, visceral yolk sac, or in both these membranes. Stained cells were invariably intimately intermixed with unstained cells in the mosaic parietal endoderms. In contrast, one or both of two distinct patterns of staining could be discerned in mosaic visceral yolk sacs. The first, a conspicuously ‘coherent’ pattern, was found to be due to endodermal chimaerism; the second, a more diffuse pattern, was attributable to chimaerism in the mesodermal layer of this membrane. The overall distribution of cells with donor staining characteristics resulting from primitive endoderm versus early ICM cell injections was consistent with findings in earlier experiments in which allozymes of glucosephosphate isomerase were used as markers. The conspicuous lack of phenotypically intermediate cells in predominantly stained areas of mosaic membranes suggested that the histochemical difference between Mod-1+/Mod-1+ and Mod-1n/Mod-ln genotypes was cell-autonomous. This conclusion was strengthened by the results of staining mixed in vitro cultures of parietal endoderm in which presence or absence of phagocytosed melanin granules was used as an independent means of distinguishing wild type from null cells. By substituting tetranitro blue tetrazolium for nitro blue tetrazolium in the incubation medium, satisfactory differential staining was obtained for both the extraembryonic endoderm and other tissues of earlier postimplantation wild type versus null embryos. Finally, absence of cytoplasmic malic enzyme activity does not appear to have a significant effect on the viability or behaviour of mutant cells.

scholarly journals Cell lineage allocation in equine blastocysts produced in vitro under varying glucose concentrations

Reproduction ◽  
2015 ◽  
Vol 150 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Young-Ho Choi ◽  
Pablo Ross ◽  
Isabel C Velez ◽  
B Macías-García ◽  
Fernando L Riera ◽  
...  
Keyword(s):  
High Glucose ◽  
Culture Medium ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Primitive Endoderm ◽  
Blastocyst Development ◽  
Embryo Culture Medium ◽  
Effect Of Glucose

Equine embryos developin vitroin the presence of high glucose concentrations, but little is known about their requirements for development. We evaluated the effect of glucose concentrations in medium on blastocyst development after ICSI. In experiment 1, there were no significant differences in rates of blastocyst formation among embryos cultured in our standard medium (DMEM/F-12), which contained >16 mM glucose, and those cultured in a minimal-glucose embryo culture medium (<1 mM; Global medium, GB), with either 0 added glucose for the first 5 days, then 20 mM (0-20) or 20 mM for the entire culture period (20-20). In experiment 2, there were no significant differences in the rates of blastocyst development (31–46%) for embryos cultured in four glucose treatments in GB (0-10, 0-20, 5-10, or 5-20). Blastocysts were evaluated by immunofluorescence for lineage-specific markers. All cells stained positively forPOU5F1. An inner cluster of cells was identified that included presumptive primitive endoderm cells (GATA6-positive) and presumptive epiblast (EPI) cells. The 5-20 treatment resulted in a significantly lower number of presumptive EPI-lineage cells than the 0-20 treatment did.GATA6-positive cells appeared to be allocated to the primitive endoderm independent of the formation of an inner cell mass, as was previously hypothesized for equine embryos. These data demonstrate that equine blastocyst development is not dependent on high glucose concentrations during early culture; rather, environmental glucose may affect cell allocation. They also present the first analysis of cell lineage allocation inin vitro-fertilized equine blastocysts. These findings expand our understanding of the factors that affect embryo development in the horse.

145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO

2009 ◽  
Vol 21 (9) ◽  
pp. 63
Author(s):  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Pluripotent Cells ◽  
Drug Induced ◽  
Developmental Potential

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.

In vitro development of inner cell masses isolated immunosurgically from mouse blastocysts

Development ◽  
1978 ◽  
Vol 45 (1) ◽  
pp. 107-121
Author(s):  
Brigid Hogan ◽  
Rita Tilly
Keyword(s):  
Outer Layer ◽  
Normal Mouse ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Giant Cells ◽  
Visceral Endoderm ◽  
Floating Structures ◽  
Parietal Endoderm ◽  
Vitro Development

This paper describes the development in culture of inner cell masses isolated immunosurgically from C3H/He mouse blastocysts immediately after collection between 3·5 and 4·0 days p.c. By 24–48 h most of the inner cell masses isolated from half-expanded blastocysts, and about 50% of those from expanded blastocysts, regenerate an outer layer of trophectoderm- like cells and so resemble mini-blastocysts. With further in vitro culture these structures attach to the substratum and give rise to trophoblast-like giant cells, together with clusters of parietal endoderm cells or inner cell masses surrounded by visceral endoderm. Many of the inner cell masses from the remaining expanded blastocysts develop into floating structures with an outer layer of endoderm cells, and by 7 days consist of a large fluid filled cyst surrounding a collapsed vesicle of epithelial cells. Mesodermal cells line the cysts and form numerous blood islands. When mechanically disrupted, and grown as attached sheets of cells, these cystic structures give rise to patches of trophoblast-like giant cells similar to those described in the previous paper. These results suggest that the inner cell mass of normal mouse blastocysts contains cells which are capable of giving rise to trophoblast in culture.

Cell interactions and endoderm differentiation in cultured mouse embryos

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 379-394
Author(s):  
Brigid L. M. Hogan ◽  
Rita Tilly
Keyword(s):  
Basement Membrane ◽  
Mouse Embryo ◽  
Cell Lineage ◽  
Giant Cells ◽  
Visceral Endoderm ◽  
Extraembryonic Endoderm ◽  
Type Iv ◽  
The Matrix ◽  
Parietal Endoderm

Morphological and biochemical evidence is presented that the visceral extraembryonic endoderm of the 6·5-day mouse embryo will differentiate into parietal endoderm when cultured in contact with extraembryonic ectoderm undergoing transition into trophoblast giant cells. Egg cylinders from 6·5-day embryos were dissected into embryonic and extraembryonic halves and cultured in suspension in vitro for up to 7 days. After 4 days, the endoderm cells of the extraembryonic fragments morphologically resemble parietal endoderm, are associated with a thick basement membrane and synthesize large amounts of the matrix proteins laminin and Type IV procollagen. A similar transition in phenotype is not seen in the endoderm of embryonic fragments, nor in visceral extraembryonic endoderm cells cultured in isolation. In another series of experiments, complete egg cylinders were dissected free of visceral endoderm ovei lying the extraembryonic ectoderm and then cultured in vitro. The visceral endoderm cells which recolonize the surface of the extraembryonic ectoderm develop a parietal endoderm phenotype and lay down a thick basement membrane. These results suggest that the differentiation of the extraembryonic endoderm of the early mouse embryo into visceral and parietal phenotypes can be influenced by local cell—cell or cell—substrate interactions, and is not determined solely by cell lineage.

scholarly journals Analysis of accessible chromatin landscape in the inner cell mass and trophectoderm of human blastocysts

2020 ◽  
Vol 26 (9) ◽  
pp. 702-711
Author(s):  
Min Yang ◽  
Xin Tao ◽  
Shiny Titus ◽  
Tianhua Zhao ◽  
Richard T Scott ◽  
...  
Keyword(s):  
Epigenetic Regulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Distal Region ◽  
Chromatin Accessibility ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Accessible Chromatin ◽  
Human Preimplantation Embryos

Abstract Early embryonic development is characterized by drastic changes in chromatin structure that affects the accessibility of the chromatin. In human, the chromosome reorganization and its involvement in the first linage segregation are poorly characterized due to the difficulties in obtaining human embryonic material and limitation on low input technologies. In this study, we aimed to explore the chromatin remodeling pattern in human preimplantation embryos and gain insight into the epigenetic regulation of inner cell mass (ICM) and trophectoderm (TE) differentiation. We optimized ATAC-seq (an assay for transposase-accessible chromatin using sequencing) to analyze the chromatin accessibility landscape for low DNA input. Sixteen preimplantation human blastocysts frozen on Day 6 were used. Our data showed that ATAC peak distributions of the promoter regions (&lt;1 kb) and distal regions versus other regions were significantly different between ICM versus TE samples (P &lt; 0.01). We detected that a higher percentage of accessible binding loci were located within 1 kb of the transcription start site in ICM compared to TE (P &lt; 0.01). However, a higher percentage of accessible regions was detected in the distal region of TE compared to ICM (P &lt; 0.01). In addition, eight differential peaks with a false discovery rate &lt;0.05 between ICM and TE were detected. This is the first study to compare the landscape of the accessible chromatin between ICM and TE of human preimplantation embryos, which unveiled chromatin-level epigenetic regulation of cell lineage specification in early embryo development.

202 INNER CELL MASS EXCHANGE IN SHEEP EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 218 ◽  
Author(s):  
P. Loi ◽  
K. Matsukawa ◽  
C. Galli ◽  
G. Ptak
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Lineage ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Linear Probe ◽  
Mammalian Embryos ◽  
Natal Mortality ◽  
Normal Offspring

The presence of a developmental axis in the mammalian oocyte/embryo is still a controversial issue (Plusa 2005 Nature 17, 391–395). However, pre-established or not, mammalian blastocysts display a clear asymmetry with distinct embryonic and abembryonic poles. The present emphasis on ‘mosaic’ development in mammalian embryos is in contrast with classical embryological work, aimed at cell lineage analysis, where manipulation procedures severely perturbed the natural blastocyst asymmetry (Gardner 2001 RBM Online 4, 46–51). However, all of the experimental work thus far has been carried out on mouse embryos. In our work, we designed experiments to determine whether sheep embryos subjected to inner cell mass (ICM) transfer retain normal developmental competence. In vitro-derived sheep blastocysts (Ptak et al. 2003 Biol. Reprod. 69, 278–285) were manipulated with a Narishige micromanipulator fitted to a inverted Nikon microscope. ICMs were dissected with a blade, and the trophoblastic vesicle and ICMs were cultured in SOFaa plus 10% FCS. After re-expansion, trophoblastic vesicles were injected with ICMs by means of a bevelled pipette and cultured overnight with SOFaa plus 10% FCS. From a total of 35 blastocysts used, 25 re-expanded following injection, and 20 of them showed ICMs adherent to the trophoblast. Seven blastocysts were transferred into synchronized ewes 7 days after estrus, and monitored every month with an Aloka linear probe (7–5 MHz; Aloka Co., Ltd., Tokyo, Japan). Twenty-one in vitro-produced (IVP) embryos were transferred as a control. Three ewes receiving ICM-exchanged blastocysts were pregnant at the first scanning, and all delivered normal offspring (two female and one male lamb; weight: 3.54 ± 0.358 kg). These data demonstrate that dramatic alteration of the blastocyst structure does not compromise its developmental potential. Our efficiency in terms of offspring is lower compared with control IVP embryos, and also compared to data obtained in mice (Papaioannou 1982 J. Embryol. Exp. Morph. 68, 199–209), but technical improvements are expected to reduce such a gap. In conclusion, we demonstrated the feasibility of ICM/trophoblastic exchange in sheep blastocysts; these results might have important application for technologies like somatic cell nuclear transfer (SCNT). Common features of SCNT clones are placental abnormalities in early (DeSousa et al. 2001 Biol. Reprod. 65, 23–30) and late pregnancies (Loi et al. 2006 Theriogenology 65, 1110–1121). The transfer of ICM from cloned embryos to normal trophoblastic vesicles, although ineffective in cattle (Murakami et al. 2006 Cloning Stem Cells 8, 51–69), might be worth trying on sheep, a species where post-natal mortality in clones is a serious issue. Table 1.Development to term of manipulated and cloned embryos Part of this work was supported by EUROSTELLS-European Science Foundation.

scholarly journals Primitive endoderm differentiation: from specification to epithelium formation

2014 ◽  
Vol 369 (1657) ◽  
pp. 20130537 ◽  
Author(s):  
Stéphanie Hermitte ◽  
Claire Chazaud
Keyword(s):  
Molecular Mechanisms ◽  
Inner Cell Mass ◽  
Embryo Implantation ◽  
Cell Mass ◽  
Cell Lineage ◽  
Primitive Endoderm ◽  
Lineage Specification ◽  
Embryo Patterning ◽  
Cell Lineages ◽  
Endoderm Differentiation

In amniotes, primitive endoderm (PrE) plays important roles not only for nutrient support but also as an inductive tissue required for embryo patterning. PrE is an epithelial monolayer that is visible shortly before embryo implantation and is one of the first three cell lineages produced by the embryo. We review here the molecular mechanisms that have been uncovered during the past 10 years on PrE and epiblast cell lineage specification within the inner cell mass of the blastocyst and on their subsequent steps of differentiation.

scholarly journals Stage specific response in early mouse embryos exposed to prednisolone in vitro

2020 ◽  
Author(s):  
Shubhashree Uppangala ◽  
Akshatha Daddangadi ◽  
Jeena Susan Joseph ◽  
Sujit Raj Salian ◽  
Riddhi Kirit Pandya ◽  
...  
Keyword(s):  
Embryo Development ◽  
Inner Cell Mass ◽  
Recurrent Miscarriage ◽  
Cell Mass ◽  
Cell Number ◽  
Early Embryo ◽  
Specific Response ◽  
Clinical Value ◽  
Developmental Potential

Corticosteroids are increasingly being used during the peri-implantation period to treat women with repeated IVF failure and recurrent miscarriage. However, the direct effects of prednisolone (PRDL), one of the commonly used corticosteroids on early embryo development is not understood. To mimic the possible clinical scenario and to understand the embryonic response to direct PRDL exposure, this pilot study was conducted in a mouse model. Cleavage stage embryos exposed to 3 and 30µM PRDL in vitro were assessed for peri-implantation developmental potential, genetic integrity, inner cell mass (ICM) proliferation and pluripotency markers in the proliferated ICM cells. Exposure to 30µM PRDL delayed the embryonic progression beyond compaction (P<0.05) in comparison to vehicle control and, had reduced total cell number (P<0.001) than all other groups. In addition, 30µM PRDL exposure resulted in poor hatching potential (P<0.05) and increased apoptosis in blastocysts (P<0.05) compared to 3µM PRDL. On the other hand, completely formed ICM outgrowths were significantly higher (P<0.05) in 3µM PRDL compared to control. However, no significant differences were observed in the expression of pluripotency genes. In conclusion, the trend observed in embryos exposed to PRDL in vitro provides important information concerning the use of this drug when treating patients at the peri-implantation phase of IVF cycles. However, the clinical value of this observation on human embryo development needs further research.

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