Genetic identification of tissue of origin of cellular populations within the mouse placenta

Development ◽  
1985 ◽  
Vol 86 (1) ◽  
pp. 177-189
Author(s):  
J. Rossant ◽  
B. A. Croy
Keyword(s):  
Genetic Markers ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Complex Mixture ◽  
Placental Tissue ◽  
Genetic Identification ◽  
Blood Capillaries ◽  
Maternal Contribution ◽  
Foetal Blood

The mouse haemochorial placenta is a complex mixture of maternal cells and foetal trophectoderm and inner cell mass (ICM)-derived cells. The majority of the placental tissue is assumed to be trophoblast in origin but the exact extent and localization of the ICM and maternal contribution has not previously been determined. Using embryo transfer and reconstituted blastocyst techniques, combined with isozymal and in situ genetic markers, we have established that about 70% of the 13 to 15-day placenta is trophectoderm-derived, 30% is maternal in origin, and 4% develops from the ICM. Nearly all of the maternal contribution was confined to the spongiotrophoblast region and all of the ICM contribution was confined to the labyrinthine trophoblast region, where it formed the foetal blood capillaries and the endodermal sinuses. Using the same genetic markers, we showed that cell suspension techniques commonly used to produce ‘trophoblast’ cell preparations from placenta do not enrich for trophoblast, and, indeed, that collagenase, the preferred dissociation technique for cell viability, produced cell suspensions in which ICM and maternal cells were preferentially dissociated. No method for producing pure trophoblast populations has yet been found. Some unusually high ICM contributions to the placenta were found in reconstituted blastocyst experiments using ICMs isolated from early 3·5-day blastocysts, suggesting that these ICMs may have contributed to the trophectoderm layer of the blastocyst. These and other experiments suggest that the inner cell mass lineage may not be closed until some time after formation of the blastocyst.

Trophectodermal processes regulate the expression of totipotency within the inner cell mass of the mouse expanding blastocyst

Development ◽  
1984 ◽  
Vol 84 (1) ◽  
pp. 63-90
Author(s):  
Tom P. Fleming ◽  
Paul D. Warren ◽  
Julia C. Chisholm ◽  
Martin H. Johnson
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Central Area ◽  
Ultrastructural Changes ◽  
Trophectoderm Cells ◽  
Junctional Complexes ◽  
Secondary Lysosomes ◽  
Cytoplasmic Processes ◽  
Cellular Cover

Mouse blastocysts, aged 0, 2, 6 and 12 h from the onset of cavitation, were examined by transmission (TEM) and scanning (SEM) electron microscopy. In TEM sections, trophectoderm cells (TE) differed morphologically from those of the inner cell mass (ICM) by their flattened shape, paler cytosol staining and polarized disposition of both junctional complexes (apicolateral) and intracellular secondary lysosomes (SL; basal). Throughout this period of development, cytoplasmic processes, characterized by abundant SLs, cover approximately 80 % of the juxtacoelic face of the ICM. These processes are shown to be derived from the basal surface of TE cells intermediately placed between polar and mural regions. In SEM preparations of the juxtacoelic ICM surface, revealed by ‘cracking open’ blastocysts, the processes appear as tongue-shaped, centripetally oriented structures which terminate collectively at a central area on the ICM surface. The potential of cultured ICMs to generate TE was demonstrated following their immunosurgical isolation from blastocysts aged up to 12 h post cavitation and by examining the sequence of ultrastructural changes associated with TE generation by ICMs from 2 h blastocysts. In contrast, the juxtacoelic cells of similarly aged ICMs observed in situ in ultrasections of intact embryos showed little or no evidence of totipotency expression as judged by the absence of TE characteristics. Since TE expression within presumptive ICM cells is thought to be generated by an asymmetry of cell contacts (Johnson & Ziomek, 1983), we propose that the juxtacoelic TE processes, by providing a cellular cover to the ICM, function in suppressing the expression in situ of ICM totipotency.

Stem cell defects in parthenogenetic peri-implantation embryos

Development ◽  
1995 ◽  
Vol 121 (7) ◽  
pp. 2069-2077
Author(s):  
E.D. Newman-Smith ◽  
Z. Werb
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Insulin Like Growth Factor ◽  
Embryonic Antigen ◽  
Parietal Endoderm

Mouse embryos containing only maternal chromosomes (parthenotes) develop abnormally in vivo, usually failing at the peri-implantation stage. We have analyzed the development of parthenote embryos by using an inner cell mass (ICM) outgrowth assay that mimics peri-implantation development. ICMs from normal embryos maintained undifferentiated stem cells positive for stage-specific embryonic antigen-1 and Rex-1 while differentiating into a variety of cell types, including visceral endoderm-like cells and parietal endoderm cells. In contrast, ICMs from parthenotes failed to maintain undifferentiated stem cells and differentiated almost exclusively into parietal endoderm. This suggests that parthenote ICMs have a defect that leads to differentiation, rather than maintenance, of the stem cells, and a defect that leads to a parietal endoderm fate for the stem cells. To test the hypothesis that the ICM population is not maintained owing to a lack of proliferation of the stem cells, we investigated whether mitogenic agents were able to maintain the ICM population in parthenotes. When parthenote blastocysts were supplied with the insulin-like growth factor-1 receptor (Igf-1r) and insulin-like growth factor-2 (Igf-2), two genes not detectable in parthenote blastocysts by in situ hybridization, the ICM population was maintained. Similarly, culture of parthenote blastocysts in medium conditioned by embryonic fibroblasts and supplemented with the maternal factor leukemia inhibitory factor maintained the ICM population. However, once this growth factor-rich medium was removed, the parthenote ICM cells still differentiated predominantly into parietal endoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of the murine cyclin B2 cDNA and characterization of the lineage and temporal specificity of expression of the B1 and B2 cyclins during oogenesis, spermatogenesis and early embryogenesis

Development ◽  
1993 ◽  
Vol 118 (1) ◽  
pp. 229-240 ◽  
Author(s):  
D.L. Chapman ◽  
D.J. Wolgemuth
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Northern Blot ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Development ◽  
Early Embryogenesis ◽  
Northern Blot Hybridization ◽  
Germ Cell Development

A cDNA encoding the murine cyclin B2 (cycB2) was isolated from an adult mouse testis cDNA library as part of studies designed to identify cyclins involved in murine germ cell development. This cycB2 cDNA was then used to examine the pattern of cycB2 expression during male and female germ cell development and in early embryogenesis, and to compare this expression with the previously characterized expression of cycB1. A single 1.7 kb cycB2 transcript was detected by northern blot hybridization analysis of total RNA isolated from midgestation embryos and various adult tissues. Northern blot and in situ hybridization analyses revealed that cycB2 expression in the testis was most abundant in the germ cells, specifically in pachytene spermatocytes. This is in contrast to the highest levels of expression of cycB1 being present in early spermatids. In situ analysis of the ovary revealed cycB2 transcripts in both germ cells and somatic cells, specifically in the oocytes and granulosa cells of growing and mature follicles. The pattern of cycB1 and cycB2 expression in ovulated and fertilized eggs was also examined. While the steady state level of cycB1 and cycB2 signal remained constant in oocytes and ovulated eggs, signal of both appeared to decrease following fertilization. In addition, both cycB1 and cycB2 transcripts were detected in the cells of the inner cell mass and the trophectoderm of the blastocyst. These results demonstrate that there are lineage- and developmental-specific differences in the pattern of the B cyclins in mammalian germ cells, in contrast to the co-expression of B cyclins in the early conceptus.

The development potential of parthenogenetically derived cells in chimeric mouse embryos: implications for action of imprinted genes

Development ◽  
1988 ◽  
Vol 104 (1) ◽  
pp. 175-182 ◽  
Author(s):  
H.J. Clarke ◽  
S. Varmuza ◽  
V.R. Prideaux ◽  
J. Rossant
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Abnormal Development ◽  
Imprinted Genes ◽  
Chimeric Mouse ◽  
Paternal Genome ◽  
Transgenic Mouse Line ◽  
Parthenogenetic Embryos

Parthenogenetic embryos of mice die shortly after implantation and characteristically contain poorly developed extraembryonic tissue. To investigate the basis of the abnormal development of parthenotes, we combined them with normal embryos to produce chimeras and examined the distribution of the parthenogenetically derived cells during preimplantation and early postimplantation development. The parthenogenetic embryos were derived from a transgenic mouse line bearing a large insert, which allowed these cells to be identified in histological sections using in situ hybridization. At the blastocyst stage, the parthenogenetic embryos contributed cells to the trophectoderm (TE) and inner cell mass (ICM) of chimeras. By 6.5 days, however, in almost every embryo, parthenogenetically derived cells were not detected in the extraembryonic trophoblast tissue descended from the TE. In contrast, parthenogenetically derived cells could contribute to all descendants of the ICM of 6.5-and 7.5-day chimeras, including the extraembryonic visceral and parietal endoderm. Quantitative analysis of the degree of chimerism in the embryonic ectoderm at 6.5-7.5 days indicated that parthenogenetically derived cells could contribute as extensively as normal cells. These results indicate that normal trophoblast development requires gene expression from the paternally inherited genome before 6.5 days of embryogenesis. Tissues of the ICM lineage, however, apparently can develop independently of the paternal genome at least to 7.5 days of embryogenesis. Comparison of these results with those of others suggests that the influence of imprinted genes is manifested at different times and in a variety of tissues during development.

An in situ transgenic enzyme marker for the midgestation mouse embryo and the visualization of inner cell mass clones during early organogenesis

Development ◽  
1989 ◽  
Vol 106 (1) ◽  
pp. 37-46 ◽  
Author(s):  
R.S. Beddington ◽  
J. Morgernstern ◽  
H. Land ◽  
A. Hogan
Keyword(s):  
Cell Fate ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Early Organogenesis ◽  
Transgenic Mouse Strain ◽  
Transgenic Cells ◽  
Actin Promoter ◽  
Cell Autonomy ◽  
Lac Z

In order to study the deployment of cells during gastrulation and early organogenesis, it is necessary to have an in situ cell marker which can be used to follow cell fate. To create such a marker a transgenic mouse strain, designated Tg(Act-lac Z)-1, which carries 6 copies of the Escherichia coli lac Z gene under the control of the rat beta-actin promoter, was made by pronuclear injection of DNA. Staining early postimplantation hemizygous mouse conceptuses, during gastrulation and early organogenesis, for beta-galactosidase activity shows that lac Z expression is ubiquitous and constitutive in all epiblast derivatives of the 10th day conceptus. No activity is seen in trophectoderm and primitive endoderm derivatives. Postimplantation grafts of [3H]thymidine-labelled transgenic cells establish the cell autonomy of this transgenic marker. Preliminary observations on the distribution of inner cell mass (ICM) descendant clones, identified in situ in midgestation conceptuses, confirm the pluripotency of individual ICM cells. The implications regarding patterns of cell growth in nascent fetal primordia are discussed.

scholarly journals Identification of embryonic cell lineages in histological sections of M. musculus ↔ M. caroli chimaeras

Development ◽  
1983 ◽  
Vol 73 (1) ◽  
pp. 179-191
Author(s):  
J. Rossant ◽  
M. Vijh ◽  
L. D. Siracusa ◽  
V. M. Chapman
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Cell ◽  
Dna Probe ◽  
Marker System ◽  
Cell Lineages ◽  
Mus Caroli ◽  
First Time ◽  
Derivatives Of

An in situ cell marker system has been developed which allows identification of Mus caroli and Mus musculus cells in interspecific chimaeras. A radioactively labelled, cloned DNA probe to M. musculus satellite DNA was hybridized in situ to sections of M. musculus and M. caroli adult tissues. Autoradiography revealed high levels of hybridization to the nuclei of M. musculus cells, but little or no label bound to M. caroli cells. The DNA probe could also distinguish M. musculus and M. caroli cells in the same tissue section. Patches of labelled and unlabelled cells were clearly identified in sections of adult chimaeric tissues and also in the embryonic ectoderm of 6·5-day embryonic chimaeras. The ability to recognize M. musculus and M. caroli cells in sections of chimaeras should provide a powerful new tool in analyses of cell lineages in both embryonic and adult mouse chimaeras. The marker system has several advantages over other marker systems so far developed, the most important of which is its ubiquity. Since it is a nuclear marker, only cells without nuclei should be unsuited to its use. The potential of the marker system has been shown by its use in demonstrating directly for the first time the postimplantation derivatives of inner cell mass and trophectoderm in blastocysts ‘reconstituted’ with M. musculus trophectoderm and M. caroli inner cell mass.

scholarly journals 181 Oct-4: A POTENTIAL MARKER FOR PLURIPOTENCY IN CATTLE

2005 ◽  
Vol 17 (2) ◽  
pp. 241
Author(s):  
M. Vejlsted ◽  
B. Avery ◽  
J. Gjoerret ◽  
M. Schmidt ◽  
T. Greve ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Bovine Embryo ◽  
Feeder Cells ◽  
Paraffin Sections ◽  
Specific Staining ◽  
Potential Marker

The POU (Pit-Oct-Unc) domain transcription factor Oct-4 is one of the most acknowledged markers for pluripotency in murine and primate embryonic cells. At the blastocyst stage, expression of Oct-4 has been shown to remain high in the inner cell mass (ICM) while being rapidly down regulated in the trophectoderm (TE). Furthermore, in these species, expression of Oct-4 is maintained in pluripotent derivatives of the ICM and in embryonic stem (ES) cells, but lost upon differentiation. In the bovine embryo, a marker with similar qualities has long been sought. The aim of this study was to investigate, using a commercially available antibody for immunohistochemistry (IHC), whether Oct-4 might serve this role. In vitro produced (IVP) embryos were transferred to synchronized recipients at Day 6 post insemination (p.i) and flushed at Day 12; in vivo-derived embryos were flushed at Day 14. Day 8 IVP embryos (n = 20) were fixed and processed for IHC in paraffin sections together with Day 12 (n = 18) and Day 14 (n = 3) embryos. From Day 8 IVP embryos, outgrowth colonies (OCs) were formed by intact blastocyst culture on mouse SNL feeder cells. OCs were photographed using a stereomicroscope on Days 12, 14, and 16 p.i., and were examined for Oct-4 expression by in situ IHC at Day 16 p.i. (n = 94). From isolated embryonic discs of Day 12 embryos, OCs were derived by similar culture and were either processed for IHC on paraffin sections at Days 16, 18, and 20 p.i. (n = 9) or used for establishment of ES-like cell lines. Of colonies formed, representative specimens from each of the initial 5 passages (n = 18) were examined for Oct-4 expression either in paraffin sections or in situ. In Day 8 embryos, Oct-4 expression was demonstrated in all nuclei of both ICM and TE cells except for presumptive apoptotic ones. Approximately one-fifth of the OCs presented a substantial amount of Oct-4 positive cells of putative ICM, but also of TE origin. Apparently, the formation of Oct-4 positive OCs was favored by initial attachment of the embryonic pole to the feeder cells. In Day 12 and 14 embryos, specific and exclusive Oct-4 staining of nuclei of the complete epiblast, but not the hypoblast and the TE, was revealed. All OCs derived from Day 12 embryonic discs showed specific staining for Oct-4 in nuclei of putative epiblast origin only. On subsequent culture of these isolated epiblast derivatives, loss of Oct-4 staining from colonies was observed by passage 3. This study has, for the first time, shown expression of Oct-4 to be limited to pluripotent cells of bovine Day 12 and 14 embryos. Compared with murine and primate embryos, down-regulation of Oct-4 expression in bovine TE cells appears to be delayed. Findings indicate that Oct-4 may be used as a marker for pluripotency in bovine ES-like cells, although TE derivatives may maintain Oct-4 expression when isolated from Day 8 embryos.

scholarly journals Murine Blastocysts Release Mature MicroRNAs Into Culture Media That Reflect Developmental Status

2021 ◽  
Vol 12 ◽  
Author(s):  
David Connor Hawke ◽  
Danyal Baber Ahmed ◽  
Andrew John Watson ◽  
Dean Harvey Betts
Keyword(s):  
Late Stage ◽  
Inner Cell Mass ◽  
Culture Media ◽  
Quantitative Polymerase Chain Reaction ◽  
Cell Mass ◽  
Conditioned Media ◽  
Non Invasive ◽  
Mature Micrornas ◽  
Extracellular Mirna

Extracellular microRNA (miRNA) sequences derived from the pre-implantation embryo have attracted interest for their possible contributions to the ongoing embryonic–uterine milieu, as well as their potential for use as accessible biomarkers indicative of embryonic health. Spent culture media microdroplets used to culture late-stage E4.0 murine blastocysts were screened for 641 mature miRNA sequences using a reverse transcription–quantitative polymerase chain reaction–based array. We report here 39 miRNAs exclusively detected in the conditioned media, including the implantation-relevant miR-126a-3p, miR-101a, miR-143, and miR-320, in addition to members of the highly expressed embryonic miR-125 and miR-290 families. Based on these results, an miRNA panel was assembled comprising five members of the miR-290 family (miR-291-295) and five conserved sequences with significance to the embryonic secretome (miR-20a, miR-30c, miR-142-3p, miR-191, and miR-320). Panel profiling of developing embryo cohort lysates and accompanying conditioned media microdroplets revealed extensive similarities in relative quantities of miRNAs and, as a biomarker proof of concept, enabled distinction between media conditioned with differently staged embryos (zygote, 4-cell, and blastocyst). When used to assess media conditioned with embryos of varying degrees of degeneration, the panel revealed increases in all extracellular panel sequences, suggesting cell death is an influential and identifiable factor detectable by this assessment. In situ hybridization of three panel sequences (miR-30c, miR-294, and miR-295) in late-stage blastocysts revealed primarily inner cell mass expression with a significant presence of miR-294 throughout the blastocyst cavity. Furthermore, extracellular miR-290 sequences responded significantly to high centrifugal force, suggesting a substantial fraction of these sequences may exist within a vesicle such as an exosome, microvesicle, or apoptotic bleb. Together, these results support the use of extracellular miRNA to assess embryonic health and enable development of a non-invasive viability diagnostic tool for clinical use.

scholarly journals The selection arena in early human blastocysts resolves the pluripotent inner cell mass

2018 ◽  
Author(s):  
Manvendra Singh ◽  
Thomas J Widmann ◽  
Vikas Bansal ◽  
Jose L Cortes ◽  
Gerald G Schumann ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Major Gene ◽  
Cell Mass ◽  
Endogenous Retrovirus ◽  
Early Embryo ◽  
Human Embryos ◽  
Pluripotent Cells ◽  
Early Human

Is the human early embryo unique in lacking an inner cell mass (ICM) and having parallel rather than step-wise development? Here we reanalyse single-cell transcriptomic data and stain human embryos in situ to reveal both classical step-wise development and the missing ICM, a transcriptomic homolog of macaque ICM, that differentiates to epiblast and primitive endoderm. This apparent classicism obscures numerous features that render our blastocyst phylogenetically distinct: unlike mice, human epiblast has hallmarks of self-renewal and we have abundant, previously unrecognized, blastocyst non-committed cells (NCCs), part of an apoptosis-mediated quality control/purging process. Comparative transcriptomics further reveals the transcriptomes of the pluripotent cells to be especially fast evolving, rendering all primate embryos unique. Rapid transcriptome turnover is in large part owing to endogenous retrovirus H (ERVH) activity, ERVH being associated with recent major gene expression gain and loss events of pluripotency-associated genes. Each species is characterised by the ERVHs that are active and the neighbour genes whose expression are in turn modulated. The current portfolio of naive cultures, putative in vitro mimics of pluripotent cells, are both developmentally and phylogenetically "confused" in part owing to a lack of HERVH expression.

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