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PubMed1991

The retinol-binding protein of the expanding pig blastocyst: molecular cloning and expression in trophectoderm and embryonic disc.

Retinol-binding protein (RBP) is a major secretory product of the porcine conceptus. Using an oligonucleotide probe corresponding to a highly conserved region of all known mammalian RBP, we have isolated an apparently full-length cDNA clone for porcine conceptus RBP from a cDNA library constructed from pig conceptuses collected between days 13-17 of pregnancy. The cDNA was 937 base-pairs in length and coded for a protein whose inferred amino-terminal sequence was identical to that reported for both porcine conceptus RBP and porcine serum RBP. Its length was consistent with the size (approximately 1 kilobase) of the RBP message in porcine conceptuses. Porcine conceptus RBP and human serum RBP share 91% amino acid sequence identity. The inferred differences in sequence were evenly distributed throughout the length of the polypeptide. RBP mRNA was detectable within the trophoblast of day 11 porcine conceptuses by in situ hybridization with a 618-basepair 35S-labeled probe corresponding to the 3' end of porcine RBP. Silver grain density was distributed relatively uniformly over the trophoblast and the inner cell mass. Western blot analysis of conceptus culture medium demonstrated that the conceptuses of cattle (on day 19) and sheep (on day 15) as well as pigs secrete RBP during early pregnancy. Secretion of large quantities of RBP by the trophoblast of preimplantation pig conceptuses suggests important roles for vitamin A and RBP near the time of conceptus elongation.

Amino Acid Sequence↗

PubMed1998

The murine Polycomb-group gene eed and its human orthologue: functional implications of evolutionary conservation.

Similar to Drosophila, murine Polycomb-group (PcG) genes regulate anterior-posterior patterning of segmented axial structures by transcriptional repression of homeotic gene expression. The murine PcG gene eed (embryonic ectoderm development) encodes a 441-amino-acid protein with five WD motifs which, except for the amino terminus, is highly homologous to Drosophila ESC (Extra Sex Combs). Here, sequence and expression analysis as well as chromosomal mapping of the human orthologue of eed is described. Absolute conservation of the human eed protein along with significant divergence at the nucleotide level reveals functional constraints operating on all residues. The human orthologue appears to be ubiquitously expressed and maps to chromsome 11q14.2-q22.3. Using the first WD motif of the beta-subunit of the bovine G protein as a structural reference, the predicted locations of two previously identified eed point mutations (A. Schumacher et al., 1996, Nature 383: 250-253) are also reported herein. The proline substitution (L196P) in the second WD motif of the l7Rn5(3354SB) null allele maps to the internal core of the inner end of the beta-propeller blade and is likely to disrupt protein folding. In contrast, the asparagine substitution (I193N) in the second WD motif of the hypomorphic l7Rn5(1989SB) allele maps onto the surface of the beta-propeller blade near the central cavity and may affect surface interactions without compromising propeller packing. These results illustrate the critical importance of all residues for eed function in mammals and support a model whereby the amino terminus might implement function(s) related to embryonic development in higher organisms.

5' Untranslated Regions↗

PubMed1993

Trophectoderm microbiopsy in murine blastocysts: comparison of four methods.

PURPOSE: This study compares four trophectoderm microbiopsy techniques for removal of blastomeres from murine blastocysts: (1) aspiration, trophectoderm pipetted through the zona; (2) incision, trophectoderm excised with a microrazor; (3) slit/excision, the zona slit and herniating trophectoderm excised; and (4) hatch/excision, trophectoderm cells excised after spontaneous hatching. RESULTS: Murine blastocysts were comparatively biopsied using one of four methods and contrasted to zona slit and nonmicromanipulated controls. Operative cellular injury was assessed by uptake of fluorescein isothiocyanate (FITC). Postoperative embryonic viability was assessed by blastocoele reexpansion and hatching inner cell mass development and trophectoderm plating. All techniques yielded cells available for genetic analysis. CONCLUSIONS: The slit/excision technique and hatch/excision techniques exhibited lower operative injury and the higher postoperative viability than aspiration or incision. The slit/excision and the hatch/excision techniques, though requiring two operative steps, appear to be the least damaging of the four methods. Therefore, they should be most applicable to human blastocysts obtained either by extended culture in vitro or by uterine lavage.

Animals↗

PubMed1996

Development of bovine and porcine embryonic teratomas in athymic mice.

Inner cell masses (ICM) and embryonic discs from bovine and porcine blastocysts of various ages were transplanted under the kidney capsule of athymic (nude) mice to evaluate growth of teratocarcinomas containing both differentiated tissues and undifferentiated stem cells. Inner cell masses were isolated immunosurgically from Day 8, Day 9 and Day 10 porcine blastocysts and from Day 8, Day 10 and Day 12 bovine blastocysts. Embryonic discs were mechanically dissected from Day 11 and Day 12 porcine embryos and from Day 14 bovine embryos. Day 6 egg cylinders were dissected from BALB/C embryos and from hybrid embryos of a cross between BALB/C and an outbred strain of mouse. Two to four ICM, embryonic discs or egg cylinders were transplanted under the kidney capsule of each athymic host. After 8 weeks, graft hosts were killed and their tumors removed, fixed and prepared for histological and immunohistochemical examination. Embryonic teratomas developed at high frequency from murine egg cylinders and from Day 11 and Day 12 porcine and Day 14 bovine embryos. Tumors were observed only infrequently from younger bovine and porcine blastocysts. Murine embryonic tumors were composed of numerous differentiated cell types of ectodermal, mesodermal and endodermal origins, but representation of the three embryonic germ layers was somewhat more restricted in bovine and porcine embryonic tumors. No undifferentiated stem cells were detected in tumors of any of the three species. These results demonstrate that teratomas will develop from bovine and porcine embryos when grafted to an immunocompromised host, but the presence of undifferentiated teratocarcinoma stem cells from these species has yet to be achieved.

Animals↗

PubMed1983

Cell position regulates endodermal differentiation in embryonal carcinoma cell aggregates.

It has been suggested that cell position regulates endodermal differentiation in mouse embryo inner cell masses and in aggregates of embryonal carcinoma (EC) cells. This hypothesis states that cells at the interface between the cell mass and blastocoel fluid or culture medium differentiate into endoderm, whereas internally located cells follow alternative developmental pathways. To test the cell position hypothesis, pluripotent PSA-1 cells were aggregated with hypoxanthine phosphoribosyltransferase-deficient, parietal-like, endodermal cells. The resulting aggregates consisted of cores of PSA-1 cells surrounded by endodermal cells. Autoradiography was used to distinguish between endodermal cells that were the products of EC cell differentiation and the exogenous endoderm. Alkaline phosphatase staining was used to distinguish EC cells from endodermal cells. As predicted by the cell position hypothesis, the PSA-1 EC cells, all of which were internally located, did not differentiate into endodermal cells. Nonspecific inhibition of differentiation did not account for the lack of PSA-1-derived endoderm since the PSA-1 cells in such aggregates did differentiate into columnar ectodermal-like cells. Similar experiments were also conducted with F9 cells. In this case, aggregation cultures contained retinoic acid to induce F9 cells to differentiate into visceral endoderm. In cultures containing F9 cells surrounded by parietal-like endodermal cells, no F9-derived endoderm was detected either autoradiographically or by assaying for alpha-fetoprotein production, a visceral endoderm marker. Thus, retinoic acid-induced endodermal differentiation was also regulated by cell position. Collectively, the above results provide strong evidence for the hypothesis that cell position regulates endodermal differentiation in aggregates of EC cells.

Alkaline Phosphatase↗

PubMed2006

The production and directed differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of early progenitors in this lineage. There are reports of enriched cultures of keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurons, hepatic progenitors, and cells that have some markers of gut tissue and pancreatic islet-like cells. The prospects for use of hESC derivatives in regenerative medicine are significant, and there is much optimism for their potential contributions to human regenerative medicine.

Biomarkers↗

PubMed1989

Impact of cellular and molecular organization on lens metabolism.

The relationship between the metabolic gradient within the ocular lens and its cellular and molecular organization is discussed. The lens shares a number of organizational similarities with other stratified ectodermal tissues. All of these tissues were avascular and therefore, dependent upon the simple diffusion of nutrients from the surrounding medium. They contain non-metabolizing cell layers which are continually produced by an irreversible maturation process. This process is not random aging but a well orchestrated sequence of events dependent upon the physico-chemical properties of specialized proteins typical of each tissue. In the lens, these proteins are the crystallins. The signal for this maturation is decreased metabolism in conjunction with cell dehydration. Metabolism of these maturing cells is decreased by limited nutrient penetration because of barriers to nutrient diffusion and the high rate of utilization of nutrients by the more metabolically active cells near the basement membrane. Additionally, the regulation of anaerobic metabolism is dependent upon the maintenance and dissolution of an organized array of enzymes and the effects of dehydration, i.e. excluded volume effects and modified macromolecular organization. In the lens, the absence of metabolism during the maturation of the inner fiber cell layers is as important as the presence of active metabolism where differentiation and active protein synthesis occur. Therefore, any stress that disrupts tissue organization will have a detrimental effect on lens integrity.

Aging↗

PubMed2005

Emerin expression in early development of Xenopus laevis.

Emerin is an integral protein of the inner nuclear membrane in the majority of differentiated vertebrate cells. In humans, deficiency of emerin causes a progressive muscular dystrophy of the Emery-Dreifuss type. The physiological role of emerin is poorly understood. By screening and sequencing of EST clones we have identified two emerin homologues in Xenopus laevis, Xemerin1 and Xemerin2. Xemerins share with mammalian emerins the N-terminal LEM domain and a single transmembrane domain at the C-terminus. As shown by immunoblot analysis with Xemerin-specific antibodies, both proteins have an apparent molecular mass of 24 kDa but differ in their isoelectric points. Xemerin1 and Xemerin2 proteins are not detectable in oocytes nor during early embryogenesis. Protein expression is first found at stage 43 and persists in somatic cells. However, RT-PCR and Northern blot analysis show Xemerin mRNAs of approximately 4.0 kb to be present in oocytes and throughout embryogenesis. During embryogenesis the level of Xemerin mRNAs increases at stage 22 and is particularly abundant in mesodermal and neuro-ectodermal regions of the embryo. These data provide the necessary background to further investigate the role of emerin in nuclear envelope assembly, gene expression and organ development of X. laevis as a model organism.

Amino Acid Sequence↗

PubMed2000

Oct-4 expression in inner cell mass and trophectoderm of human blastocysts.

The expression of the transcription factor Oct-4 is thought to be one of the decisive factors that maintain totipotency in embryonic and germ cells. In mice, oct-4 is exclusively expressed in germ cells and totipotent cells of the embryo. In humans, Oct-4 is expressed in germ cells, embryonic stem cells and whole embryos at various stages of development. However, there is limited information about the distribution of Oct-4 expression in human embryos. In an attempt to address this issue, the inner cell mass (ICM) and trophectoderm (TE) of 17 human blastocysts were separated and Oct-4 mRNA expression individually assessed by reverse transcription-polymerase chain reaction (RT-PCR). In discarded blastocysts that developed from two pronuclear zygotes, the mean Oct-4 expression was 31 times higher in totipotent ICM cells than in differentiated TE cells. This finding suggests that, in accordance with data from the mouse, Oct-4 is highly expressed in human ICM cells as opposed to TE cells; this in turn supports the hypothesis that Oct-4 plays a similar role to maintain totipotency in these two species.

Blastocyst↗

PubMed1980

The control of trophoblastic growth in the guinea pig.

The growth of mouse trophectoderm depends upon the presence of the inner cell mass. Whether this applies to other species of mammals is not known. To investigate this problem, the guinea pig was selected for two reasons. Firstly the growth of guinea-pig trophoblast resembles that of man. Secondly, earlier studies suggest that the proliferation of guinea-pig trophectoderm may not be under ICM control. Therefore, in the present study, the guinea-pig blastocyst was cut microsurgically to yield two tissue fragments. These contained roughly equal numbers of trophectodermal cells, one fragment being composed only of trophectoderm and the other containing ICM tissue as well. Subsequently, the growth of these mural and polar fragments was followed in vitro since numerous technical difficulties make an in vivo analysis of this problem impracticable. In the manner similar to the mouse, the isolated mural trophectoderm of the guinea pig stopped dividing and became giant. In contrast, guinea-pig polar fragments formed egg-cylinder-like structures. The latter contained regions structurally similar to two presumptive polar trophectodermal derivatives namely the ectoplacental and extraembryonic ectodermal tissues. These findings suggest that guinea-pig trophectodermal growth may occur in a manner similar to the mouse and thus be under ICM control.

Animals↗

PubMed1984

Characterization of a pluripotent stem cell line derived from a mouse embryo.

A pluripotent, karyotypically normal, male culture line ESC-BLC 1 of embryonal stem cells was established from delayed mouse blastocysts of strain 129/ter Sv. The cell line was isolated after cultivation of inner cell mass cells on X-irradiated feeder layer of mouse embryonal fibroblasts. The pluripotent status of the cell line was confirmed by in vivo and in vitro differentiation. For in vivo differentiation, cells were injected subcutaneously into syngeneic mice. The resulting tumors contained various tissues, derivatives of all three primary germ layers. In vitro cultivated pluripotent stem cells differentiated into endoderm-like, neuronal-like and tubular structures. Determination of alkaline phosphatase in cell line ESC-BLC 1 yielded a high specific activity; G-banding of metaphases revealed a normal, male karyotype.

Alkaline Phosphatase↗

PubMed2004

Molecular study of mouse peri-implantation development using the in vitro culture of aggregated inner cell mass.

To study the mechanisms of mouse peri-implantation development, we explored the in vitro culture of the isolated inner cell mass (ICM) of the blastocyst. As previously reported, individually cultured ICM recapitulated several early embryological events, such as the formation of primitive endoderm, epiblast, and proamniotic cavity. However, we found that the timing and efficiency of these morphogenetic processes significantly varied among the ICM. Due to this unpredictability in developmental potential, individually cultured ICM may be unsuitable for further analysis. By contrast, we found that when five ICM were fused into a single mass, such aggregates (5x ICM) underwent efficient and synchronous morphogenesis. The synchronous nature of 5x ICM development was also demonstrated by the temporal and spatial pattern of apoptotic cell death. TUNEL assay showed that a number of the epiblast cells committed apoptosis in 48 hr of culture, which took place after primitive endoderm differentiation but prior to proamniotic cavity formation. In situ hybridization analysis showed that Oct4 was downregulated and alpha-fetoprotein was upregulated in the primitive endoderm of the cultured 5x ICM. In addition, RT-PCR analysis revealed the expression of various primitive endodermal genes, but not of extraembryonic ectodermal markers in the cultured 5x ICM. Taken together, we propose that the 5x ICM is a useful in vitro tool to study the mechanisms of peri-implantation development of the mouse embryo. Mol. Reprod. Dev. 67: 83-90, 2004.

Animals↗

PubMed1998

A delayed-implantation-associated protein prevents resumption of DNA synthesis by the trophectoderm of delayed-implanting mouse blastocysts in vitro.

Objective of this study was to determine the ability of a delayed-implantation-associated protein (MW 170,000, DIAP170K) to inhibit DNA synthesis by mouse blastocysts. Mice were ovariectomized on day 3 of pregnancy and treated with daily injections with 1 mg progesterone till day 7 to induce delayed implantation. Blastocysts were collected on day 8 with or without a single injection of 25 ng estradiol-17 beta on day 7 that activates blastocyst metabolisms (activated blastocysts and delayed-implanting blastocysts respectively). DNA synthesis was determined by measuring [3H]thymidine incorporation by blastocysts. DIAP170K at 10 micrograms/m/ suppressed resumption of DNA synthesis by delayed-implanting blastocysts and suppression was maximal at 50 micrograms/m/. However, DIAP170K did not affect DNA synthesis by blastocysts obtained on day 5 of pregnancy (normal blastocysts) and activated blastocysts. Resumption of DNA synthesis in the inner cell mass (ICM) and trophectoderm from delayed-implanting blastocysts was then separately assessed. DNA synthesis resumed in the trophectoderm of intact blastocysts during 24-hr culture but not in the trophectoderm cultured apart from the ICM. DIAP170K inhibited the resumption of DNA synthesis by the trophectoderm of intact delayed-implanting blastocysts but did not affect DNA synthesis by the ICM. In conclusion, DIAP170K inhibits resumption of DNA synthesis by trophectoderm of delayed-implanting blastocysts. This action of DIAP170K may play a central role in maintaining, but not achieving, dormancy of DNA synthesis by delayed-implanting blastocysts in mice.

Animals↗

PubMed2003

Alterations in desmosome size and number coincide with the loss of keratinocyte cohesion in skin with homozygous and heterozygous defects in the desmosomal protein plakophilin 1.

Recessive mutations in the desmosomal plaque protein plakophilin 1 (PkP1) underlie ectodermal dysplasia/skin fragility syndrome (MIM 604536). We undertook an immunohistochemical and quantitative electron microscopic examination of suprabasal desmosomes from 4 skin samples from 3 PkP1 deficient patients, an unaffected carrier with a PKP1 heterozygous acceptor splice site mutation and 5 healthy control subjects. Desmosomal plaque size (>50 desmosomes per individual) and frequency (>20 high power fields, HPF) were assessed. Compared with controls, desmosomes were reduced dramatically both in size (49%) and frequency (61%) in the lower suprabasal layers (LSB) in PkP1 null patients (P<0.01). In the LSB compartment of the heterozygous carrier, corresponding reductions were 37% and 20%, respectively (P<0.01). Surprisingly, the PkP1 null patient's upper suprabasal layer, (USB), desmosome size was larger (59%, P<0.01) than the control value, and showed increased desmoglein 1 and PkP2 USB staining. The USB desmosome frequency in PKP1 null patients was similar to the LSB compartment (but reduced by 43% compared to USB controls). The carrier showed no difference in the USB desmosome size and frequency compared with the controls (P>0.05). The PKP1 null patients showed poorly developed inner and outer desmosomal plaques. Thus, both the patients and unaffected carrier showed reductions in the LSB desmosome size and number; despite only PkP1 null patients exhibiting any phenotype. These findings attest to the molecular recruiting and stabilizing roles of PkP1 in desmosome formation, particularly in the LSB compartment.

Adolescent↗

PubMed1991

An investigation of the fate of cells transplanted orthotopically between morulae/nascent blastocysts in the mouse.

A technique has been devised for selectively replacing some inside cells (ICs) or outside cells (OCs) of late morulae/nascent blastocysts with corresponding cells from genetically dissimilar, synchronous embryos. The main purpose was to determine whether the inner cell mass (ICM) contributes cells to the overlying polar trophectoderm at any stage during the blastocyst phase of development. Notwithstanding the high incidence and level of chimaerism in ICM derivatives of post-implantation conceptuses obtained in the IC transplantation experiments, trophoblast tissue was composed entirely of host cells in the majority of cases. Even where a donor IC contribution to trophoblast was detected there were strong grounds for suspecting that it was due to tissue contamination rather than genuine chimaerism. Thus, not only did such contributions differ in both level and distribution from those produced by transplantation of OCs but they also varied markedly in frequency according to the day of gestation on which conceptuses were dissected. The possibility that ICs regularly colonize the polar trophectoderm but fail to persist there was excluded by the results of short-term transplantation experiments using an in-situ genetic marker. These findings offer no support for the hypothesis that the ICM serves as a population of stem cells for the trophectoderm as well as the primitive endoderm and ectoderm during normal development. The frequency of chimaerism was lower in OC than IC transplantation experiments. Nevertheless, in a substantial proportion of chimaeras, OCs colonized derivatives of the ICM. This is consistent with evidence from other studies that some outside cells divide differentially at 5th cleavage to produce an OC plus an IC rather than two OCs.

Animals↗

PubMed2000

Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells.

Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of in vitro fertilized human blastocysts. We examined the potential of eight growth factors [basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-beta1), activin-A, bone morphogenic protein 4 (BMP-4), hepatocyte growth factor (HGF), epidermal growth factor (EGF), beta nerve growth factor (betaNGF), and retinoic acid] to direct the differentiation of human ES-derived cells in vitro. We show that human ES cells that have initiated development as aggregates (embryoid bodies) express a receptor for each of these factors, and that their effects are evident by differentiation into cells with different epithelial or mesenchymal morphologies. Differentiation of the cells was assayed by expression of 24 cell-specific molecular markers that cover all embryonic germ layers and 11 different tissues. Each growth factor has a unique effect that may result from directed differentiation and/or cell selection, and we can divide the overall effects of the factors into three categories: growth factors (Activin-A and TGFbeta1) that mainly induce mesodermal cells; factors (retinoic acid, EGF, BMP-4, and bFGF) that activate ectodermal and mesodermal markers; and factors (NGF and HGF) that allow differentiation into the three embryonic germ layers, including endoderm. None of the growth factors directs differentiation exclusively to one cell type. This analysis sets the stage for directing differentiation of human ES cells in culture and indicates that multiple human cell types may be enriched in vitro by specific factors.

Cell Differentiation↗

PubMed2002

Embryonic stem cell differentiation and the analysis of mammalian development.

Molecular and cellular analysis of early mammalian development is compromised by the experimental inaccessibility of the embryo. Pluripotent embryonic stem (ES) cells are derived from and retain many properties of the pluripotent founder population of the embryo, the inner cell mass. Experimental manipulation of these cells and their environment in vitro provides an opportunity for the development of differentiation systems which can be used for analysis of the molecular and cellular basis of embryogenesis. In this review we discuss strengths and weaknesses of the available ES cell differentiation methodologies and their relationship to events in vivo. Exploitation of these systems is providing novel insight into embryonic processes as diverse as cell lineage establishment, cell progression during differentiation, patterning, morphogenesis and the molecular basis for cell properties in the early mammalian embryo.

Animals↗

PubMed1988

Fate of the inner cell mass in mouse embryos as studied by microinjection of lineage tracers.

We microinjected horseradish peroxidase and rhodamine-conjugated dextran into single inner cell mass (ICM) cells of preimplantation mouse embryos to study their fate in culture. Simultaneous iontophoresis of both lineage markers allowed immediate localization of the injected cell by epifluorescence, followed by microdrop culture of individual embryos. After 24 hr in culture, labeled descendants were found in the polar trophectoderm, ICM, and parietal endoderm, providing direct evidence that the ICM contributes descendants to the trophectoderm and the endoderm in the intact mouse embryo. Our results substantiate the totipotency of the ICM during the expanding blastocyst stage and further demonstrate that the ICM is a stem cell population from which cells are recruited into these tissue lineages during growth of the blastocyst.

Animals↗