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PubMed2002

Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo.

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.

Amino Acid Sequence↗

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↗

PubMed1988

Production of oestrone and oestradiol-17 beta by different regions of the filamentous pig blastocyst.

Pig blastocysts aged 14, 16 and 18 days were divided into 15 cm segments representing tissue adjacent to the embryonic disc, an intermediate section and the tip region. Whenever total blastocyst length allowed, the intermediate segment was divided into proximal and distal portions for separate culture. All were rinsed with buffer and incubated with dehydroepiandrosterone for 3 h. Rinsing buffer and incubation medium were subsequently assayed for concentrations of oestrone and oestradiol-17 beta. The highest production of oestrogen was found in the embryonic disc region. The intermediate regions had the lowest synthetic ability, while the tip region produced more oestrogens than the intermediate regions but less than the disc region. The production of oestrone was higher (P less than 0.05) in 18-day-old blastocysts than in younger ones while oestradiol-17 beta production was lower (P less than 0.05) on Day 16. The proportional role of the embryonic disc region as oestrogen-producing tissue increased over time. On Day 14, each intermediate region produced over 70% as much oestrogen as the disc region. These proportions declined on Days 16 and 18 to about 50 and 30% respectively. The regional variation in the ability of blastocysts to produce oestrogens may have some influence on the ability of the blastocyst to create an adequate microenvironment within the uterus which permits successful differentiation and placentation.

Animals↗

PubMed1975

The "imaged-desmosome": a component of intercalated discs in embryonic guinea pig myocardium.

A heretofore undescribed structural variation of the desmosomes of the intercalated disc is found in myocardial cells of the embryonic guinea pig. These desmosomes consist of the usual pair of opaque leaflets, each of the pair contributed by one of the apposed muscle cells. In addition, in the cytoplasm of one of the cells there appears a pair of linear densities (facsimile-lines) parallel to the nearest desmosomal plaque and separated from it by a 60 mm space. The facsimile lines superficially resemble the desmosomal leaflets in length and thickness, thus forming a cytoplasmic "image" of the desmosome. These "imaged-desmosomes" are found predominantly in the longitudinally-running portions of the intercalated discs and are common in 7-week embryos. Their incidence drops sharply by eight weeks of gestation, and they are virtually absent from the heart of the newborn animal. Often tubules of the sarcoplasmic reticulum (SR) are found in apposition to the facsimile-lines; thus it appears that association of SR tubules with desmosomes is responsible for the formation of imaged-desmosomes.

Animals↗

PubMed1993

Alkaline phosphatase staining of pig and sheep epiblast cells in culture.

To define better the characteristics of pig and sheep epiblast cells in culture, the cells were tested for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of mouse embryonic stem cells. Pig and sheep epiblast cells were positive for AP staining both at isolation from the blastocyst and after primary in vitro culture. The innermost portion of the attendant endoderm surrounding the epiblast was also positive for AP staining during primary culture. AP staining was lost upon differentiation or senescence of the epiblast cells. Also, all differentiated epiblast-derived cell cultures were negative for AP staining, with the exception of neuron-like cultures. Epiblast-like cells were cultured from day 10 (pig) and day 13 (sheep) embryonic discs, and these cells were also AP positive until they differentiated. Trophectoderm-endoderm-like cells from embryonic discs were AP negative or weakly positive. AP is a convenient marker for undifferentiated pig and sheep epiblast cells in culture when used in conjunction with cell morphology analysis.

Alkaline Phosphatase↗

PubMed1993

Culture of pig embryos.

Pig embryos can be cultured using a number of different strategies including complex approaches like culture in vivo in a surrogate oviduct (rabbit, sheep, mouse), culture in mouse oviducts in organ culture, and co-culture of embryos with cells in addition to simple approaches like culture in defined media or salt solutions. Addition of serum to medium has been of particular importance where blastocyst development and hatching are required. Pig conceptuses (day 10-15), embryonic discs or cell lines derived from conceptuses can be cultured in complex media like Eagle's minimal essential medium or Dulbecco's modified Eagle's medium with serum, although embryonic discs can be cultured in the absence of serum. In contrast, early stage pig embryos (one-cell to blastocyst) are best cultured in simpler media such as those used for mouse embryos. The media that have been used are all relatively similar in composition. They contain salts and one or more energy sources such as glucose, lactate, or pyruvate with BSA as a macromolecular component. Early attempts to culture pig embryos were not very successful, but some embryos did develop to the blastocyst stage. More recent reports indicate a much higher rate of development with relatively little or no change in media composition. Some workers have reported improved development in medium lacking glucose, which is consistent with findings with laboratory animals such as hamsters. Glutamine can serve as the sole exogenous energy source in medium lacking glucose, lactate and pyruvate. Addition of taurine and hypotaurine to culture medium enhances development of pig embryos in vitro. We suggest, where possible, adoption of a standard medium that could be used by different laboratories and, perhaps, with different species. Use of one medium for different species would simplify experimental protocols, enhance studies of comparative embryonic physiology and metabolism, and expedite transfer of information obtained in different species.

Animals↗

PubMed1995

Promotion of gastrulation by maternal growth factor in cultured rabbit blastocysts.

Rabbit blastocysts of day 6 post coitus were cultured in a chemically defined, protein-free medium for 24 h. Although the trophoblast continued to grow, the embryonic disc degenerated. Addition of basic fibroblast growth factor (FGF-2, of human recombinant or bovine origin, 10 ng/ml) to the culture medium resulted in significant developmental progress. The embryonic disc became pear-shaped showing a round anterior edge and a posterior node. The primitive streak and Hensen's node indicated that gastrulation had begun. Mesoderm formation was confirmed from histological sections and by localization of the expression of T-gene transcripts in whole-mount preparations. FGF-2 mRNA was detected in both day-6 endometrium and day 6-blastocysts using in vitro translation followed by immunoprecipitation with a monoclonal antibody to FGF-2. In the uterine secretions of day-6 pregnant and pseudopregnant animals, several proteins exhibiting FGF-2 antigenicity were detected on Western blots following two-dimensional gel electrophoresis. As day-6 blastocysts required exogenous FGF-2 in vitro and as FGF-2 of uterine origin is present in the uterine secretion, the maternal growth factor can promote gastrulation in vivo.

Animals↗

PubMed1996

Changes in structure of the trophectoderm of a marsupial in Mid-pregnancy up to the time of implantation.

Pre- and peri-implantation embryos of the dasyurid marsupial Sminthopsis crassicaudata were examined for morphological differentiation of the trophectoderm. The cells of unilaminar blastocysts were all squamous and stained intensely with toluidine blue. In bilaminar blastocysts and embryos at the early embryonic-disc stage, the trophectoderm was similar in appearance to, but stained more lightly than, the underlying endoderm. Trophoblast differentiation did not appear to occur until the mesoderm had begun to migrate between the trophoblast and endoderm beyond the embryonic disc. At this stage, trophoblasts had three distinct morphologies: (1) vacuolated, tall and columnar cells in the trilaminar region; (2) large cuboidal cells in the adjacent bilaminar region; and (3) squamous cells in the abembryonic pole of the bilaminar region. These variations in cell structure correlate with differences in subsequent functional activity in these three regions of the yolk sac placenta.

Animals↗

PubMed1992

Conjoined twins: theoretical embryologic basis.

A theoretical basis for the embryology of conjoined twins was formulated from clinical experience with ten cases and extensive review of pertinent embryologic and clinical literature, including over 500 cases. Regarding the age old question of fusion or fission, it is concluded that there is no known embryologic process by which conjoined twins can be formed by fission but firm evidence to support fusion in all cases. Whether the fusion occurs between embryos on one embryonic disc or on two is of no consequence since they are all monovular. Intact ectoderm will not fuse to intact ectoderm, and all seven types of conjoined twins are explained by seven possible sites of union in the early embryo. One new term is proposed: parapagus, from the Greek para, meaning "side," combined with pagus, meaning "fixed"; this is the group formerly called dicephalus or diprosopos. These anterolaterally united parapagus twins must result from two nearly parallel notochords in close proximity; craniopagi and pygopagi from fusion at the cranial and caudal neuropores, respectively; cephalopagi and ischiopagi from union at the pharyngeal and cloacal membranes, respectively; thoracopagi from merging of the cardiac anlage; and omphalopagi from fusion of the umbilicus or of the edges of two embryonic discs in any area not including the above sites. Parasitic twins result from embryonic death of one twin, leaving various portions of the body vascularized by the surviving autosite. The rarity of cases (2) not easily explained by the above theories, and the nearly 6% of twins with two umbilical cords arising from the placenta would seem to support these conclusions. Should one wish to learn the methods of a conjurer, he might vainly watch the latter's customary repertoire, and, so long as everything went smoothly, might never obtain a clue to the mysterious performance, baffled by the precision of the manipulations and the complexity of the apparatus; if, however, a single error were made in any part or if a single deviation from the customary method should force the manipulator along an unaccustomed path, it would give the investigator an opportunity to obtain a part or the whole of the secret.(ABSTRACT TRUNCATED AT 400 WORDS)

Humans↗

PubMed1987

Analysis of X-chromosome inactivation in horse embryos.

To define the time of X-chromosome inactivation in the horse, 122 conceptuses were collected transcervically between Days 6 and 28 (ovulation = Day 0) and subjected to cytogenetic analysis: 59 of the embryos were divided and in 41 of these separate cytogenetic analysis of the embryonic disc and remaining tissues was possible. Conceptuses were measured and photographed before capsule removal, culture in the presence of 5-bromodeoxyuridine and subsequent fixation for cytogenetic analysis. On average, 15 slides were prepared per conceptus. C-banding was used to determine the sex of each conceptus and, in the females, the effects of 5-bromodeoxyuridine incorporation were revealed by u.v. irradiation which produced R-bands and allowed identification of the inactive X-chromosome. Inactivation of the X chromosome began gradually in the trophoblastic (+/- attached endoderm and mesoderm) cells around Day 7.5 and in the embryonic disc around Day 11.5. Cells with an inactive X-chromosome predominated in the trophoblast by Day 10.5 and in the disc by Day 12.5.

Animals↗

PubMed1988

The dynamic structure of rabbit blastocyst coverings. II. Ultrastructural evidence for a role of the trophoblast in neozona formation.

The neozona is the innermost layer of the complex blastocyst coverings of the rabbit and is located between the mucoprotein layer and the trophoblast. As shown previously the neozona begins to replace the zona pellucida from the 4th day post coitum (d p.c.) on (Denker and Gerdes 1979). In the present study, rabbit blastocyst coverings were checked for regional differences in their composition, comparing the embryonic and the abembryonic pole of the blastocyst, at 5 and 6 d p.c. These two stages were chosen because at 5 d p.c. a complete trophoblast layer is still present at both the embryonic disc (Rauber's layer) and the extraembryonic regions (mural and abembryonic pole trophoblast), whereas at 6 d p.c. Rauber's layer has largely degenerated. Correlation of regional differences in blastocyst coverings structure with presence or absence of an intact trophoblast is taken as suggestive evidence for a role of the trophoblast in the formation or the structural modification of blastocyst coverings components. Blastocysts of both stages were fixed in glutaraldehyde with and without ruthenium red and processed for TEM. The neozona was found to be almost equally well developed in all regions at 5 d p.c. On contrast, at 6 d p.c. (Rauber's layer defective) the neozona is consistently found to be much thinner at the embryonic disc than in the extraembryonic regions where the trophoblast is still intact. This is the first report on regional differences of the structural composition of blastocyst covering within the same blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Critical periods for alcohol teratogenesis in mice, with special reference to the gastrulation stage of embryogenesis.

Studies directed towards determining a 'critical' developmental period for the induction of neural, ocular and craniofacial malformations typical of those in severe forms of the fetal alcohol syndrome (FAS) are described here. Sequential developmental analyses of C57Bl/6J mice whose mothers had been given alcohol either intraperitoneally or by gastric intubation on the seventh day of pregnancy (a time corresponding to three weeks after fertilization in humans) illustrate that early deficiencies in the anterior aspect of the embryonic disc of embryos at the gastrulation stage lead to defects characteristic of FAS. These include microcephaly, small nose, long upper lip with deficient philtral component, short palpebral fissures, microphthalmia and tortuous retinal vessels, as well as other eye defects involving primarily the anterior segment. Brain malformations are most marked in the derivatives of the ventromedial forebrain. Gastrulation is a period of intense mitotic activity (particularly in the developing mesoderm) in mammalian embryos. This, together with other studies showing that alcohol suppresses rates of cell division, suggests that alcohol-induced deficiencies in gastrulating mesodermal cells (cells responsible for inducing and maintaining neuroepithelial differentiation), as well as cellular deficiencies in the other germ layers in the anteromedial aspect of the embryonic disc, may play a significant role in the subsequent developmental abnormalities observed.

Abnormalities, Drug-Induced↗

PubMed1995

Morphology of incipient mesoderm formation in the rabbit embryo: a light- and retrospective electron-microscopic study.

Mesoderm formation is a hallmark of vertebrate gastrulation and, at the same time, one of the prime examples for epithelio-mesenchymal transformation. Recent advances in experimental embryology and molecular biology have clarified the role of growth factors and genes in this process; however, its microscopic anatomy in higher vertebrates is still far from clear. Therefore, the present study describes the morphology of mesoderm formation in the rabbit embryo, a species which may be representative for both the avian and the mammalian embryo in this respect. Serial semithin sections were correlated with topographical landmarks in surface views of embryonic discs at 6.4, 6.5, and 6.6 days post conceptionem, and selected semithin sections were reembedded for ultrastructural analysis. Mesoderm cells are shown to be generated by ingression of bottle-shaped epiblast cells in the area of the posterior node and the primitive streak. Here, basal endocytotic pits and absence or discontinuity of the basal lamina are taken as suggestive evidence for specific removal of extracellular matrix material. Within the bottle-shaped cells most organelles are concentrated in a narrow apical neck which will subsequently constitute the 'trailing end' of the ingressing mesoderm cells. These features support the assumption that most principles of epithelio-mesenchymal transformation seen during primary mesenchyme formation in the sea urchin also apply to mesoderm formation in vertebrates. However, transient tripartite zonula adherens-type junctions are formed apically between ingressing mesoderm cells and the neighboring epiblast cells. They are interpreted here as being responsible for maintaining supracellular integrity of the embryonic disc during the shedding of mesoderm cells in the amniote embryo.

Animals↗

PubMed2006

Tissue-specific laminin expression facilitates integrin-dependent association of the embryonic wing disc with the trachea in Drosophila.

The interaction of heterologous tissues involves cell adhesion mediated by the extracellular matrix and its receptor integrins. The Drosophila wing disc is an ectodermal invagination that contacts specific tracheal branches at the basolateral cell surface. We show that an alpha subunit of laminin, encoded by wing blister (wb), is essential for the establishment of the interaction between the wing and trachea. During embryogenesis, wing disc cells present Wb at their basolateral surface and extend posteriorly, expanding their association to more posteriorly located tracheal branches. These migratory processes are impaired in the absence of the trachea, Wb, or integrins. Time-lapse and transmission electron microscopy analyses suggest that Wb facilitates integrin-dependent contact over a large surface and controls the cellular behavior of the wing cells, including their exploratory filopodial activity. Our data identify Wb laminin as an extracellular matrix ligand that is essential for integrin-dependent cellular migration in Drosophila.

Animals↗

PubMed2001

Headless flies generated by developmental pathway interference.

Ectopic expression of transcription factors in eye-antennal discs of Drosophila strongly interferes with their developmental program. Early ectopic expression in embryonic discs interferes with the developmental pathway primed by Eyeless and generates headless flies, which suggests that Eyeless is necessary for initiating cell proliferation and development of both the eye and antennal disc. Interference occurs through a block in the cell cycle that for some ectopic transcription factors is overcome by D-CycE or D-Myc. Late ectopic expression in cone cell precursors interferes with their differentiation. We propose that this developmental pathway interference is a general surveillance mechanism that eliminates most aberrations in the genetic program during development and evolution, and thus seriously restricts the pathways that evolution may take.

Animals↗

PubMed1989

Immunocytochemical demonstration of the binding of growth-related polypeptide hormones on chick embryonic tissues.

The presence of endogenous growth-related polypeptide hormones, such as growth hormone (GH), somatomadin-C/insulin-like growth factor-1 (SM-C/IGF-1), prolactin (PRL) and Mullerian inhibiting substance (MIS) on chick embryonic tissues have been detected by electron microscopic (EM) immunocytochemistry. Antiserum against GH, anti-SM-C/IGF-1, anti-PRL and anti-MIS were used respectively as primary antibodies for immunolabeling probes by peroxidase (PO) and avidin-biotin complex (ABC)-gold ligands. Cross-reaction studies by ELISA showed negative or weak antigen-antibody interactions. Chick embryos, gonads, and Mullerian ducts (Mds) of various ages were fixed in 2.5% glutaraldehyde for 30 min. Washes in phosphate buffer were administered between each of the following incubations: (i) 2% BSA; (ii) primary antibody; (iii) biotinylated or PO-conjugated secondary antibody; (iv) avidin conjugated with gold particles. SM-C/IGF-1 bindings were negative on 1d embryonic disc, heavily stained on 2d endoderm. However, the GH bindings were found on the embryonic layers of 1d and 2d embryos, and increasing on the luminal epithelial cells of Mds during development. PRL was found in parallel with GH, but in less amount. The 10d Mds were double labeled with anti-SM-C/IGF-1-gold and anti-MIS-PO (MIS-PO), and the results showed SM-C/IGF-I negative, but MIS-PO positive bindings. This study provides the first immunocytochemical evidences for: (i) The presence of GH, SM-C/IGF-1, PRL and MIS bindings on chick embryonic tissues, and further supports their potential role as growth mediators during embryonic development. (ii) The amount of GH and MIS bindings were found correspondingly to their physiological status of Md growth or regression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila.

Extracellular factors such as FGF and EGF control various aspects of morphogenesis, patterning and cellular proliferation in both invertebrates and vertebrates. In most systems, it is primarily the distribution of these factors that controls the differential behavior of the responding cells. Here we describe the role of Sprouty in eye development. Sprouty is an extracellular protein that has been shown to antagonize FGF signaling during tracheal branching in Drosophila. It is a novel type of protein with a highly conserved cysteine-rich region. In addition to the embryonic tracheal system, sprouty is also expressed in other tissues including the developing eye imaginal disc, embryonic chordotonal organ precursors and the midline glia. In each of these tissues, EGF receptor signaling is known to participate in the control of the correct number of neurons or glia. We show that, in all three tissues, the loss of sprouty results in supernumerary neurons or glia, respectively. Furthermore, overexpression of sprouty in wing veins and ovarian follicle cells, two other tissues where EGF signaling is required for patterning, results in phenotypes that resemble the loss-of-function phenotypes of Egf receptor. These results suggest that Sprouty acts as an antagonist of EGF as well as FGF signaling pathways. These receptor tyrosine kinase-mediated pathways may share not only intracellular signaling components but also extracellular factors that modulate the strength of the signal.

Animals↗

PubMed1996

Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts.

We report the derivation of eight pluripotent cell lines from common marmoset (Callithrix jacchus) blastocysts. These cell lines are positive for a series of markers (alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that characterize undifferentiated human embryonal carcinoma cells and rhesus embryonic stem cells. All eight cell lines had a modal chromosome number of 46; seven cell lines were XX and one was XY. Two cell lines (Cj11 and Cj62) were cultured continuously for over a year and remained undifferentiated and euploid. In the absence of fibroblast feeder layers, these cell lines differentiated to multiple cell types, even in the presence of leukemia inhibiting factor. Differentiated cells secreted bioactive CG into the culture medium and expressed alpha-CG, beta-CG, and alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. Bioactive CG secretion in differentiating cells was increased substantially in the presence of GnRH agonist D-Trp6-Pro9-NHEt. When grown at high densities, these cells formed embryoid bodies with a close resemblance to early postimplantation embryos, including the formation of a yolk sac, amnion, and an embryonic disc with an early primitive streak. These results make these pluripotent cells strong candidates for marmoset embryonic stem cells.

Animals↗