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PubMed1996

Diastematomyelia and spina bifida can be caused by the intraspinal grafting of somites in early avian embryos.

OBJECTIVE: In this experimental study, an embryological model was created to reproduce diastematomyelia and spina bifida and to investigate new aspects of the origin of spinal cord malformations. METHODS: A somite was implanted from a donor quail embryo into the neural tube of a 2-day-old chick embryo. The somite was chosen because the septum that characteristically separates the two hemicords consists exclusively of mesodermal derivatives. RESULTS: After 2 days of reincubation, diastematomyelia, spina bifida, or a normal embryo without a graft was observed. If the graft persisted in the neural tube, it formed a septum between the floor and roof plates but never made contact with the lateral walls of the tube. Otherwise, the graft was extruded from the neural tube. In this case, the quail cells often were found in dorsal or dorsolateral positions in the surrounding tissue. Sometimes, the wall of the neural tube formed an extrusion in the direction of the eliminated graft. On many occasions, however, spina bifida aperta was produced and no quail cells could be found in the host. CONCLUSION: The results suggest that diastematomyelia may be the result of abnormal mesodermal invasion of the neural tube. The development of a septum in the neural tube after implantation of a somite may mimic the process during spontaneous diastematomyelia formation, which could be the consequence of abnormal gastrulation, the process by which the two early germ layers of the blastodisc are converted into the three definitive germ layers.

Animals↗
PubMed2001

Patterning and lineage specification in the amphibian embryo.

Xenopus has been widely used to study early embryogenesis because the embryos allow for efficient functional assays of gene products by the overexpression of RNA. The first asymmetry of the embryo is initiated during oogenesis and is manifested by the darkly pigmented animal hemisphere and lightly pigmented vegetal hemisphere. Upon fertilization a second asymmetry, the dorsal-ventral asymmetry, is established, with the sperm entry site defining the prospective ventral region. During the cleavage stage, a vegetal cortical cytoplasm (VCC)/beta-catenin signaling pathway is differentially activated on the prospective dorsal side of the embryo. The overlapping of the VCC/beta-catenin and transforming growth factor beta (TGF-beta) pathways in the dorsal vegetal quadrant specifies dorsal-vental axis formation by regulating formation of the Spemann organizer, including the anterior endomesoderm. The organizer initiates gastrulation to form a triploblastic embryo in which the mesoderm layer is located between the ectoderm layer and the endoderm layer. The interplay between maternal and zygotic TGF-beta s and the T-box transcription factors in the vegetal hemisphere initiates the specification of germ-layer lineages. TGF-beta signaling originating from the vegetal region induces mesoderm in the equatorial region, and initiates endoderm differentiation directly in the vegetal region. The ectoderm develops from the animal region, which does not come into contact with the vegetal TGF-beta signals. A large number of the downstream components and transcriptional targets of early developmental pathways have been identified and characterized. This review gives an overview of recent advances in the understanding of the functional roles and interactions of the molecular players important for axis determination and germ-layer specification during early Xenopus embryogenesis.

Amphibians↗
PubMedDate not supplied

Human embryonic stem cells: culture, differentiation, and genetic modification for regenerative medicine applications.

Human embryonic stem (hES) cells can proliferate extensively in culture and can differentiate into representatives of all three embryonic germ layers in vitro and in vivo. The undifferentiated hES cells have now been cultured for more than 50 passages in vitro, yet maintain a normal karyotype. The hES cells express a series of specific surface antigens, as well as OCT-4 and human telomerase, proteins associated with a pluripotent and immortal phenotype. On differentiation, OCT-4 and human telomerase expression decreases with the emergence of a maturing population of cells. During hES cell differentiation, modulation of the expression of many genes has been evaluated using microarray analysis. To improve the ease, reproducibility, and scalability of hES culture, methods have been developed to propagate the cells in the absence of mouse embryonic cell feeders. hES cells maintained in culture using extracellular matrix factors together with mouse embryonic cell conditioned medium proliferate indefinitely while maintaining a normal karyotype, proliferation rate, and complement of undifferentiated cell markers. hES cells cultured without feeder layers retain their capacity to differentiate into cells of all three germ layers in vitro and in teratomas. The hES cells can also be genetically modified transiently or stably using both plasmid and viral gene transfer agents. These analyses and technological developments will aid in the realization of the full potential of hES cells for both research and therapeutic applications.

Animals↗
PubMed2004

Pluripotency of cryopreserved blastomeres of the goldfish.

To examine the pluripotency of cryopreserved blastomeres, we transplanted them into blastula. Donor blastomeres were prepared from blastula of goldfish (Carassius auratus) and cryopreserved in liquid nitrogen for two months. Fifty-five percent and 44% of blastomeres survived after thawing. Cryopreserved blastomeres were transplanted to the blastula of triploid crucian carp (C. a. longsdorfii), which reproduces gynogenetically in nature. At four days after the operation, resultant chimeric embryos transplanted with cryopreserved blastomeres showed a survival rate (41.6%) lower than that of embryos transplanted with unfrozen blastomeres (57.1%). Transplanted blastomeres were histologically identified in various organs derived from all three germ layers. A primordial germ cell differentiated from a cryopreserved blastomere was detected in one of the 32 chimeric fish examined. These results suggest blastomeres that survive after cryopreservation retain their pluripotency and are able to differentiate into both somatic and germ cell lines.

Animals↗
PubMed2005

The fate of isolated blastomeres with respect to germ cell formation in the amphipod crustacean Parhyale hawaiensis.

Germ cells may be specified through the localization of germ line determinants to specific cells in early embryogenesis, or by inductive signals from neighboring cells to germ cell precursors in later embryogenesis. Such determinants can be produced and localized during or after oogenesis, either autonomously by oocytes or by associated nutritive cells. In Drosophila, each oocyte is connected to nurse cells by cytoplasmic bridges, and determinants synthesized in nurse cells are transported through these bridges to the oocyte. However, the Drosophila model may not be applicable to all arthropods, since in many species of all four extant arthropod classes, gametogenesis functions without nurse cells. In this paper, I use immunodetection of Vasa protein to study germ cell development in the amphipod crustacean Parhyale hawaiensis, a species whose ovaries lack nurse cells and whose eggs lack obvious polarity. Previous cell lineage analyses have shown that all three germ layers and the germ line are exclusively specified by third cleavage. In the present study, I use a molecular marker to follow germ cell development during P. hawaiensis embryogenesis. I determine the capacity of individual blastomeres to form germ cells by isolating blastomeres at early cleavage stages and provide experimental evidence for localized germ cell determinants at the two-cell stage in P. hawaiensis. These experiments indicate that many aspects of early amphipod development, including timing and symmetry of cell division, the transition from holoblastic to superficial cleavage, and possibly some gastrulation movements, are cell autonomous following first cleavage.

Amphipoda↗
PubMed2001

The allocation and differentiation of mouse primordial germ cells.

Analysis of the lineage potency of epiblast cells of the early-streak stage mouse embryo reveals that the developmental fate of the cells is determined by their position in the germ layer. Epiblast cells that are fated to become neuroectoderm can give rise to primordial germ cells (PGCs) and other types of somatic cells when they were transplanted to the proximal region of the epiblast. On the contrary, proximal epiblast cells transplanted to the distal region of the embryo do not form PGCs. Therefore, the germ line in the mouse is unlikely to be derived from a predetermined progenitor population, but may be specified as a result of tissue interactions that take place in the proximal epiblast of the mouse gastrula. The initial phase of the establishment of the PGC population requires, in addition to BMP activity emanating from the extraembryonic ectoderm, normal Lim1 and Hnf3beta activity in the germ layers. The entire PGC population is derived from a finite number of progenitor cells and there is no further cellular recruitment to the germ line after gastrulation. The XX PGCs undergo X-inactivation at the onset of migration from the gut endoderm and re-activate the silenced X-chromosome when they enter the urogenital ridge. Germ cells that are localised ectopically in extragonadal sites do not re-activate the X-chromosome, even when nearly all germ cells in the fetal ovary have restored full activity of both X-chromosomes. XXSxr germ cells can re-activate the X-chromosome in the sex-reversed testis, suggesting that the regulation of X-chromosome activity is independent of ovarian morphogenesis.

Animals↗
PubMed2006

Evolutionary origins of Notch signaling in early development.

Notch signaling is known to play key roles in early embryonic development and in the specification or patterning of germ layers in several lower organisms. Thus it is surprising that the removal of maternal and zygotic protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway, does not affect early embryogenesis in the mouse. Mouse embryos lacking Pofut1 develop normally through blastogenesis to E8.0 when all three germ layers have formed. Here we summarize roles for Notch signaling in early embryonic development of several species, and discuss the potential evolutionary origins of these roles. We propose that Notch signaling might have first been used in the common ancestor of the metazoa for certain advanced developmental processes such as segmentation, and only later in evolution been coopted for use at early stages of embryogenesis in some organisms.

Animals↗
PubMed2004

Bves expression during avian embryogenesis.

Bves (blood vessel/epicardial substance) is a transmembrane protein postulated to play a role in cell adhesion. While it is clear that Bves and gene products of the same family are expressed in adult striated muscle cells, the distribution of these proteins during development has not been critically examined. An understanding of the expression pattern of Bves is essential for a determination of protein function and its role in embryogenesis. In this study, we present an expression analysis of Bves during chick gastrulation and germ layer formation. Our data show that Bves is expressed in epithelia of all three germ layers early in development. Furthermore, Bves protein is observed in epithelial tissues during organogenesis, specifically the developing epidermis, the gut endoderm, and the epicardium of the heart. These data support the hypothesis that Bves may play a role in cell adhesion and movement of epithelia during early embryogenesis.

Amino Acid Sequence↗
PubMed1989

Embryonic cellular organization: differential restriction of fates as revealed by cell aggregates and lineage markers.

Cleavage-stage Lytechinus variegatus embryos were dissociated and the cells were aggregated in an experimental system designed to address questions of embryonic organizational capability. Using monoclonal antibodies against stage- and structure-specific antigens, it was determined that germ-layer specific molecules were expressed in the expected locations in aggregates and that the germ layers differentiated in the normal temporal sequence, but the dorsoventral axis of the embryo was not reestablished properly. In other experiments individual rhodamine-labeled blastomeres were incorporated into unlabeled aggregates. Micromeres localized and differentiated normally. Mesomeres, however, which in normal embryos form only ectoderm, were found to change their specified fate and participate in gut formation. The sequence of aggregate organization revealed other properties of the embryo. Ectodermal and endodermal epithelia formed via two temporally distinct epithelialization events. Ectoderm separated from a mass of interior cells at about 12 hrs, and endoderm compacted from the interior cells at about 20 hr. A lathrytic agent that prevents gut formation in normal embryos also prevented gut formation in aggregates; however, it did not affect formation of the ectoderm. Hence, formation of the triploblastic structure in aggregates appears to be dependent upon developmentally regulated, distinct cell adhesion events.

Animals↗
PubMed2005

Human embryonic stem cells and their spontaneous differentiation.

Human embryonic stem cells (hESCs) usually grow in saucer-shaped colonies with thickened rims and can form spherical human embryoid bodies (hEBs) under non-adherent conditions. A problem associated with ES cell culture is the spontaneous differentiation of cells into a variety of cell types representing all three germ layers, which is evident in both hESC colonies and hEBs. This presentation deals with the precise origins of hESCs and their spontaneous differentiation in vitro. We have used advanced digital microscopy, including transmission electron microscopy (TEM) to define the fine structure of these cells. We present images of undifferentiated hESCs and their spontaneous differentiation into basic embryonic cell types such as nerve, muscle, connective tissue, epithelium, and digestive tract progenitors, representing all three primary germ layers: embryonic ectoderm, mesoderm and endoderm. It appears that hESCs work in concert and interact with one another, as in tissue formation of the embryo. Our fine structural observations agree mostly with those of the Thomson group. Digital microscopy of plastic sections and TEM are invaluable tools in the precise characterization of cells forming these tissues and a combined study with immunofluorescent markers is most desirable.

Cell Communication↗
PubMed2002

The essential role of RI alpha in the maintenance of regulated PKA activity.

Cloning of the individual regulatory (R) and catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) and expression of these subunits in cell culture have provided mechanistic answers about the rules for PKA holoenzyme assembly. One of the central findings of these studies is the essential role of the RI alpha regulatory subunit in maintaining the catalytic subunit under cAMP control. The role of RI alpha as the key compensatory regulatory subunit in this enzyme family was confirmed by gene knockouts of the three other regulatory subunits in mice. In each case, RI alpha has demonstrated the capacity for significant compensatory regulation of PKA activity in tissues where the other regulatory subunits are expressed, including brain, brown and white adipose tissue, skeletal muscle, and sperm. The essential requirement of the RI alpha regulatory subunit in maintaining cAMP control of PKA activity was further corroborated by the knockout of RI alpha in mice, which results in early embryonic lethality due to failed cardiac morphogenesis. Closer examination of RI alpha knockout embryos at even earlier stages of development revealed profound deficits in the morphogenesis of the mesodermal embryonic germ layer, which gives rise to essential structures including the embryonic heart tube. Failure of the mesodermal germ layer in RI alpha knockout embryos can be rescued by crossing RI alpha knockout mice to C alpha knockout mice, supporting the conclusion that inappropriately regulated PKA catalytic subunit activity is responsible for the phenotype. Isolation of primary embryonic fibroblasts from RI alpha knockout embryos reveals profound alterations in the actin-based cytoskeleton, which may account for the failure in mesoderm morphogenesis at gastrulation.

Animals↗
PubMed2008

Insights from diploblasts; the evolution of mesoderm and muscle.

The origin of both mesoderm and muscle are central questions in metazoan evolution. The majority of metazoan phyla are triploblasts, possessing three discrete germ layers. Attention has therefore been focused on two outgroups to triploblasts, Cnidaria and Ctenophora. Modern texts describe these taxa as diploblasts, lacking a mesodermal germ layer. However, some members of Medusozoa, one of two subphyla within Cnidaria, possess tissue independent of either the ectoderm or endoderm referred to as the entocodon. Furthermore, members of both Cnidaria and Ctenophora have been described as possessing striated muscle, a mesodermal derivative. While it is widely accepted that the ancestor of Eumetazoa was diploblastic, homology of the entocodon and mesoderm as well as striated muscle within Eumetazoa has been suggested. This implies a potential triploblastic ancestor of Eumetazoa possessing striated muscle. In the following review, I examine the evidence for homology of both muscle and mesoderm. Current data support a diploblastic ancestor of cnidarians, ctenophores, and triploblasts lacking striated muscle.

Animals↗
PubMed1979

[Morphologic characteristics of definitive and provisional structures of normal 17-day-old human embryos].

The structure of the presomite human embryo was investigated at embryogenesis. The embryonic shield is a three-layer gastrula 810 mkm long in the anteroposterior direction and 855 mkm wide (at the level of the primitive nodule). The primitive streak is 200 mkm long; the primitive nodule is well pronounced. All three germ layers are separately followed only in the cranial end of the embryo. The chordo-mesodermal process, 80 mkm long, is seen and is situated anterior to the primitive nodule, between ecto- and endoderm; in its zone, as well as in the area of the primary nodule and the primary streak, along the middle line, the germ layers are in close contact with each other. In the caudal end the mesoderm grows thin, and the external and internal layers come into contact forming the cloacal membrane. Extraembryonic formations are described: amniotic vesicle, yolk sac, amniotic peduncle, allantois and chorionic membrane wall. Together with the extraembryonic ecto- and endoderm, exocoelomic mesoderm participates in the formation of walls of the primitive germ vesicles. The yolk sac wall contains blood islets. Primary blood vessels are detected in the connective tissue matrix of the chorionic layer and in the amniotic peduncle. According to the anamnesis, morphological data and comparing to the data of the literature on presomitic human embryos, the age of the embryo "Krym" is determined as old as 17 days.

Adult↗
PubMed2003

Differentiation and genetic manipulation of human embryonic stem cells and the analysis of the cardiovascular system.

Human embryonic stem (ES) cells are pluripotent cells derived from blastocyst-stage embryos. The cells are characterized by their self-renewal capability and by their ability to differentiate into a wide range of cell types. In vivo, injection of the human ES cells into immune-deficient mice generates teratomas harboring derivatives of all three embryonic germ layers. In vitro, spontaneous aggregation of human ES cells results in the formation of embryoid bodies (EBs) comprised of differentiated cells from the three embryonic germ layers. Induced differentiation of ES cells into specific subsets of cells may be generated by treatment with several growth factors. Cardiomyocytes and endothelial cells were among the tissue types identified in vitro, and one of the most dramatic examples for the differentiation of human ES cells is the formation of rhythmic contractions of EBs containing pulsing cardiac muscle cells. Cells of the cardiovascular system were characterized by many molecular markers and by their structural and functional properties. The ability to genetically manipulate human ES cells now allows for the purification of specific cell types. Human ES cells have tremendous value as an in vitro model to study embryonic differentiation and as a source of cells for cellular transplantation in various pathologies among them cardiovascular diseases.

Animals↗
PubMed1976

Development of teratomas from the ectoderm of mouse egg cylinders.

We studied the developmental capacities of the primary ectoderm and endoderm of 6-day embryos of hybrids between strains 129/Sv-SIJ C P and A/He mice by grafting these germ layers into the testes of adult mice for 30 days. Grafts of embryonic ectoderm gave rise to teratocarcinomas composed of undifferentiated embryonal cells and derivatives of all three germ layers including respiratory and alimentary epithelium. Grafts of extraembryonic ectoderm gave rise to invasive trophoblastic giant cells. Grafts of endoderm did not develop.

Animals↗
PubMed2006

In vitro osteogenesis from human skin-derived precursor cells.

Embryonic tissue and organ development are initiated from three embryonic germ layers: ectoderm (skin and neuron), mesoderm (blood, bone, muscle, cartilage and fat) and endoderm (respiratory and digestive tract). In former times, it was believed that cell types in each germ layer are specific and do not cross from one to another throughout life. A new finding is that one tissue lineage can differentiate across to another tissue lineage, and this is termed transdifferentiation. We were interested in studying the transdifferentiation of skin-derived precursor cells (ectoderm layer) to osteoblastic cells (mesoderm layer). Human skin-derived precursor cells (hSKP) were isolated and induced into an osteoblastic lineage using osteogenic induction medium (alpha-MEM plus 10% fetal bovine serum supplemented with ascorbic acid, beta-glycerophosphate and dexamethasone). The specific characteristics of osteoblastic cells, including the expression of enzyme alkaline phosphatase, the deposition of mineral and the expression of osterix, bone sialoprotein and osteocalcin, were detected only from the inductive group. The results in our study show that SKP from human skin are a practically available source for osteogenesis. The samples are easily obtainable for autologous use with a high expansion capacity.

Adult↗
PubMed1999

The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo.

ActRIA (or ALK2), one of the type I receptors of the transforming growth factor-beta (TGF-beta) superfamily, can bind both activin and bone morphogenetic proteins (BMPs) in conjunction with the activin and BMP type II receptors, respectively. In mice, ActRIA is expressed primarily in the extraembryonic visceral endoderm before gastrulation and later in both embryonic and extraembryonic cells during gastrulation. To elucidate its function in mouse development, we disrupted the transmembrane domain of ActRIA by gene targeting. We showed that embryos homozygous for the mutation were arrested at the early gastrulation stage, displaying abnormal visceral endoderm morphology and severe disruption of mesoderm formation. To determine in which germ layer ActRIA functions during gastrulation, we performed reciprocal chimera analyses. (1) Homozygous mutant ES cells injected into wild-type blastocysts were able to contribute to all three definitive germ layers in chimeric embryos. However, a high contribution of mutant ES cells in chimeras disrupted normal development at the early somite stage. (2) Consistent with ActRIA expression in the extraembryonic cells, wild-type ES cells failed to rescue the gastrulation defect in chimeras in which the extraembryonic ectoderm and visceral endoderm were derived from homozygous mutant blastocysts. Furthermore, expression of HNF4, a key visceral endoderm-specific transcription regulatory factor, was significantly reduced in the mutant embryos. Together, our results indicate that ActRIA in extraembryonic cells plays a major role in early gastrulation, whereas ActRIA function is also required in embryonic tissues during later development in mice.

Activin Receptors, Type I↗
PubMed2005

Shield formation at the onset of zebrafish gastrulation.

During vertebrate gastrulation, the three germ layers, ectoderm, mesoderm and endoderm are formed, and the resulting progenitor cells are brought into the positions from which they will later contribute more complex tissues and organs. A core element in this process is the internalization of mesodermal and endodermal progenitors at the onset of gastrulation. Although many of the molecules that induce mesendoderm have been identified, much less is known about the cellular mechanisms underlying mesendodermal cell internalization and germ layer formation. Here we show that at the onset of zebrafish gastrulation, mesendodermal progenitors in dorsal/axial regions of the germ ring internalize by single cell delamination. Once internalized, mesendodermal progenitors upregulate E-Cadherin (Cadherin 1) expression, become increasingly motile and eventually migrate along the overlying epiblast (ectodermal) cell layer towards the animal pole of the gastrula. When E-Cadherin function is compromised, mesendodermal progenitors still internalize, but, with gastrulation proceeding, fail to elongate and efficiently migrate along the epiblast, whereas epiblast cells themselves exhibit reduced radial cell intercalation movements. This indicates that cadherin-mediated cell-cell adhesion is needed within the forming shield for both epiblast cell intercalation, and mesendodermal progenitor cell elongation and migration during zebrafish gastrulation. Our data provide insight into the cellular mechanisms underlying mesendodermal progenitor cell internalization and subsequent migration during zebrafish gastrulation, and the role of cadherin-mediated cell-cell adhesion in these processes.

Animals↗