Monensin inhibits the first cellular movements in early chick embryo

Summary In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.     

Alpha-fetoprotein expression in intra- and extraembryonic fluids of developing chick embryo

The ontogeny of alpha-fetoprotein (AFP) has been studied in the chicken (from 7 days of incubation until 2 days after hatching) using (1) the two-dimensional immunoelectrophoresis technique, (2) the polyacrylamide gel electrophoresis, and (3) the high resolution two-dimensional polyacrylamide gel electrophoresis. The molecular weight of AFP was estimated at 71,000. AFP was seen as a heterogeneous population composed of four isoforms which slightly differ by their isoelectric points. Up to the 18th day of development, qualitative changes in AFP heterogeneity do not occur. Only traces of the two alkaline isoforms were observed in plasma of 2 days post-hatching chickens. AFP has been identified in allantoic and cerebrospinal fluids but is not present in amniotic fluid. At 7 days of embryonic age, all the plasma AFP species are present in cerebrospinal fluid.     

Lectin receptors on the peri- and early post-implantation mouse embryo

Summary Mouse embryos at the blastocyst, blastocyst outgrowth, and primitive streak (day 7.5) stages of development were analysed for expression of lectin receptors using a panel of six FITC-conjugated lectins with affinities for five distinct saccharides (BSL, ConA, DBA, LTL, UEA and WGA). Blastocyst trophoblast expressed receptors for all the lectins but later tissues of the trophectoderm lineage lost receptors for distinct but overlapping subsets of the lectin panel. The inner cell mass (ICM) of the early blastocyst lacked receptors only for UEA. Differentiation of primary endoderm was accompanied by the aquisition of UEA receptors but subsequent differentiation into visceral and parietal endoderm involved the loss of receptors for both fucose binding lectins (UEA and LTL). Embryonic ectoderm in the day 7.5 egg cylinder retained receptors only for ConA and WGA. Thus, in general, differentiation during the peri- and early post-implantation period was associated with a differential loss of lectin receptors in all cell lineages of the mouse conceptus.     

N-glycosylated proteins interfere with the first cellular migrations in early chick embryo.

Cell adhesion and migration properties which are known to play a crucial role in developmental events seem to be modulated by variations in glycosylation of glycoproteins. In the chick embryo, the extracellular matrix (ECM) appears as a loose meshwork of fibrillar material in the space between the epiblast and the hypoblast shortly before the first major cell migrations start. Chick embryos treated with tunicamycin (TN), a specific inhibitor of N-linked glycosylation of proteins, show little or no ECM, diminished cell adhesion and a dramatic alteration in the architecture of the epiblast and of the hypoblast. The first major cell migrations which signal the onset of PS and gastrula formation are inhibited irreversibly in these embryos. Tunicamycin induces a substantial change in the labeling pattern with change in mobility of some polypeptides and with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from these already present in the control blastoderm. The N-linked glycosylation of protein(s) that are synthesized during the interaction of the epiblast and of the hypoblast seem to play a critical role in cell adhesion and in the morphogenetic movements of gastrulation in the early chick embryo.     

Differential expression of Eya1 and Eya2 during chick early embryonic development

Eyes absent is essential for compound eye formation in Drosophila. Its mammalian homologues of Eya are involved in the development of sensory organs, skeletal muscles and kidneys. Mutations of EYA1 in human cause branchio-oto-renal syndrome, with abnormalities in branchial derivatives, ear and kidney. For an insight into the function of Eya1 and Eya2 in early development, we performed whole-mount in situ hybridization and compared the expression patterns of these two genes in the developing chick embryos. Eya1 was first expressed in the primitive streak at Hamburger and Hamilton stage 4 (HH4) and appeared in the ectoderm and head mesenchyme distinct from migrating neural crest cells at HH6-HH11. At HH15 and HH17, the olfactory, otic and vagal/nodose placodes and cranial ganglia were positive for Eya1. In contrast, Eya2 was already expressed in the endoderm at HH4, and appeared in the endoderm and prospective placodal region at HH6-HH11. Eya2 expression was observed in pharyngeal clefts and pouches as well as cranial placodes at HH15 and HH17. These results indicate differential expression of Eya1 and Eya2, both spatially and temporally, in chick during early development. The expression patterns are somewhat different from those of other species such as Xenopus, zebrafish and mouse. The results suggest distinct and unique functions for Eya1 and Eya2 in early chick development.     

Development of the basal lamina and extracellular materials in the early chick embryo

Summary Chick embryos at developmental stages up to primitive streak formation were fixed in a mixture of tannic acid and glutaraldehyde. A basal lamina was present in the unincubated embryo and consisted of a lucent lamina interna and a lamina densa. At the primitive streak stage the lamina densa showed a periodicity of stained elements. Densely stained materials were present on the cell surfaces lining the cavity between the epiblast and endoblast, and on the mesoderm cells within this cavity. Considerable amounts of extracellular material were observed in the cavity. Hyaluronidase treatment removed the cell surface and extracellular material, indicating that hyaluronic acid is a major component. This enzyme disrupted the basal lamina, leaving a fibrillar remnant with no periodic structure. It is therefore suggested that the dense periodicities consist of glycosaminoglycan built on an enzyme-resistant framework which is probably collagen. Enzyme-resistant fibrils, presumably collagen precursors, are present elsewhere within the tissue spaces.     

Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development

This paper shows stage- and tissue-specific global demethylation and remethylation occurring during embryonic development. The egg genome is strikingly undermethylated and the sperm genome relatively methylated. Following a loss of genomic methylation during preimplantation development, embryonic and extraembryonic lineages are progressively and independently methylated to different final extents. Methylation continues postgastrulation and hence could be a mechanism initiating, or confirming, differential programming in the definitive germ layers. It is proposed that much of the methylation observed in somatic tissues acts to stabilize and reinforce prior events that regulate the activity of specific genes, chromosome domains or the X chromosome (in females). Fetal germ cell DNA is markedly undermethylated and we favour the idea that the germ lineage is set aside before the occurrence of extensive methylation of DNA in fetal precursor cells.     

Patterns of cell division and interkinetic nuclear migration in the chick embryo hindbrain

Early in its development, the chick embryo hindbrain manifests an axial series of bulges, termed rhombomeres. Rhombomeres are units of cell lineage restriction, and both they and their intervening boundaries form a series that reiterates various features of neuronal differentiation, cytoarchitecture, and molecular character. The segmented nature of hindbrain morphology and cellular development may be related to early patterns of cell division. These were explored by labeling with BrdU to reveal S-phase nuclei, and staining with basic fuchsin to visualise mitotic cells. Whereas within rhombomeres, S-phase nuclei were located predominantly toward the pial surface of the neuroepithelium, at rhombomere boundaries S-phase nuclei were significantly closer to the ventricular surface. The density of mitotic figures was greater toward the centres of rhombomeres than in boundary regions. Mitotic cells did not show any consistent bias in the orientation of division, either in the centres of rhombomeres, or near boundaries. Our results are consistent with the idea that rhombomeres are centres of cell proliferation, while boundaries contain populations of relatively static cells with reduced rates of cell division.     

The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages

The cell movements underlying the morphogenesis of the embryonic endoderm, the tissue that will give rise to the respiratory and digestive tracts, are complex and not well understood. Using live imaging combined with genetic labeling, we investigated the cell behaviors and fate of the visceral endoderm during gut endoderm formation in the mouse gastrula. Contrary to the prevailing view, our data reveal no mass displacement of visceral endoderm to extraembryonic regions concomitant with the emergence of epiblast-derived definitive endoderm. Instead, we observed dispersal of the visceral endoderm epithelium and extensive mixing between cells of visceral endoderm and epiblast origin. Visceral endoderm cells remained associated with the epiblast and were incorporated into the early gut tube. Our findings suggest that the segregation of extraembryonic and embryonic tissues within the mammalian embryo is not as strict as believed and that a lineage previously defined as exclusively extraembryonic contributes cells to the embryo.     

Male diploid embryonic cell line ofDrosophila virilis

Summay A new established cell line 79f7Dv3g, ofDrosophila virilis consisting initially of male and female cells and represented now, after 6 yr of cultivation, only by male cells is described. The population doubling time is 36 h at 25° C. The cell culture is also able to grow in serum-free media for an indefinite time without special selection and has a population doubling time of 2 d.     

The role of DE-cadherin during cellularization, germ layer formation and early neurogenesis in the Drosophila embryo

The Drosophila E-cadherin homolog, DE-cadherin, is expressed and required in all epithelial tissues throughout embryogenesis. Due to a strong maternal component of DE-cadherin, its early function during embryogenesis has remained elusive. The expression of a dominant negative DE-cadherin construct (UAS-DE-cad(ex)) using maternally active driver lines allowed us to analyze the requirements for DE-cadherin during this early phase of development. Maternally expressed DE-cad(ex) result in phenotype with variable expressivity. Most severely affected embryos have abnormalities in epithelialization of the blastoderm, resulting in loss of the blastodermal cells' apico-basal polarity and monolayered structure. Another phenotypic class forms a rather normal blastoderm, but shows abnormalities in proliferation and morphogenetic movements during gastrulation and neurulation. Mitosis of the mesoderm occurs prematurely before invagination, and proliferation in the ectoderm, normally a highly ordered process, occurs in a random pattern. Mitotic spindles of ectodermal cells, normally aligned horizontally, frequently occurred vertically or at an oblique angle. This finding further supports recent findings indicating that, in the wild-type ectoderm, the zonula adherens is required for the horizontal orientation of mitotic spindles. Proliferation defects in DE-cad(ex)-expressing embryos are accompanied by the loss of epithelial structure of ectoderm and neuroectoderm. These germ layers form irregular double or triple layers of rounded cells that lack zonula adherens. In the multilayered neuroectoderm, epidermal precursors, neuroblasts and ganglion mother cells occurred intermingled, attesting to the pivotal role of DE-cadherin in delamination and polarized division of neuroblasts. By contrast, the overall number and spacing of neuroblasts was grossly normal, indicating that DE-cadherin-mediated adhesion is less important for cell-cell interaction controlling the ratio of epidermal vs. neural progenitors.     

家蝇胚胎细胞系的建立及其生物学特性

昆虫细胞系在许多领域得到广泛的应用。以家蝇Musca domestica L.卵(胚胎)为材料,用含20%胎牛血清的M3培养基培养,原代培养35d左右形成单层,每7d传代1次,连续传代60代次。细胞群体倍增时间为44h,染色体2n=12,可在液氮及4℃冰箱中保存复苏,命名为MDEC-07114。     

Mechanisms of adhesion among cells of the early chick blastoderm. Role of calcium ions in the adhesion of extraembryonic endoderm cells

Summary Cells from the extraembryonic endoderm of the gastrulating chick embryo adhere to one another in the absence of divalent cations. The addition of Mg²⁺ ions to the medium has no effect on the aggregation kinetics but the addition of Ca²⁺ ions increases the number of cells which aggregate and also stabilizes adhesion. Some aggregation also occurs when cells are suspended in saline devoid of Ca²⁺ and Mg²⁺ ions and supplemented with EGTA, a Ca²⁺ ion complexing agent, but adhesion is not stabilized. Shear sensitive and shear resistant bonds form in Ca-containing as well as in EGTA-containing saline. These results suggest that extraembryonic endoderm cells have Ca²⁺ indepedent and Ca²⁺ dependent mechanisms of adhesion.     

Developmentally regulated lectin in embryonic chick muscle and a myogenic cell line.

Soluble extracts of embryonic chick pectoral muscle and myoblast clone L6 agglutinated trypsin treated glutaraldehyde fixed rabbit erythrocytes. Agglutination activity was blocked by thiodigalactoside, lactose and related saccharides but not by many other saccharides. Agglutination activity of chick pectoral muscle extracts increased at least one order of magnitude between 8 and 16 days of chick embryo development, as the pectoral muscle differentiated. With L6 myoblasts there was a three-fold increase in activity of the extracts as the myoblasts fused to form multinucleated myotubes.     

Glycoprotein differences among cells of the 14-day embryonic chick neural retina

In order to test the hypothesis that the progressive layering and differentiation of cell types during the development of the neural retina are associated with cell surface alterations we have separated distinct cell populations from the 14-day embryonic chick retina. Cells of these populations have been shown to differ in associative behavior and intramembrane particle content. We now report that these cells differ in cell surface glycoproteins. Proteins were labeled with two different extrinsic labels and one metabolic label. We used enzymatic transfer of galactose from UDP-gal to cellular acceptors, and borotritide reduction after galactose oxidation as extrinsic labels. Glucosamine incorporation was used as the metabolic label. In all these cases, we were able to identify bands on electrophoretic gels which were unique to individual populations.     

Method for electroporation for the early chick embryo

In vitro whole-embryo culture of chick embryos, originally invented by New, has been widely used for studies of early embryogenesis. Here, a method for electroporation using the New culture and its derivatives is described, to achieve misexpression of exogenous gene in a temporally and spatially controlled manner in gastrulating chick embryos. Detailed information for the devices and procedures, and some experimental examples are presented.