Chick Blastoderm Research Articles

Hemoglobin synthesis in isolated erythroid colonies from the chick embryo

Primary cultures derived from mechanically dissociated definitive streak chick blastoderms were grown in a warm air stream on the stage of inverted phase microscope, through which in vitro erythroid development could be observed. Proerythroid cells divide three or four times in 48 hr to give rise to erythroid colonies ranging from 10 to 1000 cells, depending on the size of the blastoderm fragments from which they were derived. Erythroid cell development follows a similar course in cultures grown in a carbon dioxide incubator. Colonies consisting of about 50 cells, derived from blastoderm fragments containing 5 to 10 cells, were isolated and labeled with [ 3H]leucine, and their labeled hemoglobins were analyzed by isoelectric focusing. Both early hemoglobins (E,M,P,P′, and P″) and late hemoglobins (A and D) are made in colonies derived from single blastoderm fragments. The ratio of late to early hemoglobins is about 1.7 in all colonies analyzed. The implications of this finding for the clonal model of erythroid development are discussed.

The regulation of haemoglobin synthesis in cultured chick blastoderms by steroids related to 5beta-androstane.

1. After 24h of incubation, the blastoderm may be dissected from the early developing chick embryo and successfully maintained under conditions of organ culture in vitro. 2. Low concentrations of steroids related to 5beta-androstane stimulate the synthesis of foetal haemoglobins, types E and P, in a highly steroid- and tissue-specific manner.

Comparison of early embryonic and differentiating cell surfaces: Interaction of lectins with plasma membrane components

1. 1. Cells of the unincubated as well as those of primitive streak chick blastoderm, which are preparing for or are involved in morphogenetic movements, are agglutinated by wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A, but not by fucose-binding protein. 2. 2. Agglutination of these cells with soybean agglutinin occurs only after neuraminidase treatment, while that induced by concanavalin A, wheat germ and Ricinus communis agglutinins is not affected. 3. 3. Trypsin treatment of blastoderm cells had no effect on lectin-mediated agglutination. 4. 4. In contrast, cells derived from 10- and 12-day differentiating chick liver were agglutinated by wheat germ agglutinin only after trypsinization. 5. 5. Mechanically dissociated embryonic liver cells, which are not agglutinated, more 3H-labelled wheat germ agglutinin per cell than trypsinized cells, suggesting that during differentiation there may be a spatial reorganization of wheat germ agglutinin receptors within the plasma membrane. 6. 6. Membranes isolated from the above cell types were examined by analytical polyacrylamide gel isoelectric focusing and, in combination with affinity chromatography using wheat germ agglutinin conjugated to agarose, membrane material in the differentiating liver membrane, which binds to this lectin, was identified.

Mitotic cells and their microappendages in the primitive streak of the chick embryo.

Fresh pullet eggs (White Leghorn Strain) were incubated to the primitive streak stage of development. Blastoderms were fixed in situ with isotonic aldehyde fixatives and prepared for scanning electron miscropy by means of post-osmication, critical point drying and gold-palladium coating. Cells judged to be in various stages of mitosis by their surface contours were numerous on the ventral surface of the chick blastoderm. Cells which were in the late preparatory stages for mitosis had rounded up from their surroundings. Microvilli dominated the surface. The degree of separation and number of microvilli increased until late metaphase or anaphase. Mitotic cells did not completely separate themselves from adjacent cells. Ruffles and blebs were not prominent during mitotis and long filopodia were absent. A definite localization of microappendages (microvilli, blebs, ruffles) to the area of cytokinesis was evident in early telophase and persisted through daughter cell formation.

Studies on cell differentiation: inducing capacity of sulfhydryl-containing amino acids on post-nodal pieces of chick blastoderms.

The inducing capacity of sulfhydryl (SH)-containing amino acids (cysteine and glutathione) on post-nodal pieces (PNPs) of stage 4 chick blastoderms was investigated. PNPs were treated for different lengths of time with chick Ringer's solution (control group) or chick Ringer's solution containing cysteine or glutathione, followed by culturing for 2-10 days on Spratt-Haas agar medium or on the chorioallantoic membrane of 8-day chick embryos. Control PNPs rarely showed differentiation, but those treated with the amino acids for six hours or longer developed structures such as neural tissue, notochord, somite mesoderm, and nephric tubules. The pulsatile tissue was only seen in the PNPs cultured for four days or longer. Two-four hours of treatment was too short to provoke induction in a statistically significant number of PNPs. The highest frequency of induction was noted in those pretreated with glutathione (8 mug/ml) for eight hours, followed by culturing for four days. The magnitude of the inducing capacity and toxicity of the amino acids were concentration dependent: a deleterious effect was observed at 14 mug/ml; the highest frequency of induction occurred at 8 mug/ml, but the frequency decreased as the concentration decreased; at 2 mug/ml all PNPs remained viable, but only a few (9-14%) showed differentiation. The inducing capacity of the amino acids was counteracted by equimolar concentrations of rho-chloromercuribenzoic acid or omega-chloroacetophenone. The effects of glutathione (8 mug/ml) differed from those of Hensen's node grafts in that the former caused sublethal cytolysis and inhibited H-3-uridine uptake in competent ectodermal cells during the first 18 hours of cultivation.

Analysis of DNA isolated from intracellular yolk granules in the early chick blastoderm

DNA isolated from intracellular yolk granules of early chick blastoderms was analysed in the electron microscope and in the analytical ultracentrifuge. The yolk DNA molecules were found to be linear and comparatively short, with a buoyant density identical to that of nuclear DNA.

Characterization of Spontaneous, Chemical, and Viral Transformants of a C3H/3T3-Type Mouse Cell Line by Transplantation Into Young Chick Blastoderms<xref ref-type="fn" rid="fn2">2</xref>

Cells from a C3H/3T3-type cell line were transplanted into defects of the lower layer of stage-4 chick blastoderms before and after "spontaneous," chemical, and viral transformation. To check the validity of inhibition of lower-layer defect closure as a criterion of malignancy, we compared the behavior of the lower layer toward these cells with their in vitro growth pattern, their capacity to invade embryonic chick skin explants that were organotypicaly cultured, and their tumorigenicity in syngeneic mice. No false-positive results were observed with either test. The study of the in vitro growth pattern gave false-negative results during the early phase of spontaneous transformation, whereas these cells, exhibiting an untransformed growth pattern, were shown to be malignant by the other tests. We concluded that the inhibition of lower-layer defect closure is a reliable, sensitive and rapid test for the detection of malignancy in tissue-cultured cells from any source.

Effects of L-tryptophan on morphogenesis and growth in the early chick blastoderm

Chick blastoderms, suppliedin vitro andin ovo with L-tryptophan at the primitive streak stage, showed in their continued development typical retardation of brain formation and somitogenesis in the embryo, whereas heart formation remained unaffected. In contrast to an overall reduction in size observed at the higher L-tryptophan concentrations, a moderate enlargement of the area opaca, compared with the controls, was found at the lower concentrations. This enlargement was combined with an increased flattening of the ectodermal area opaca cells and a reduction of the number of microvilli covering these cells. As a simultaneous supply of glucose could reduce, to some extent, the morphogenetic disturbances, these might partly be ascribed to a blocking of gluconeogenesis from L-tryptophan, but the overall reduction in size mentioned, together with the observation of a reduced decomposition of intracellular yolk granules in the L-tryptophan-treated blastoderms, indicates that impairment of intracellular yolk granule decomposition was the principal disturbance. The possible role of serotonin-probably formed from the L-tryptophan supplied-is suggested as a regulating factor in this connexion.

Changes in the differentiation tendencies of the hypoblast-free Hensen's node during "Gastrulation" in the chick embryo.

Hensen's node was isolated from chick blastoderms of medium-streak to headfold stages from which the endodermal layer had previously been removed. The isolates were culturedin vivo by means of the intra-coelomic grafting technique. Node pieces with the endodermal layer intact served as controls.Endodermal differentiation tendencies gradually decreased from the medium-streak to the pre-head-process stage and completely disappeared at the head-process stage, whereas the controls gave rise to endodermal structures throughout all stages. "Cranial" structures such as oesophagus and trachea, often together with thyroid, parathyroid and/or thymus, were only found in grafts of younger stages, while gizzard, intestine and/or pancreas were observed in many gut-containing grafts throughout all stages.There was a constant high incidence of notochord, muscle, and cartilage formation. The incidence of mesonephric structures, sometimes accompanied by adrenal gland, rose steadily throughout all stages both in experimentals and controls.Neural differentiation tendencies (rhombencephalon and/or spinal cord) were always present in the nodes isolated (with or without endoderm) from the definitive primitive-streak stage onwards, but in nodes from earlier stages the incidence of neural differentiation was significantly lower.The results are discussed in relation to the possible location and determination of the prospective endoderm and mesoderm.

The onset of differentiation in the epiblast of the chick blastoderm (SEM and TEM).

Chick embryos have been examined by scanning and transmission electron microscopy from the unincubated to the late head process stages (Hamburger and Hamilton stages 1–5). Regional variations in cell structure have been found to develop very shortly after incubation begins and these become more clear cut with further development. In particular, those cells which are destined to invaginate possess fewer microvilli but more globular projections and more vesicles, than the cells which are destined to form the ectodermal covering of the embryo.

Microappendages on the ventral surface of the primitive streak chick embryo.

AbstractFresh pullet eggs (White Leghorn Strain) were incubated to obtain the primitive streak stage of development. Blastoderms were fixed in situ with isotonic aldehyde fixatives and prepared for scanning electron microscopy by means of postosmication, critical point drying and gold‐palladium coating.The ventral surface of the chick blastoderm exhibits extreme pleomorphism. There is variation among microappendages (microvilli, blebs, ruffles) which tend to define cellular outlines. In recessed areas adjacent to the ventrolateral aspect of the primitive streak microappendages are present in great profusion. Here clusters of them possess intricate physical relationships. Microvilli may arise from either blebs or ruffles. Other microvilli may terminate in blebs. Extensive connecting structures are present. The primitive streak chick embryo is discussed as a model for the study of microappendages of the plasmalemma.

Morbidity of aging non-incubated chicken blastoderms: further cytological evidence and interpretation

ABSTRACTCytological analysis was carried out on the blastodermal cells of White Leghorn eggs subjected to several pre-incubation treatments. These treatments were storage, ranging from 0 days to 4 weeks, together with one or more of the following: (1) variation of ambient temperature (5 °C, 15·5 °C, 24 °C), (2) variation of carbon dioxide level (normal air, 1·5% CO2-enriched air), (3) increased humidity (wrapping in plastic bags). To facilitate analysis, the chromosomal configuration of metaphase cells was classified into four types, I–IV, according to their increasingly ‘abnormal’ appearance, which included condensation, dispersal and/or clumping of chromosomes. In interphase cells, the degree of ‘abnormality’ was rated on the staining capacity and shape of the nuclei.The study yielded the following results.Temperature of storage was the most important single factor determining the state of ‘normality’ of the nuclei. The CO2 level in the storage chamber or the use of plastic bags (to provide the eggs in storage with a special ‘ mini-environment ‘, believed largely due to increased humidity) had little effect on the cytological picture of the affected blastoderms. As the blastoderms of eggs stored at 15·5 °C aged, the proportion of Type IV chromosomal configurations steadily rose. At 24 °C the aging process frequently followed a different route: both metaphase and post-metaphase chromosomes often simply disintegrated; the progression from Type I to Type IV was not in evidence. Aging at 5 °C resulted in an early appearance of uniformly dark-stained, spherical or oval nuclei. These were similar to those observed in the terminal stages of retrogression seen in the interphase nuclei. During extended storage at 15·5 °C, mitosis was shown to be blocked at metaphase. No active mitoses were observed in the blastoderms of eggs stored at 5 °C. At 24 °C however, limited mitotic activity was present, up to and including anaphase. The presence or absence of a high level of mitotic activity during pre-incubation storage was not crucial to the survival of the blastoderm. However, an environment that permitted limited mitosis was important if the cells were to have the best possible chance for remaining alive during storage. The CO2 content of the air or the use of plastic bags played no role in this respect. Two explanations, at the nuclear level, are suggested for the observed chain of events in the blastoderm of a stored chicken egg.

Investigation on the origin of the definitive endoderm in the rat embryo

ABSTRACT Single germ layers (or combinations of two of them) were isolated from the primitive streak and the head-fold stage rat embryos and grown for 15 days under the kidney capsule of syngeneic adult animals. The resulting teratomas were examined histologically for the presence of mature tissues, with special emphasis on derivatives of the primitive gut. Ectoderm isolated together with the initial mesodermal wings at the primitive streak stage gave rise to tissue derivatives of all three definitive germ layers. Derivatives of the primitive gut were regularly present in these grafts. At the head-fold stage, isolated ectoderm (including the region of the primitive streak) differentiated into ectodermal and mesodermal derivatives only. Endoderm isolated at the primitive streak stage did not develop when grafted and was always completely resorbed. At the head-fold stage, however, definitive endoderm differentiated into derivatives of the primitive gut if grafted together with adjacent mesoderm. These findings indirectly suggest the migration of prospective endodermal cells from the primitive ectoderm, and therefore a general analogy with the course of events during gastrulation in the chick blastoderm.

Poly(A)-attached RNA as activator in embryonic differentiation.

SummaryPoly(A)-attached RNA (mRNA) was isolated from chicken and calf hearts. Both mRNAs were found to induce the development of explanted postnodal pieces (PNP) of stage 4 chick blastoderm into beating tubes and/or tissues. Heart formation is specific and the frequency was found to depend entirely upon the concentration of mRNA used. In contrast, the concentration had no influence on other tissue formation e.g., neural tube, tubules, myoblasts, and/or notochord.The uptake of heart [3H]RNA by PNP cells was analyzed using high-resolution autoradiography. Silver grains were localized more in nucleus than in cytoplasm. The effect of heart mRNA on PNP was sensitive to actinomycin D and bromodeoxyuridine. It appears, therefore, that exogenous mRNA plays an important role in nuclear function. This RNA may affect the nuclei of intact cells in two ways: (1) as a derepressor, and (2) as a primer of new DNA synthesis. These alternatives were tested experimentally using the isozyme, lactate dehydrogenase, as marker....

Development of Embryonic Structures from Dispersed Chick Blastoderm Cells

A study of the in vitro growth of embryonic structures from cellular material obtained from chicken embryos is reported. Cells dispersed from a single donor blastodisc and incubated in tissue culture medium were observed to undergo mitotic division and to give rise to spherical structures phylogenetically more primitive than the normal avian blastodisc. At 6, 12, 18 and 24 hours the diameter of the spheres and the average cell sizes were measured in order to estimate the number of cells per “blastula.” Based on the assumption of synchronous mitoses it was calculated that the cells had undergone 12 mitoses during the first 24 hours, a rate slower than that of embryos in vivo.

Selective Cellular Affinities in the Unincubated Chick Blastoderm

Cell suspensions from whole unincubated chick blastoderms (stage 1,4) were obtained by EDTA or trypsin dissociation. They were cultured in rotating flasks and aggregates were examined after 1 to 7 days in culture. Early aggregates were solid and consisted of a continuous phase of loosely packed cells with abundant yolk granules, among which groups of tightly packed and less yolky cells were present. Loosely packed cells became peripheral while the tightly packed cells formed continuous cords traversing the aggregate. Aggregates also developed a cavity surrounded mainly by the loosely packed cells. The latter gave rise to cells similar to those of the columnar endoderm of the yolk sac. Our data suggest that sorting-out occurs in aggregates prepared from unincubatedblastoderms, before the onset of gastrulation.

THE EFFECTS OF EMBRYONIC HEART RNA AND ACTINOMYCIN D BOTH ON THE ISOLATED TISSUE-PIECES OF THE CHICK BLASTODERM AND ON THE ISOLATED PRIMITIVE TUBULAR HEART OF THE EARLY CHICK EMBRYO.

The effects of embryonic heart RNA extracted from 18-day chick embryos were studied both on the isolated parts of the chick blastoderm and on the isolated primitive tubular heart of the early chick embryo. The embryonic heart RNA accelerated in vitro the beating of the heart-forming area and the pulsing of the primordial heart organ. In addition, a phenomenon of cardiac transformation was observed.

A possibility of distinction between normal and neoplastic cells through transplantation into chick blastoderms.

Transplantation of certain types of cells and tissues into young chick blastoderms suggested a new method for distinguishing between normal and cancer cells. Different kinds of neoplastic and non-neoplastic cells and tissues were transplanted into defects of the lower layer of stage-4 blastoderms. Histologic examination of the blastoderms after 24 hours' incubation showed the lower layer was completely repaired at the level of certain types of non-neoplastic grafts; on the contrary, repair of the lower layer was inhibited by certain types of neoplastic grafts. Interference of neoplastic cells with migration of cells of the lower layer is proposed as the mechanism responsible for such inhibition. The experimental device might be useful as a diagnostic tool.

The development of tubular heart in RNA-treated post-nodal pieces of chick blastoderm

ABSTRACT Post-nodal pieces of the stage-4 chick blastoderms were transected 0·6 mm posterior to Hensen’s node and cultured in vitro with and without chicken-heart RNA. After 24 h the expiants were fed daily with fresh egg-extract medium for 4 days. On the 4th day pulsating heart was found in the RNA-treated but not in the untreated series. Histological examination revealed that cell differentiation other than of erythrocytes and epithelial tissue seldom occurred in the control and, in contrast, pulsating heart and cardiac myoblasts were present in most of the RNA-treated expiants that had differentiated.

Culture of erythropoietic cells from chick blastoderms.

Culture of erythropoietic cells from chick blastoderms.