Tracing of cell lineage in embryonic development of Phallusia mammillata (ascidia) by vital staining of mitochondria

Vital staining of mitochondria with a fluorescent dye 3,3′-diethyloxacarbocyanine was used to follow cell lineage in embryos of Phallusia mammillata. The results agree in general with the plan established by Conklin in 1905. Strong fluorescence migrated after fertilization similarly to the pigment of the “yellow crescent” in Styela. Later, fluorescence segregated into muscle cell primordia, but not into mesenchyme cells. An animal hemisphere cell, b 8.17 also exhibited strong fluorescence and joined a group of muscle primordia, very likely becoming a muscle cell itself. In the tadpole, all the tail muscle cells were fluorescent. Fluorescence was also noticed in nerve cell primordia of the vegetal hemisphere, particularly in the cell A 8.16 whose descendants appeared to become part of the sensory vesicle which was strongly fluorescent in the tadpole. The usefulness of this type of vital staining in following cell lineage of colorless embryos is stressed.     

A lymphocyte cell surface molecule that is antigenically related to actin

When viable murine lymphocytes are incubated sequentially with a saturating amount of affinity-purified, rabbit anti-actin and highly conjugated FITC-goat anti-rabbit Ig, about 52% of mesenteric lymph node (MLN) lymphocytes and 36% of thymocytes exhibit a faint, but sharply punctate surface fluorescence. Cell surface actin (CSA) can be distinguished from staining of cytoplasmic actin in permeable cells, which are identified by their uptake of ethidium bromide. Staining of actin in ethidium bromide-permeable cells is 10-fold more intense than staining of actin on ethidium bromide-impermeable cells and is seen as uniformly fluorescent rings or crescents at the periphery of the cell and as dimmer, diffuse fluorescence centrally. Binding of rabbit anti-actin and goat anti-rabbit Ig to the lymphocyte cell surface is not mediated by Fc receptors; F(ab')2 fragments of these antibodies detect the same number of positive cells as do the intact molecules, and affinity-purified anti-KLH does not bind significantly. The cell surface stain, measured by flow cytometry or fluorescence microscopy, can be absorbed by pretreatment of the anti-actin with immobilized actin but not with IgG-Sepharose. Double-label experiments show that about 70% of the non-B cells and 30% of the MLN B cells bear detectable CSA. Although we have not ascertained the origin of CSA, we find that the number and brightness of cells exhibiting CSA cannot be increased by preincubating the cells with exogenous native skeletal muscle actin or with supernatant from dissociated MLN, indicating that there are no free binding sites for exogenous actin. The findings imply that either there is a developmentally expressed binding site(s) for actin, or that at various stages of development lymphocytes express a protein antigenically related to actin on their surface.     

Zymographic demonstration of lactate and malate dehydrogenases isoenzymes in the rodent salivary glands

Summary Analysis of lactate and malate dehydrogenase zymograms of rodent salivary glands showed species and organ specific patterns.Lactate dehydrogenase isoenzyme patterns occupied the middle positions in relation to those of skeletal and heart muscle. Activities of the major salivary glands were in the order submaxillary gland>parotid>sublingual gland. Zymogram of the mouse and rat showed LDH₄ and LDH₅ high activity patterns, while that of the rabbit was the fast moving active one. Hamster salivary gland exhibited a neutral type of the former and the latter.Malate dehydrogenase isoenzyme exhibited very similar patterns for the mouse, rat and hamster. Malate dehydrogenase zymogram of rabbit showed 3 active bands, which was different from the other rodents.     

Hatching enzyme immunolocalization during embryonic development of the ascidians Ciona intestinalis and Phallusia mammillata

Summary

Different stages of the embryonic development of the ascidians, Ciona intestinalis and Phallusia mammillata, were observed by confocal microscopy after treating embryos with polyclonal antibody raised against C. intestinalis hatching enzyme and after staining with FITC-conjugated second antibody. In both species fluorescence is localized, at the gastrula stage, in the ectoderm. At the subsequent neurula and tail bud stages, in C. intestinalis, the enzyme is localized in the anterior region and tail region, while in P. mammillata it is only present in the anterior region.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

5'-nucleotidase activity in developing chick pectoral muscle.

The activity of the plasma membrane enzyme 5′-nucleotidase varies dramatically during the embryonic development of chick pectoral muscle. The specific activity is greatest at early stages of differentiation (8-day embryos), falls to a minimum on days 12–14, then rises again in older embryos. In cultured muscle cells obtained from embryonic chick muscle the 5′-nucleotidase activity is essentially absent. Muscle cells grown in the presence of bromodeoxyuridine, an inhibitor of muscle differentiation, contain enhanced levels of 5′-nucleotidase activity. These results indicate that 5′-nucleotidase may be absent in muscle fibers, but present in other cells of muscle tissue.     

The effect on preimplantation embryo development of non-specific inflammation localized outside the reproductive tract

The aim of this study was to evaluate the possible effect of non-specific acute inflammation localized outside the reproductive tract on the quality of preimplantation embryos. In fertilized female mice two experimental models of inflammation were used—trinitrobenzene sulfonic acid colitis and carrageenan paw oedema. Inflammation was induced during the cleavage period of embryo development and embryos were collected at 92 h post hormonal synchronization. Stereomicroscopical evaluation of in vivo derived embryos showed that the presence of inflammation in the maternal body did not affect their basic developmental abilities, i.e. there were no significant differences in the proportion of early blastocysts, morulas, slowly developing embryos and degenerates between embryonic pools obtained from mothers with induced inflammation and control mothers. In the next step, non-degenerated embryos from all mothers were cultured in vitro under standard conditions for another 24 h, and the average cell number (fluorescence DNA staining) and the incidence of cell death (fluorescence viability staining combined with TUNEL assay) were evaluated. The majority of cultured embryos reached expanded blastocyst stage. There were no significant differences in the average cell numbers of blastocysts, but blastocysts derived from mothers with induced inflammation showed a significantly higher incidence of dead cells in both experiments. The majority of dead cells were of apoptotic origin. These results show that non-specific inflammation localized outside the reproductive tract has no detrimental effect on the preimplantation embryo growth; however it can affect the embryo quality.     

Detection of glycophorin A-like glycoproteins on the surface of cultured human cells

1. In previous studies we have isolated and characterized mucin-type glycopeptides from mouse and human melanoma cells. 2. These glycopeptides have clusters of oligosaccharides of the type (NeuNAc)0-2----[Gal----GalNAc] linked to serine and or threonine suggesting an apparent similarity to glycophorin. 3. We now report the interaction of polyclonal anti-glycophorin antibodies with various cultured cells. Antisera to highly purified glycophorin A were raised in rabbits. 4. Human melanoma cells (HM7), human breast cells (HBL-100) and two lines of human breast cancer cells (MCF-7 and MDA-MB-231) showed medium to very strong cell surface fluorescence pattern after staining with rabbit anti-glycophorin F(ab')2 and FITC-conjugated goat anti-rabbit F(ab')2. 5. Immunodiffusion, immunoelectrophoresis and affinity chromatography on anti-glycophorin IgG-Sepharose 4B of detergent extracts of metabolically labeled cultured cells gave further evidence for the presence of glycophorin-like components in these cells. 6. Glycoproteins of MCF-7 cells interacting with anti-glycophorin antibodies were affinity purified and partially characterized.     

Immunohistochemical localization of cyclic AMP during normal and abnormal chick and mouse limb development

This paper describes the immunohistochemical localization of cAMP during limb chondrogenesis in talpid3 chick, brachypod mouse, and normal embryos. Comparisons were made between chick wing buds at Stages 22, 25, and 30, and mouse hind limb buds at Days 11, 12.5 and 14. At Stage 22, the normal mesenchyme in the chick displayed areas of bright fluorescence compared to a lesser intense and more evenly distributed fluorescence in talpid3. Sections of the central region from normal Stage 25 limb buds exhibited an intense fluorescence that was uniformly distributed, whereas, in talpid3 staining was more mosaic with some areas fluorescing brightly and others showing little fluorescence. At Stage 30 the staining pattern was similar between normal and talpid3, with the fluorescence being brighter in the cartilage tissue than in the surrounding soft tissue. Difference in the staining patterns of normal and brachypod limb tissue were not detectable. At Days 11 and 12.5, tissue from both genotypes displayed a very bright, uniform fluorescence. In the 14-day hind limb buds, the staining patterns were comparable to those observed in Stage 30 chick wing buds. However, under in vitro conditions conducive for the expression of the chondrogenic phenotype, differences in the intensity and extensiveness of fluorescent staining were detectable in cultures derived from 12-day normal and brachypod hind limb mesenchyme. Compared to the control, the uneven distribution of immunofluorescence in the talpid3 limb buds and the differences in intensity and extensiveness of fluorescence in the brachypod cultures support the hypothesis that cAMP is involved in limb cartilage differentiation.     

Effect of embryo developmental stage and culture conditions on number and quality of ovine in vitro produced blastocysts

This study evaluated the final output and quality of in vitro produced blastocysts derived from in vivo recovered sheep embryos cultured at various early developmental stages to blastocyst. A total of 270 embryos were recovered from the oviduct, at different days of the early luteal phase, and were classified into three different developmental stages: 2- to 4-cell (n = 93); 5- to 8-cell (n = 92) and 9- to 12-cell (n = 85). The effect of culture conditions was studied, at the same time, by randomly allocating the embryos to one of four groups: three groups of culture with fresh oviduct monolayers (2, 4 and 5 days old) and a fourth group with 2-day monolayers derived from frozen-thawed oviduct cells. Two control groups were established: first, embryos cultured in semi-defined medium (n = 29) and, second, blastocysts obtained in vivo and cryopreserved (n = 43). Influence on blastocyst yield of embryo developmental stage at the start of culture was statistically significant (p < 0.001). Two- to four-cell embryos showed a significantly lower developmental rate (67.7%) than the 5- to 8-cell (83.6%; p < 0.001) and 9- to 12-cell groups (90.5%; p < 0.0001) and lower quality in terms of blastocyst cryotolerance (56.0 vs. 83.7%; p < 0.005). There were no detected effects relating to the age or handling of the monolayer on the embryo developmental rate, but the day of blastocyst appearance was different between embryos cultured on monolayers derived from fresh or frozen-thawed cells (p < 0.0001); the main influence was on the group of 9- to 12-cell embryos (p < 0.0001). Current results confirm the temporal sensitivities of sheep embryos to in vitro culture, regardless of the culture conditions.     

Heterogeneity of rabbit aortic endothelial cells in primary culture

Factor VIII-related antigen (F8-RAg) and angiotensin-converting enzyme (ACE) are accepted diagnostic markers of endothelial cells in culture. However, when we isolated cells from rabbit thoracic aorta (after collagenase treatment and gentle scraping of the intima) and examined them with immunoperoxidase techniques, we observed two cell types which stained specifically for either F8-RAg or ACE, but not both. Each cell type was morphologically distinguishable in primary culture. F8-RAg-positive cells were recognizable in distinct patches as more elongated, tightly apposed, and firmly adherent cells; they exhibited only faint or no staining for ACE and no accumulation of a fluorescent, acetylated low-density lipoprotein probe (DiI-Ac-LDL), another endothelial cell marker. In contrast, ACE-positive cells were more rounded, less closely apposed, and grew as strict monolayers that exhibited a characteristic cobblestone appearance at confluence; ACE-positive cells were F8-RAg negative, but demonstrated intense labeling with DiI-Ac-LDL. Subcultures of ACE-positive cells were also stained by anti-rabbit thrombomodulin.     

水稻胚胎发育与萌发过程中凝集素的生物合成及其对转录与翻译抑制剂的敏感性

稻胚凝集素(RGL)在开花后7—13天之间合成活性很强,15天以后明显下降。7—13天之间RGL合成相当旺盛是由于这一时期编码RGL的mRNA得到迅速转录,而在开花后15—30天之间以及萌发4小时内RGL的合成主要是由胚分化发育期间(7—13天)所形成的mRNA所指导的。RGL主要在水稻胚胎发育过程中合成、积累,在萌发过程中几乎不表达,所以RGL是胚胎特异的蛋白质。     

Migratory and organogenetic capacities of muscle cells in bird embryos

Summary The migratory and organogenetic capacities of muscle cells at different stages of differentiation were tested in heterospecific chick/quail recombinants. Grafts containing muscle cells were taken from the premuscular masses from 4- to 5-day quail embryos, from the limb or trunk muscles of 12-day embryonic and 4-day post-natal quails, and from experimentally produced bispecific premuscular masses in which the myoblasts are of quail origin and the connective tissue cells of chick origin. Grafts were implanted into 2-day chick embryos in place of the somitic mesoderm at the limb level. Hosts were examined 4 to 7 days after operation.After implantation of a piece of premuscular mass, quail cells were found at and around the site of the graft in the truncal region and within the limb as far as the autopod. Quail cells participated predominantly in the trunk and limb musculature, which contained a number of quail myotubes and of bispecific quail/chick myotubes. Apart from skeletal muscles, quail cells contributed sporadically to nerve envelopes and blood vessel walls in the limb.When the graft was of bispecific constitution, quail nuclei in the limb and the trunk were found exclusively in monospecific and bispecific myotubes.After implantation of differentiated embryonic or post-natal muscle tissue, quail cells in the limb contributed only sporadically to nerve envelopes and blood vessel walls, while in the trunk they also participated in the formation of muscles and tendons.It is concluded that the myogenic cells in 4 to 5-day quail premuscular masses are still able to undergo an extensive migration into the limb buds and there participate in the formation of myotubes and anatomically normal muscles. They display developmental potentialities equivalent to those of the somitic myogenic stem cells. These capacities are lost in 12-day embryonic muscles.     

Comparison of different staining procedures for the flow cytometric analysis of U-937 cells infected with different Leishmania-species.

The human macrophage cell line U-937 infected with different Leishmania species, Leishmania mexicana amazonensis (Lma), Leishmania donovani (Ld) and Leishmania infantum (Li), was analyzed by flow cytometry (FCM). Leishmania spp. were labeled with different stains prior to the infection of the U-937 cells (BCECF-Am, PKH2-GL and SYTO 17) or after the infection (AO, FITC-conjugated monoclonal antibodies, PI). Infected cells were analyzed by flow cytometry, fluorescence microscopy and in parallel microscopically after Giemsa staining. The data obtained by these two methods were compared to decide which method is mostly appropriate for detection and estimation of the infection rate. Three fluorescent stains were suitable: BCECF-Am, SYTO 17 and FITC-conjugated MoAb with 0.02% digitonin. None of the vital stains gave evaluable results after 3 days of incubation.     

Quantitative morphologic studies on the myocardium in early stages of ontogenesis and after application of various cardioactive agents

The authors have morphometrically studied the differentiation of the myocardium in dynamic phases of the embryonic and postnatal development in chickens and Syrian Hamsters. Moreover, they investigated the action of the beta-adrenalytic substances Practolol and Trimepranol on ultrastructure of the cardiac muscle in adult animals. The volume of mitochondria in myocardial cells in 6-day old chicken embryos amounts to 5.65% of the total cell volume, in 12-day old embryos 14.35%, in 18-day old embryos 19.60%, in 1-day old chickens 23.24% which is nearly as much as in adult animals. The volume of myofibrils in 6-day old embryos is about 3.2%, in 12-day old embryos about 7.4%, in 18-day old embryos about 16.4% and in 1-day old chickens about 21.2%. The differences between individual groups are statistically significant. The dynamics of differentiation of the myocardium in Syrian Hamsters was studied in 5 phases, namely in 14-day old embryos and in postnatal phases on the 2nd, 5th, 14th and 21st days after birth. Most cells in 14-day old embryos are rather immature. Participation of the volume of mitochondria, myofibrils, equipment of mitochondria with cristae etc. considerably increase in postnatal phases. These findings suggest that the heart of mammals is rather immature at birth and will differentiate mainly in the postnatal developmental phases. Many morphometric findings, as regards the action of beta-adrenalytic drugs on the ultrastructure of the myocardium in adult rabbits, point to the fact that application of these substances will give rise to degenerative alterations in approximately 10% of myocardial cells. Theoretical explantation of these mechanisms is being discussed.     

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.     

Microspectrographic analysis of formaldehyde-induced fluorescence in the quail adenohypophysis after injection of l-dopa and 5-hydroxytryptophan

Summary The spectral distribution of the formaldehyde-induced fluorescence emitted by model solutions and by adenohypophyses after intraperitoneal injection of l-dopa or 5-hydroxytryptophan was analyzed microspectrographically. Based on previously reported studies and on present findings, it seems that dopamine is stored in the strongly fluorescent cells after injection of l-dopa, and that a compound closely related to 5-hydroxytryptophan or serotonin is present in most of the cells after injection of 5-hydroxytryptophan. A non-specific, granular fluorescence appeared after 5-hydroxytryptophan and, to a lesser extend, l-dopa treatment. It probably represents autofluorescence of lysosomes, which are numerous in these circumstances.     

Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP

The CaMV 35S promoter is the most commonly used promoter for driving transgene expression in plants. Though it is presumed to be a constitutive promoter, some reports suggest that it is not expressed in all cell types. In addition, the information available on its expression profile in all possible cell and tissue types and during early stages of development is incomplete. We present here a detailed expression profile of this promoter investigated using the green fluorescent protein (GFP) gene as a reporter system in cotton during embryo development, and in all the vegetative and floral cell and tissue types. GFP expression was not detected during the early stages of embryogenesis. The first perceptible GFP expression was observed in a small area at the junction of hypocotyl and cotyledons in embryos at around 13 days after anthesis. The GFP fluorescence progressively became stronger and expanded throughout the cotyledon and hypocotyl as embryo development advanced. After germination, varying levels of promoter activity were observed in all cell and tissue types in the hypocotyl, cotyledon, stem, leaf, petiole, and root. The promoter was also expressed in all floral parts. Although cotton pollen exhibited a low level of greenish autofluorescence, it was possible to discern GFP-dependent fluorescence in some of the pollen from all the T₀ plants examined. Developing cotton fibers also exhibited GFP fluorescence suggesting that the 35S promoter was active in these specialized epidermal cells. Thus, we show that the expression of the 35S promoter was developmentally regulated during embryogenesis and that beyond a certain stage during embryogenesis, the promoter was expressed in most cell and tissue types in cotton albeit at different levels.     

肌动蛋白存在于车蝗精母细胞核和染色体中

以兔抗肌动蛋白抗体为一抗,FITC偶联的羊抗兔IgG抗体为二抗进行间接免疫荧光实验,观察到车蝗（Oedaleus asiaticus）精母细胞核及减数分裂Ⅰ细线期、终变期、减数分裂Ⅱ中期染色体上均发出明亮的黄经发色荧光,说明其中含有肌动蛋白。本文结果证明肌动蛋白是车蝗减数分裂细胞核和染色体的组成成分。