Changes of localization of lectin-binding sites on the intestinal epithelial cell surface membrane during differentiation of the columnar intestinal cells

Light and electron microscopically localizations of Concanavalin A, Soybean Agglutinin, and Asparagus Pea Lectin-binding sites on the intestinal microvillous membrane were studied by means of horseradish peroxidase labelled lectins. The distribution of the lectin-binding sites was composed with the cell differentiation from the crypts to the tip of the intestinal villi. The visualization of lectin receptors in the mucin vesicles in the goblet cells was established as well. The controls for the determination of the specificity of reactions were carried out.     

The Golgi apparatus in the acinar cells of the developing embryonic pancreas: II. Localization of lectin-binding sites

The reaction patterns of the Golgi apparatus following staining with the lectins concanavalin A (ConA), Ricinus communis I agglutinin (RCA I), and Helix pomatia lectin (HPA) were studied in the pancreas acinar cells of rat embryos in the course of cell differentiation from day 13 through day 20 of gestation. The binding reactions were localized by means of pre-embedment incubation of 10-microns-thick cryosections of pancreas tissue, prefixed in a mixture of 4% formaldehyde/0.5% glutaraldehyde, using horseradish peroxidase for electron microscope visualization. ConA, which preferentially binds to alpha-D-mannosyl residues, consistently stained the cisternae of the cis Golgi side. The majority of the stacks also showed ConA staining of medial cisternae. The reaction of the trans side was variable; in each stage of development, the cisternae of the trans Golgi side either were devoid of labeling or appeared intensely stained. The reactions obtained with RCA I, which recognizes terminal beta-D-galactosyl residues, changed in the course of cell differentiation; in the protodifferentiated and early differentiated states, the system of "rigid lamellae," located at the trans side of the Golgi stacks, was intensely labeled, but became unreactive after production of secretion granules had started, the reaction then being restricted to the stacked saccules. In regard to the Golgi stacks in each of the developmental stages, RCA I binding sites either were confined to the trans cisternae, or, in addition, were found distributed across elements of the medial and cis compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of lectin-binding sites on the surface of Trypanosoma cruzi grown in chemically defined conditions

 The transformation of Trypanosoma cruzi epimastigotes to mammal-infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions (TAU 3AAG medium). During this process, changes in the nature of cell surface sugar composition and sugar distribution was evaluated using FITC and gold-labeled lectins and observed by flow cytometry and transmission electron microscopy. The pattern of labeling with the lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Limax flavus (LFA), Canavalia ensiformis (Con-A), and Ricinus communis (RCA-I) significantly changed during the metacyclogenic process. The results obtained are discussed in relation to the role played by T. cruzi cell surface carbohydrate residues on the process of parasite–host cell interaction. Accepted: 26 May 1998     

Trypsin-induced cell surface changes in ascidian embryonic cells: regulation of differentiation of a tissue-specific protein.

Developing animal quartets removed surgically from 8-cell stage of Ascidia malaca and Phallusia mamillata have been treated for a short time with a low concentration of trypsin. The result is a differentiation of some neural structures, of pigment spots and of a tissue-specific enzyme, brain pigment cell tyrosinase. Tyrosinase activity, as detected histochemically, appeared in the pigment cells some hours before the normal time independently of any inductive interactions with related embryonic tissues. A study with the electron microscope has given evidence of the presence of presumed nervous cells and melanin granules related to them. An autoradiographic study using [³H]uridine has demonstrated presumed RNA synthesis which suggests gene activation. The results are discussed in relation to the possible role of the plasma membrane during embryonic development.     

Role of cell interactions in ascidian muscle and pigment cell specification

Summary Muscle and brain pigment cell specification was studied by disrupting cell adhesion, cell dissociation, and reaggregation in embryos of the ascidianStyela clava. Treatment of embryos with Ca²⁺-free sea water between the 2-cell and gastrula stages disrupted blastomere adhesion but did not prevent acetylcholinesterase or muscle actin expression in presumptive muscle cells. Similar treatments initiated between the 2- and 32-cell stages caused more ectoderm cells to express tyrosinase and develop pigment granules than expected from the cell lineage. Whereas 2 pigment cells become the otolith and ocellus sensory organs in normal embryos, up to 33 pigment cells could differentiate in embryos after disruption of cell adhesion. Replacement of Ca²⁺-free sea water with normal sea water restored cell adhesion and usually resulted in development of embryos containing the conventional number of pigment cells. Dissociation of embryos into single cells between the 2- and 64-cell stages and culture of these cells beyond the fate restricted stage had no effect on the accumulation of muscle actin mRNA and muscle actin synthesis, but blocked pigment cell differentiation. Reaggregation of the dissociated cells did not enhance the number of cells that developed muscle features, but rescued pigment cell development. The results indicate that ascidian muscle cell specification occurs by an autonomous mechanism, whereas pigment cell specification occurs by a conditional mechanism involving cell interactions. In addition, the results suggest that negative cell interactions may restrict the potential for pigment cell development in the ectoderm of cleaving ascidian embryos.     

Proteomic profiles of embryonic development in the ascidian Ciona intestinalis

We report here proteomics-based protein profiles of three embryonic stages of the ascidian Ciona intestinalis. Two-dimensional gel electrophoresis revealed 416, 539, and 695 protein spots in the unfertilized eggs, 16 cell-stage embryos, and tadpole larvae, respectively. Comparative and quantitative analyses of the spot patterns identified proteins showing an increase or decrease in amount during embryonic development. Protein identification by MALDI-TOF/MS indicated not only the abundance and importance of metabolic enzymes and translation elongation factors but also the functional importance of actin-binding proteins and molecular chaperones during ascidian development. Global changes in spots for vitellogenin-like protein suggested post-translational modification or proteolytic digestion of this protein during embryogenesis. Comparison between mRNA and protein levels among unfertilized eggs, 16 cell-stage embryos and tadpole larvae indicated nonparallel expression patterns of genes and proteins. Ascidians provide an excellent system for studying gene expression and cell differentiation during development, and the present study should shed light on the associated molecular mechanism at the protein level.     

Requirement of cell division for muscle actin expression in the primary muscle cell lineage of ascidian embryos

The role of cell division in the expression of muscle actin and its relationship to acetylcholinesterase (AChE) development was examined in cleavage-arrested embryos of the ascidian Styela. Muscle actin expression was detected by two-dimensional gel electrophoresis of radioactively labelled proteins and by in situ hybridization with a cDNA probe, whereas AChE activity was assayed by enzyme histochemistry. In the majority of cases, muscle actin expression was first detected in embryos arrested after the 16-cell stage. Some embryos showed muscle actin expression after arrest at the 8-cell stage, however, muscle actin mRNA did not accumulate in embryos arrested at earlier cleavages. The cells that expressed muscle actin in 8- to 64-cell cleavage-arrested embryos belonged to the primary muscle lineage; secondary muscle cell precursors did not express muscle actin. Zygotic muscle actin mRNA appeared to accumulate with myoplasmic pigment granules in the perinuclear region of cleavage-arrested embryos, suggesting that the myoplasm may have a role in the organization of muscle cells. In contrast to muscle actin, AChE was detected in a small proportion of embryos treated with cytochalasin as early as the 1- or 2-cell stage, and most embryos treated with cytochalasin at later cleavages expressed this enzyme in some of their cells. Most primary muscle lineage cells expressed both muscle actin mRNA and AChE, however, some cells expressed only muscle actin mRNA or AChE. The results suggest that at least three cleavages are required for muscle actin expression and that muscle actin and AChE expression can be uncoupled in cleavage-arrested embryos.     

Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.     

Search for the presence of lectin-binding sites on Toxoplasma gondii

Evidence for the presence of carbohydrate on the surface membrane of Toxoplasma gondii trophozoites and on the cell wall of toxoplasma brain cysts was sought by fluorescent lectin staining. Using FITC-conjugated preparations of Concanavalin A (Con A), wheat germ agglutinin (WGA), or soy bean agglutinin (SBA), we have failed to obtain evidence for the binding of these lectins on the surface of T. gondii trophozoites. In contrast, the three test lectins bound effectively and specifically to the wall of toxoplasma brain cysts. Prefixation of cysts with glutaraldehyde or brief trypsinization of cysts did not affect the intensity of cyst wall fluorescence when stained with FITC-conjugated Con A, SBA, or WGA. The results are interpreted to indicate that whereas exposed Con A, SBA, and WGA binding sites are associated with the wall of toxoplasma brain cysts, such lectin-binding saccharide residues are not present on the surface of trophozoites in exposed or reactive form.     

On the developmental regulation of acetylcholine receptor mobility in the Xenopus embryonic muscle membrane

We have investigated the possible mechanisms underlying a developmental decrease in acetylcholine (ACh) receptor mobility in the membrane of cultured, spherical, mononucleate Xenopus embryonic muscle cells (myoballs) utilizing the method of in situ electrophoresis. We observed that between 1 and 4 days in culture, a substantial redistribution of ACh receptors can be induced by the externally applied electric field which resulted in highly asymmetrical ACh sensitivities at the cathode- and anode-facing poles of the cell. Between 5 and 8 days in culture, the extent of ACh receptor redistribution induced by the field declined to a lower level. Pretreatment with cytoskeletal disrupting agents or with a disulfide bond reducing agent before in situ electrophoresis had no effect on 2-day-old cultures but enhanced receptor mobility in 6-day-old cultures. Pretreatment with Ca²⁺-Mg²⁺-free saline (CMF), which releases cell coat material in other systems, substantially increased receptor mobility when tested on days 2, 6, and 8. On day 6, pretreatment with CMF containing cytochalasin B (CB) and colchicine produced an even greater increase in receptor mobility as compared to treatment with CB and colchicine alone. Our findings suggest that the developmental decrease in ACh receptor mobility is accounted for by at least two different mechanisms: (1) An early-developing, CMF-sensitive restriction possibly mediated by the cell coat; (2) a later-developing restriction possibly dependent on cytoskeletal elements and disulfide linkages. The recovery of high ACh receptor mobility in the older cultures following some of the pretreatments indicates that factors determining ACh receptor mobility can arise from molecular interactions external to the lipid bilayer.     

Induction of anterior neural fates in the ascidian Ciona intestinalis

The sensory vesicle of ascidians is thought to be homologous to the vertebrate forebrain and midbrain (Development 125 (1998) 1113). Here we report the isolation of two sensory vesicle markers in the ascidian Ciona intestinalis, which are homologs of vertebrate otx and gsx homeobox genes. By using these markers to analyze the induction of anterior neural tissue in Ciona, we find that the restriction of anterior neural fate to the progeny of the anterior animal blastomeres is due to a combination of two factors. The vegetal blastomeres show a differential inducing activity along the anterior-posterior axis, while the competence to respond to this inducing signal is markedly higher in the anterior animal blastomeres than in the posterior animal blastomeres. This differential competence to respond is also observed in response to bFGF, a candidate neural inducer in ascidians (J. Physiol. 511.2 (1998) 347) and can be detected by the gastrula stage. Our results, however, indicate that bFGF can only induce a subset of the responses of the endogenous inducer, suggesting that additional signals in the embryo are necessary to induce a fully patterned nervous system.     

Isolation of developmental mutants of the ascidian Ciona savignyi

Ascidians have been used extensively as model animals for experimental embryology. We report here the results of a pilot study with the aim of developing genetic methods for the ascidian Ciona savignyi. The chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to induce point mutations. F1 animals, produced by using sperm from ENU-treated animals to fertilize untreated eggs, were grown to reproductive age. Sperm and eggs collected from the hermaphrodite F1 adults were used to generate self-fertilized F2 broods, which were then screened for recessive, zygotically acting mutations. Animals carrying potential mutations were outcrossed to wild type to test for the heritability of the phenotypes. We report on a number of mutants isolated using this method, including several with abnormalities in tail and notochord development. Received: 15 March 1999 / Accepted: 6 May 1999     

Population genetic structure of the ascidian Styela rustica at Kongsfjorden, Svalbard, Arctic

The actual Arctic biota shows a strong affinity with that of the Boreal Atlantic and Pacific ones, as a result of an active recolonization process after the Quaternary glaciations. The geographic distribution of sessile species is usually linked to larvae dispersive capabilities which can be directly related with time spent in the plankton. Ascidians larvae are lecitothorphic and short-lived, which suggest that ascidians could be not efficient dispersers. However, the solitary ascidian Styela rustica (Linnaeus, 1767) (Tunicata, Ascidiacea) shows a wide distribution pattern from the North Atlantic to the Arctic that, together with the relatively recent colonization of the Arctic system could indicate that this species efficiently disperses and colonizes new habitats. In this study we used ISSR-PCR markers to study the genetic structure of five populations of the ascidian Styela rustica at Kongsfjorden, west Spitsbergen (Svalbard archipelago). We analyzed whether this species presents a low genetic structure, as can be expected due to the historical process of recent post glaciations colonization, or if there is genetic differentiation at a local scale, caused by short-lived larvae and limited dispersal potential. The genetic diversity in each population assessed using the marker diversity index (M) ranged from 0.288 to 0.324. Population HN, situated close to a fast retreating glacier, showed the lowest diversity. Processes associated with deglatiation (icebergs calving from the glacier that scour the benthos and the increment of inorganic particulate matter on the water column) would drive to reduced population sizes and explain the reduced genetic variability observed in the HN population with respect to the others in the fjord. This suggests a possible linkage with the global warming process. Although the weak genetic structure found among the studied populations could indicate a founder effect, the genetic landscape shape analysis together with a positive relationship between genetic and geographic distances also suggest possible current gene flow among populations in the fjord.     

Changes in surface charge density on liposomes induced by Escherichia coli endotoxin

Changes in surface charge density of liposomes induced by E. coli endotoxin were studied by microelectrophoresis. Endotoxin altered the surface charge of phosphatidylcholine liposomes from neutral to negative. The negative charge of the endotoxin-phosphatidylcholine complex was neutralized electrostatically by binding with Ca²⁺ (2 mM). Phosphatidylcholine liposomes were made positive by addition of the positively charged detergent, hexadecyltrimethylammonium chloride. Endotoxin made the positively charged liposomes less charged. On the other hand, phosphatidylserine liposomes which were negatively charged became less charged in the presence of high concentration of endotoxin (8 mg/ml). The endotoxin effect on phosphatidylserine liposomes was abolished by EDTA (1 mM) but potentiated by CaCl₂ (0.1–2 mM). These results indicate that endotoxin interacts with liposomes both hydrophobically and electrostatically.     

Autonomous muscle cell differentiation in partial ascidian embryos according to the newly verified cell lineages

Recent analysis of cell lineages in ascidian embryos by the intracellular injection of a tracer enzyme has clearly demonstrated that muscle cells are derived not only from the B4.1-cell pair of the eight-cell stage embryo, as has hitherto been believed, but also from both the b4.2- and A4.1-cell pairs (H. Nishida and N. Satoh, 1983, Dev. Biol.99, 382–394). In order to reexamine the developmental autonomy in muscle lineage cells, the B4.1 pair was isolated from the eight-cell stage embryo. The progeny cells of the B4.1 pair, as well as those of the six other blastomeres, were then allowed to develop in isolation into partial embryos. Autonomous muscle cell differentiation not only in partial embryos originating from the B4.1 cells but also in those from the six other blastomeres was substantiated by (a) occurrence of localized histospecific muscle acetylcholinesterase and (b) development of myofibrils. These results support the validity of the recent cell lineage study and confirmed the self-differentiation potency of muscle lineage cells in ascidian embryos according to the newly verified cell lineages.     

Onset of heartbeat revealed by successive structural changes in the developing single embryonic heart of the waterstrider (Gerris paludum insularis)

Videofilm images of the heartbeat in the living embryos of the waterstrider, Gerris paludum insularis, were analyzed to demonstrate successive changes in the width of the contractile heart at the different developmental stages. This information is graphically represented and termed structural cardiogram. Onset of the embryonic heartbeat characterized by anteriorly spreading peristaltic movement of the heart wall occurs at about 55% HL (percent heart length) as early as at about 81 h after katatrepsis (K+81 h embryo). This peristaltic wave occurs almost always following swinging movement of abdominal tip observed exclusively at this stage. Similar peristaltic wave of the heart wall may also be observed at later stages, exclusively in the anterior two-fifth of the heart. Conduction velocity of the peristaltic wave estimated from structural cardiogram of K+81 h embryo was approximately 0.57 mm/s; it was approximately 1.33 mm/s in the K+102 h embryos. In the posterior three-fifth of the heart, however, rhythmic movement was not peristaltic. Development of heartbeat generator in the specific region of the heart was discussed in relation to the onset of embryonic heartbeat.