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.     

Cytochemical localization of vanadium(III) in blood cells of ascidian Phallusia mammillata Cuvier,and its relevance to hematic cell lineage determination

When the blood cells of ascidians Phallusia mammillata are stained with the ligand 2,2′-bipyridine, those cells which contain vanadium(III), in an easily sequestered form, take up the stain producing in situ, a purple complex. This material extracted displays spectral characteristics consistent with the formation of an oxo-bridge vanadium(III) bipyridine dimer. The staining is localized in the signet ring cell, a bivacuolated cell, a cell type with numerous darkly staining compartments, and also by the vacuolated amoebocyte. The possible ramifications of these observation are discussed in relation to the delineation of the signet ring cell lineage.     

Serotonin localization in Phallusia mammillata larvae and effects of 5-HT antagonists during larval development

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an important role in a wide range of non-neural processes. Using immunofluorescence with an antiserotonin antibody, 5-HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5-HT on development, we treated embryos with two different 5-HT receptor subtype antagonists. Treatment at the gastrula stage with 10 microM ondansetron, an antagonist of the 5-HT(3) receptor, induced anterior truncation and a short tail. At 10 microM, ritanserin, a 5-HT(2B) receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5-HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5-HT is suggested.     

Localization of mitochondria in plant cells by vital staining with rhodamine 123

The positively-charged fluorescent dye rhodamine 123 (r-123) specifically stains mitochondria in living plant protoplasts, suspensionculture cells, and root hairs. This dye functions as a vital stain and permits visualization of the localization, distribution and movement of the mitochondria. Dehydration of root hairs caused mitochondria to aggregate into clumps. Mitochondria were either homogenous or heterogeneous and were frequently seen to accumulate in the perinuclear regions of suspension-culture cells but not in those of protoplasts or root-hair cells. Dinitrophenol and high concentrations of ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid and KCl immediately eliminated fluorescence in r-123-stained mitochondria whereas ionomycin enhanced it. Treatment of seedlings with r-123 resulted in differential brightness of fluorescence in different tissues. Meristematic tissues, such as root and shoot tips, exhibited the brightest fluorescence. The cytotoxicity of r-123 in both germinating seedlings and suspension-culture cells was low. The specificity, sensitivity and low toxicity of r-123 should make it a useful tool in experiments designed to examine agents and conditions which affect the location, the physiological status or the viability of mitochondria.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - DAPI 46-diamidino-2-phenylindole - r-123 rhodamine 123     

Glycoprotein constituents of the vitelline coat of Phallusia mammillata (Ascidiacea) with fertilization inhibiting activity.

Vitelline coats (VCs) of Phallusia mammillata were isolated and purified following homogenization of live eggs in order to investigate the molecular basis of sperm-egg recognition. Clean VCs were partly solubilized by sonication in H2O and the soluble fraction (SFVC), derived from the outer surface of VCs, was used for further characterization. Electrophoretic analyses of radioiodinated VCs revealed that SFVC consists of two major glycoprotein components with apparent average Mr's of 450,000 and 180,000, respectively. The 450,000 Mr component is composed of several charge isomers, whereas the 180,000 Mr component is supposed to consist of two oligomers, both with acidic pI, held together by a disulfide linkage(s). Each of the two components possesses WGA-binding sites as shown by transblotting followed by WGA-peroxidase treatment. The amino acid composition of SFVC was determined after acid hydrolysis and its carbohydrate composition was analyzed and quantified by GLC. GlcNAc and GalNAc were found to predominate with 86% by weight of total sugar content and fucose, mannose, and glucose accounted for the remaining 14%. The susceptibility of SFVC to enzymatic (N-glycosidase F) and chemical (TFMS) deglycosylation as well as to protease (trypsin and chymotrypsin) digestion was investigated. Furthermore, sperm receptor activity of SFVC was tested in a fertilization assay. The fertilization rate decreased in a concentration-dependent manner when sperm were preincubated with SFVC. Additionally, sperm treated with SFVC showed binding for FITC-WGA or WGA-gold at the apical portion of the sperm head. Therefore, we strongly assume that one or both of the identified glycoprotein macromolecules of SFVC are involved in sperm-egg recognition.     

Dopamine and serotonin modulate the onset of metamorphosis in the ascidian Phallusia mammillata

Neurotransmitters play an important role in larval metamorphosis in different groups of marine invertebrates. In this work, the role of dopamine and serotonin during metamorphosis of the ascidian Phallusia mammillata larvae was examined. By immunofluorescence experiments, dopamine was localized in some neurons of the central nervous system and in the adhesive papillae of the larvae. Dopamine and serotonin signaling was inhibited by means of antagonists of these neurotransmitters receptors (R(+)-SCH-23390, a D(1) antagonist; clozapine, a D(4) antagonist; WAY-100635, a 5-HT(1A) antagonist) and by sequestering the neurotransmitters with specific antibodies. Moreover, dopamine synthesis was inhibited by exposing 2-cell embryos to alpha-methyl-l-tyrosine. Dopamine depletion, obtained by these different approaches, caused early metamorphosis, while serotonin depletion delayed the onset of metamorphosis. The opposite effects were obtained using agonists of the neurotransmitters: lisuride, a D(2) agonist, inhibited metamorphosis, while DOI hydrochloride and 8-OH-DPAT HBr, two serotonin agonists, promoted it. So, it is possible to suppose that dopamine signaling delayed metamorphosis while serotonin signaling triggers it. We propose a mechanism by which these neurotransmitters may modulate the timing of metamorphosis in larvae.     

Protease activity in fractionated blood cells of the vanadium accumulating ascidian Phallusia mammillata

Proteolytic activity was studied in the fractionated blood cells of the vanadium accumulating ascidian P. mammillata by separating the cells before measuring their activity. Cells were separated to avoid vanadocyte breakdown and subsequent vanadium diffusion into the assay medium. Our study revealed activity in the morula cell extract that was obtained by sonication and Centricon concentration. After removing part of the extract for enzyme activity assay the remainder was kept at 0 degrees C; it was later found that much of the protein in this latter fraction formed a sediment whereas the protease remained in solution. The serine-protease substrate specificity of the enzyme was measured and the results are discussed in relation to serine protease involvement in immune defense.     

Tracing the lineage of tracing cell lineages

The study of cell lineages has been, and remains, of crucial importance in developmental biology. It requires the identification of a cell or group of cells and of all of their descendants during embryonic development. Here, we provide a brief survey of how different techniques for achieving this have evolved over the last 100 years.     

The roles of ERAS during cell lineage specification of mouse early embryonic development

Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with its expression pattern, the increased expression of Eras promotes cell proliferation through controlling AKT activation and the commitment from ground to primed state through ERK activation in mouse embryonic stem cells; and the reduced expression of Eras facilitates primitive streak and mesoderm formation through AKT inhibition during gastrulation. The expression of Eras is finely regulated to match its roles in mouse early embryonic development during which Eras expression is negatively regulated by the β-catenin pathway. Thus, beyond its well-known role on cell proliferation, ERAS may also play important roles in cell lineage specification during mouse early embryonic development.     

Specific inhibition of sperm β-N-acetylglucosaminidase by the synthetic inhibitor N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime inhibits fertilization in the ascidian, Phallusia mammillata

Increasing evidence has evolved from studies in ascidians and mammals that sperm β- N -acetylglucosaminidase (GlcNAc'ase) plays a crucial role in fertilization. In the ascidian Phallusia mammillata , GlcNAc'ase is the predominant sperm-bound glycosidase and N-acetylglucosamine (GlcNAc) is the prevailing glycoside residue on the vitelline coat. We report here that the GlcNAc'ase inhibitor O -(2-acetamido-2-deoxy-D-glucopyrano-sylidene)-amino- N -phenylcarbamate (PUGNAC) is a potent competitive inhibitor of sperm-bound GlcNAc'ase in P. mammillata . The inhibitor constant K_i for the isolated enzyme is 47 nmol/L. Fertilization of eggs is inhibited by PUGNAC in a dose dependent competitive manner with 50% inhibition at an inhibitor concentration of 85 μmol/L. Further experiments, in which intact eggs possessing an egg coat were mixed with eggs from which the coat had been removed, showed that only fertilization of intact eggs was inhibited by PUGNAC. This finding suggests that PUGNAC prevents the binding of the sperm-associated GlcNAc'ase to terminal GlcNAc residues on the vitelline coat, thus inhibiting sperm binding and subsequently fertilization. Furthermore and most importantly, it shows that treatment with PUGNAC does not affect the viability of sperm and that the process of sperm-egg fusion is not affected.     

Flow-cytometric determination of glutathione alterations in vital cells by o-phthaldialdehyde (OPT) staining

A flow-cytometric method for the detection of changes of cellular glutathione content in vital cells is described. The reaction is based on formation of a fluorescent product between o-phthaldialdehyde (OPT) and reduced glutathione (GSH). OPT is a more GSH-specific dye than other thiol-specific dyes (e.g., bromobimanes), because it forms a cyclic compound with GSH. Changes of GSH induced by oxidation or thiol-blocking agents are visualized in vital cells after a 5-min staining at room temperature.     

Melanoma Cell Adhesion Molecule (MCAM) expression in the myogenic lineage during early chick embryonic development

We describe the expression pattern of cMCAM, a cell adhesion molecule of the immunoglobulin superfamily, in early chick embryonic development by in situ hybridisation. An initial ectodermal domain of expression is subsequently expanded, and cMCAM is expressed in the neural crest cells, otic vesicle, heart primordium, notochord and endoderm. In addition, cMCAM expression localises in the myotome once the somite cells have been specified. An in vitro murine cellular system allowed us to confirm that MCAM expression coincides with the onset of myogenic cell determination.     

Isolation, characterization, and localization of a sperm-bound N-acetylglucosaminidase that is indispensable for fertilization in the ascidian, Phallusia mammillata

N-Acetylglucosaminidase (GlcNAc'ase), which possesses by far the highest activity of all Phallusia mammillata sperm glycosidases, was isolated and purified using DEAE-cellulose, phenyl-Sepharose, and concanavalin A affinity chromatography. The molecular size of the native enzyme estimated by G-200 gel permeation was 158 kDa. On SDS-PAGE, the denatured enzyme migrated as a single band with a Mr of 78 kDa. This indicates that under nondenaturing conditions the GlcNAc'ase prevails as a dimer. The molecular activity of the enzyme was determined to be 3.7 x 10(5) U/mumole, the Km for p-NP-GlcNAc was 0.65 mM, and the Ki for GlcNAc was 5.5 mM. It has been suggested that gamete binding in ascidians might be mediated by an enzyme-substrate complex established between a sperm glycosidase and corresponding glycosides on the vitelline coat. Thus, the GlcNAc'ase should be present as an exoenzyme at the proper place on the sperm surface membrane, i.e., on the sperm tip and possibly over the mitochondrial region. We localized the enzyme with fluorescence and electron microscopy using the neoglycoprotein BSA-p-aminophenyl-N-acetyl-beta-D-glucosaminide (BSA-GlcNAc) or concanavalin A coupled either to fluorochromes or gold particles. Labeling of unreacted and activated sperm revealed three distinct binding sites, namely at the sperm tip, over the mitochondrion, and at the head-tail junction. In reacted sperm strong labeling was observed over the translocated mitochondrion as well as at the sperm tip. An intensive binding was observed along the rim which borders the cap-like structure at the sperm tip. The distribution of the enzyme reflected by these binding patterns accounts well for the suggested function. Using N-acetylglucosaminono-1,5-lactone oxime, a novel, highly specific inhibitor of GlcNAc'ase, we were able to show that this enzyme is indispensable for fertilization of intact eggs, but not of eggs deprived of their vitelline coat. These observations are discussed in terms of functional relationships which may exist between this enzyme, sperm binding, gamete recognition, and penetration of the vitelline coat.     

The embryonic cell lineage of the nematode Caenorhabditis elegans

The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.