Differentiation of tropomyosin-containing myofibrils in cleavage-arrested ascidian zygotes expressing acetylcholinesterase

Two muscle differentiation programs, acetylcholinesterase and tropomyosin-containing filaments and fibrils, occur together in the same cleavage-arrested zygotes (1-celled) of the ascidian Ciona intestinalis. Coexpression in such undivided but developing 'embryos' is consistent with the idea that separate elements of muscle differentiation are related at some regulatory level, perhaps through a single multi-gene regulatory factor. Fertilized Ciona eggs were exposed to cytochalasin B for 20 h and then briefly reacted histochemically for acetylcholinesterase activity. Strongly reacting specimens were selected and processed for transmission electron microscopy to reveal regions of muscle ultrastructure. Every acetylcholinesterase-reactive zygote tested contained muscle contractile elements; no example lacking acetylcholinesterase was found with myofilaments and myofibrils. As demonstrated by immunogold labelling, a polyclonal antibody to tropomyosin from Ciona adult body wall reacted differentially with the presumed ultrastructural muscle elements in cleavage-arrested zygotes. Site-specific reactions were also observed in larval tail muscle and the siphon muscles of postmetamorphic zooids.     

Differentiation without cleavage: multiple cytospecific ultrastructural expressions in individual one-celled ascidian embryos

Multiple states of differentiation developed within the same undivided egg cytoplasm of ascidian zygotes cleavage-arrested with cytochalasin B. Complex ultrastructural traits of up to four quite diverse cell lineage components were observed in regions of the common cytoplasm in such multinucleate homokaryons of Ciona intestinalis: epidermal, muscle, notochordal, and neural. Almost all specimens among those selected as showing differentiation contained two such features, half of them had at least three, and a few expressed all four. The histospecific morphological characteristics noted were the extracellular test material of epidermal cell origin, muscle myofilaments and myofibrils, sheath components (leaflets and filaments) associated with notochordal cells, and the particular localized combinations of microtubules, filamentous structures, and cilia indicative of neural tissues. Cleavage-arrested one-celled embryos of Ascidia ceratodes served to demonstrate that those which were found cytochemically to contain muscle acetylcholinesterase always had myofibrils and myofilaments. Other arrested zygotes of Ascidia (unstained specimens) also had quite fully formed test material as well as myofilaments and myofibrils. The occurrence within the same cell of so many specific markers of diverse pathways of development is consistent with a theory about a primary level of regulation based on autonomous gene activation factors already present in the fertilized egg. If further investigation substantiates a real cytoplasmic continuity within these cleavage-arrested embryos, other theories that invoke cell interactions, temporal sequences of metabolically distinct microenvironments, and gradients of substances as causes of determinative change seem inadequate to account for the coexisting expressions of differentiation described here.     

Effects of alpha-latrotoxin on the early developmental events of the sea urchin Paracentrotus lividus: a histochemical study

The effects of the spider toxin alpha-latrotoxin (alpha-LTX) on gametes, zygotes, and early embryos of the sea urchin Paracentrotus lividus have been investigated by in vivo experiments and by histochemical studies of acetylcholinesterase (AChE) activity. Treatment of unfertilized eggs with nanomolar amounts (1 to 0.3 nmol/l) of alpha-LTX neither triggered cortical granule exocytosis, nor prevented the elevation of the fertilization layer by sperms. Instead, fertilized eggs exposed to alpha-LTX showed noticeable alterations in cell surface topography, including the appearance of prominent membrane-limited blebs. Moreover, the zygotes treated with 1 nmol/l alpha-LTX failed to cleave. The histochemical staining of treated zygotes revealed a very strong AChE activity in the cortical region, including blebs. An enzyme reaction was also found in the perivitellin space. Our results suggest the hypothesis that some alpha-LTX receptors may appear after fertilization, supporting the awareness that fertilized eggs display excitable cell features.     

Effects of egg size reduction and larval feeding on juvenile quality for a species with facultative-feeding development

In free-spawning marine invertebrates, larval development typically proceeds by one of two modes: planktotrophy (obligate larval feeding) from small eggs or lecithotrophy (obligate non-feeding) from relatively large eggs. In a rare third developmental mode, facultative planktotrophy, larvae can feed, but do not require particulate food to complete metamorphosis. Facultative planktotrophy is thought to be an intermediate condition that results from an evolutionary increase in energy content in the small eggs of a planktotrophic ancestor. We tested whether an experimental reduction in egg size is sufficient to restore obligate planktotrophy from facultative planktotrophy and whether the two sources of larval nutrition (feeding and energy in the egg) differentially influence larval survival and juvenile quality. We predicted, based on its large egg size, that a reduction in egg size in the echinoid echinoderm Clypeaster rosaceus would affect juvenile size but not time to metamorphosis. We reduced the effective size of whole (W) zygotes by separating blastomeres at the two- or four-cell stages to create half- (H) or quarter-size (Q) “zygotes” and reared larvae to metamorphosis, both with and without particulate food. Larvae metamorphosed at approximately the same time regardless of food or egg size treatment. In contrast, juveniles that developed from W zygotes were significantly larger, had higher organic content and had longer and more numerous spines than juveniles from H or Q zygotes. Larvae from W, H and Q zygotes were able to reach metamorphosis without feeding, suggesting that the evolution of facultative planktotrophy in C. rosaceus was accompanied by more than a simple increase in egg size. In addition, our results suggest that resources lost by halving egg size have a larger effect on larval survival and juvenile quality than those lost by withholding particulate food.     

Segregation during cleavage of a factor determining endodermal alkaline phosphatase development in ascidian embryos.

Localized alkaline phosphatase activity (EC 3.1.3.1) develops progressively in endodermal tissues of the presumptive digestive system in Ciona intestinalis embryos. It was first detected histochemically at late gastrulation, and a puromycin sensitivity period coincident with this time suggests that new alkaline phosphatase is synthesized. Embryos in which cell division was blocked with cytochalasin B at early cleavage stages up to the 64-cell stage, eventually differentiated strong alkaline phosphatase activity in certain cells at each cleavage-arrested stage. The maximum cell numbers and their positions were identical to those of the previously known endodermal cell lineage. Actinomycin D did not prevent development of endodermal alkaline phosphatase when administered from fertilization onwards, nor did other inhibitors of RNA synthesis (chromomycin A3, cordycepin, and daunomycin). There is probably a preformed maternal mRNA for endodermal alkaline phosphatase present in the unfertilizec Ciona egg. Either this RNA itself, or some related translation factor, is localized in the egg cytoplasm and segregated during early cleavages into the endodermal cell lineage of the embryo.     

Development of myoplasm-enriched ascidian embryos

The fertilized ascidian egg is thought to be comprised of distinct regions of tissue-specific cytoplasmic determinants. This idea was tested by bisecting fertilized eggs into egg fragments and culturing them until the unoperated controls developed into larvae. Fertilized eggs were bisected using a microsurgical method in which part of the uncleaved zygote was extruded through a hole made in the follicular envelope and the cytoplasmic bridge between the two egg regions was severed. One egg fragment contained all of the egg myoplasm (termed myoplasm-enriched or ME fragment), while the other fragment lacked myoplasm. ME fragments consisting of 40-50% of the total egg volume in many cases cleaved normally and developed into larvae. In a few cases, ME larvae initiated metamorphosis and developed into normal juveniles. Triton-extraction of ME embryos and larvae showed that the myoplasm was redistributed into nonmuscle lineage cells at each stage of development. Despite the redistribution of myoplasm into many of the endoderm cells situated in the head region of ME larvae, the expression of the muscle-specific enzyme acetylcholinesterase (AchE) and a muscle-specific antigen (Mu-2) was restricted to the tail muscle cells. The endoderm cells situated in the head region of ME larvae expressed an endoderm-specific enzyme alkaline phosphatase (AP) as in the controls. Furthermore, cleavage-arrested four- and eight-cell ME embryos expressed AchE activity in the expected number of blastomeres. When a greater quantity of myoplasm was redistributed into cells that normally do not express AchE activity by producing 10-30% ME embryos, in a few cases more than the expected number of blastomeres expressed AchE activity. In conclusion, the main finding of the present investigation, based on the development of ME fragments comprising 40-50% of the total egg volume, is that ascidian embryos are capable of regulative development.     

Effects of NeemAzal on marker enzymes and hemocyte phagocytic activity of larvae and pupae of the vector mosquito Aedes aegypti

Many of the neem based botanical biocides are currently studied to a greater extent because of the possibility of their use in eco-friendly control of pests and vectors. However, no report was available to assess the impact of neem based formulation, NeemAzal on marker enzymes and hemocyte mediated cellular immune responses of important vector mosquito A. aegypti. The NeemAzal found to exert larvicidal and pupicidal activities against A. aegypti developmental stages. The pupae appear to be more susceptible to the treatment. Further, a significant increase in the level of total protein (31%), α-carboxylesterase (121%), β-carboxylesterase (46%), acid phosphatase (62%) and alkaline phosphatase (37%) was observed in larvae upon exposure to NeemAzal. Moreover, treated pupae showed increased level of acetylcholinesterase (116%) and acid phosphatase (43%) while α-carboxylesterase (34%), β-carboxylesterase (12%) levels were simultaneously decreased, and no significant changes in alkaline phosphatase were noticed. Qualitative analysis also revealed that the exposure considerably modulated the larval β-carboxylesterase isoenzyme profile whereas little changes were noticed on phosphatases. On the other hand hemocyte viability of larvae (18%) and pupae (16%) as well as phagocytic ability of larval (48%) and pupal hemocytes (44%) against yeast target was significantly reduced upon NeemAzal exposure. We demonstrated for the first time that the NeemAzal differentially affected the marker enzymes and created immuno-suppressive state by reducing the phagocytic ability of hemocytes of larvae and pupae of A. aegypti.     

Improved localization of intracellular sites of phosphatases using cerium and cell permeabilization

A simple permeabilization method has been developed that allows for intracellular localization of acid phosphatase in neutrophils and several types of tissue culture cells with cerium. This permeabilization procedure also facilitates intracellular alkaline phosphatase localization in neutrophils without the loss of cell surface reaction in this cell type. Only the cell surface reaction was detected in the absence of permeabilization. Glutaraldehyde-fixed cells were permeabilized with detergent during the cytochemical reaction. Triton X-100 at 0.0001-0.0002% gave the best results for the enzymes and cell types tested.     

Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation

The exposure of mouse zygotes pre-stained with Hoechst 33342 to u.v. irradiation for 20-30 sec significantly or completely inhibited development to blastocysts in vitro. However, development to the blastocyst stage of enucleated eggs receiving pronuclei from untreated eggs was as good as that of control reconstituted eggs when the cytoplasm originated from eggs exposed to u.v. irradiation for 20-30 sec, but was significantly lower when the cytoplasm was from eggs exposed for 40 sec. The chromosomes at the second metaphase stage could be removed with 15 sec of exposure to u.v. irradiation under a fluorescence microscope. Most eggs enucleated at the second metaphase that received a single inner cell mass nucleus (75%) showed pronuclear formation 6 h after activation; 23% of them developed to morphologically normal 2-cell eggs and 5% developed to blastocysts. These results demonstrate that the cytoplasm of mouse zygotes is more resistant to u.v. irradiation after Hoechst staining. Eggs at the second metaphase, from which chromosomes have been removed under a fluorescence microscope, can therefore be used as cytoplasm recipients for nuclear transplantation of inner cell mass nuclei.     

Plasma membrane homing of tissue nonspecific alkaline phosphatase under the influence of 3-hydrogenkwadaphnin, an antiproliferative agent from Dendrostellera lessertii

Several mammalian enzymes are anchored to the outer surface of the plasma membrane by a covalently attached glycosylphosphatidylinositol (GPI) structure. These include acetylcholinesterase, alkaline phosphatase (AP) and 5'-nucleotidase among other enzymes. Recently, it has been reported that these membrane enzymes can be released into the serum by the GPI-dependent phospholipase D under various medical disturbances such as cancer and/or by chemical and physical manipulation of the biological systems. Treatment of MCF-7 cells with two consecutive effective concentrations of 3-hydrogenkwadaphnin (3-HK, 3 nM) for 48 h enhanced membrane AP activity by almost 330% along with a 40% reduction in the AP activity of the cell culture medium. In addition, our data indicate that 3-HK is capable of inducing mainly the tissue-nonspecific alkaline phosphatase (TNAP) isoenzyme, along with enhancing its thermostability. These findings, besides establishing a correlation between the antiproliferative activity of 3-HK and the extent of plasma membrane AP activity, might assist in the development of new diagnostic tools for following cancer medical treatments.     

Studies on the alkaline phosphatase and 5'-nucleotidase of Dictyostelium discoideum.

The alkaline phosphatase and 5′-nucleotidase activities of Dictyostelium discoideum are due to two distinct enzymes. Both enzymes are membrane bound, but over 90% of the 5′-nucleotidase activity is solubilized when the crude membrane fraction of the cell is treated with phospholipase C under conditions that release only 10% of the alkaline phosphatase.Part of the alkaline phosphatase activity can be detected in whole cells, suggesting that some of the enzyme molecules are located on the exterior surface of the plasma membrane. In contrast very low 5′-nucleotidase activity can be detected in whole cells. When membrane preparations, isolated from cells that had been surface labeled with ¹²⁵I, were subjected to sedimentation equilibrium on sucrose density gradients, the majority of the ¹²⁵I-radioactivity cosedimented with the alkaline phosphatase and 5′-nucleotidase activites, suggesting that both enzymes are plasma membrane components.The two enzymes have distinctly different pH optima, but otherwise their properties are remarkably similar. Both enzymes are inhibited by cyanide, sulfhydryl inhibitors and sulfhydryl reagents, although in each case the 5′-nucleotidase is slightly more susceptible. Both enzymes are inhibited by the levamisole analogue, R 8231, but the alkaline phosphatase is inhibited to a somewhat greater extent. Both enzymes are activated by incubation at 50 °C but inactivated by higher temperatures.The two enzymes increase in activity at identical times during differentiation, suggesting that they are under coordinate developmental control.     

Studies on the Cytoplasmic Determinant for Muscle Cell Differentiation in Ascidian Embryos: An Attempt at Transplantation of the Myoplasm

The 8-cell stage embryos of the ascidian Halocynthia roretzi which had been prevented from undergoing further divisions by continuous treatment with cytochalasin B could develop histospecific muscle acetylcholinesterase in two blastomeres (B4.1 and B4.1 cells). If the cytoplasm of a B4.1 or B4.1 cell was transplanted by microinjection into either an A4.1 or A4.1 cell of recipient embryos and the transplanted embryos were permanently cleavage-arrested with cytochalasin B, a few eventually developed AChE in three blastomeres instead of in just the two blastomeres found in cleavage-arrested control embryos. Judging from the relative positions of the blastomeres, the third AChE-producing cells appeared to be the A4.1 or A4.1 cells injected with the cytoplasm of B4.1 or B4.1. Although the success rate was considerably low, this result might indicate the presence in the cytoplasm of a determinant for the muscle-specific enzyme development.     

Ultrastructural localization of phosphohydrolases in gametes, zygotes and zoospores of Ulva mutabilis Foyn.

Gametes, zoospores, and zygotes of the multicellular, green alga Ulva mutabilis showed acid phosphatase reaction product in Golgi vesicles and on the membrane lining the vacuole. In addition gametes and zoospores showed enzyme reaction product on the entire surface membrane including the flagellar membrane. The surface membrane enzyme activity disappears from the zygote shortly after copulation and at the same time lysosome-like bodies start to appear in the cytoplasm. No alkaline phosphatase activity could be detected. The distribution of acid phosphatase is discussed in relation to the events taking place during and shortly after fertilization.     

Membrane anchors of alkaline phosphatase and trehalase associated with the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori

The larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and trehalase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.     

Effect of cell contact on regionalization of mouse embryos

Cytochemical demonstrations of 5'-nucleotidase and alkaline phosphatase reveal the activity of these enzymes on regions of cell apposition from the late four-cell stage onward. These enzyme activities also appear on regions of artificial cell contact between aggregated embryos having more than four cells. Cytochemistry of single two-cell embryos does not reveal 5'-nucleotidase nor alkaline phosphatase activity, however, these enzyme activities appear at both the artificial and natural contacts in chimaeras of two two-cell embryos. We interpret these results as meaning: (1) that cell contact causes the regionalization of 5'-nucleotidase and alkaline phosphatase activity on the cell surface, (2) that these enzyme activities can be induced or enhanced by contact between two two-cell embryos, (3) that a signal is transmitted from the artificial to the natural contact.     

Production of fetal-like alkaline phosphatase by HeLa cells

Alkaline phosphatase produced by HeLa cells differs in its chemical and physical properties from the enzyme found in adult organs and tissues (Cox and Griffin, 1967). In the present study HeLa cell alkaline phosphatase was compared to a fetal form of the enzyme found in human placenta. Both enzymes have approximately the same molecular weight as judged by sucrose density gradients, and the chemical and physical properties of these alkaline phosphatases are similar. The electrophoretic pattern of the HeLa cell enzyme resembles the placental alkaline phosphatase of the heterozygous FS phenotype except that it is slower moving. Double immunodiffusion using an antibody against HeLa cell alkaline phosphatase and placental and HeLa cell enzymes as antigens shows a single line of partial identity between the two enzymes, with a small spur suggesting additional antigenic sites on the HeLa cell enzyme. The data suggest that malignant cells in culture, HeLa, are producing a fetal-like alkaline phosphatase probably by derepression of the genome. However, the electrophoretic and immunological characteristics of the enzyme are altered sufficiently so that it can be distinguished from the normally produced fetal enzyme.This work was supported by U.S. Public Health Service Grant GM 15508 and the Health Research Council of the City of New York.Fourth-year student; Honors Program.Career Scientist Health Research Council of the City of New York.     

Removal of covalently bound inositol from Torpedo acetylcholinesterase and mammalian alkaline phosphatases by deamination with nitrous acid. Evidence for a common membrane-anchoring structure.

Our earlier evidence suggested that both acetylcholinesterase and alkaline phosphatase are anchored to the cell surface via covalently-attached phosphatidylinositol [Low, Futerman, Ferguson & Silman (1986) Trends Biochem. Sci. 11, 212-215]. We now present chemical data, based upon a nitrous acid deamination reaction, showing that in both proteins the phosphatidylinositol moiety is attached through a glycosidic linkage to a sugar residue bearing a free amino group.     

Ultraviolet irradiation during ooplasmic segregation prevents gastrulation, sensory cell induction, and axis formation in the ascidian embryo

The effect of ultraviolet (uv) light on embryonic development was examined in the ascidian Styela clava. uv irradiation (3.0 x 10(-3) J mm-2) of the entire surface of fertilized eggs during ooplasmic segregation prevented gastrulation, sensory cell induction, and embryonic axis formation. The uv-irradiated embryos completed ooplasmic segregation and cleaved normally, but vegetal blastomeres did not invaginate at the beginning of gastrulation, sensory cells in the larval brain did not develop tyrosinase or melanin pigment, and the larval tail did not develop. Endoderm, epidermis, and muscle cells differentiated in the uv-irradiated embryos, however, as evidenced by expression of endodermal alkaline phosphatase (AP), an epidermal-specific antigen, and alpha-actin, myosin heavy chain, and acetylcholinesterase (AChE) in muscle cells. Higher doses of uv light (6.0-9.0 x 10(-3) J mm-2) suppressed expression of the epidermal antigen and muscle cell markers, whereas the development of endodermal AP was insensitive. Irradiation at various times between fertilization and the 16-cell stage revealed that gastrulation, sensory cell differentiation, and axis formation are sensitive to uv light only during ooplasmic segregation. Irradiation of restricted regions of the zygote during ooplasmic segregation showed that the uv-sensitive components are localized in the vegetal hemisphere. The absorption characteristics of the uv-sensitive components suggest that they are nucleic acids. The results show that uv-sensitive components that specify gastrulation, sensory cell induction, and embryonic axis formation are localized in the vegetal hemisphere of Styela eggs.     

Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.