The site of action of lithium ions in morphogenesis of Lymnaea stagnalis analyzed by secondary ion mass spectroscopy

Abstract. Exogastrulation as a disturbance of development in eggs of Lymnaea stagnalis is caused by the action of LiCl at the second cleavage stage and not at the first or third. The percentage of exogastrulae formed is strongly concentration dependent. To determine the site of action of lithium ions, the cellular contents of Li, C, Na, Mg, P, K, and Ca were analyzed by secondary ion mass spectroscopy (SIMS). The mean elemental concentrations of Na, Mg, K, and Ca are close to those found earlier by electron probe microanalysis and atomic absorption spectroscopy. Lymnaea eggs at the first, second, and third cleavage stage were treated with LiCl in a series of concentrations ranging from 50 to 0.1 mM. In all cases the cells contained a few mM lithium after treatment. After treatment at the insensitive first cleavage stage the lithium content is carried over by the cells through the sensitive second cleavage to the insensitive third cleavage stage. These data allow the conclusion that it is the external lithium concentration which is responsible for the specific effect. This presents direct analytical evidence that the primary action of lithium ions is located at the cell membrane.     

Cytological and morphological studies of the action of lithium on the development of the sea urchin embryo

Summary The effect of lithium ions on cleavage and development of sea urchin larvae was investigated. Lithium was found to interfere with the movements of the chromosomes at mitosis, which is also very delayed in the presence of lithium. The disturbances inflicted by lithium were observed during the course of development up to the pluteus stage.The lithium sensitive period coincides with the period in which the mitotic activity reaches its maximum.The rate of cleavage is reduced by lithium. In the normal untreated larva there is during the early blastula stage an animal trend in which the formation of cells of prospective ectodermal significance preponderates. The mesomeres were found to be relatively more affected by lithium than were the macromeres. This shift in the cleavage pattern influences the numbers of cells of ectodermal and endo-mesodermal significance.Studies were also made of morphological changes following treatment with lithium.     

Injection of myo-inositol reverses the effects of lithium on sea urchin blastomeres

Lithium is known to cause sea urchin blastomeres destined to give rise to epithelium rather than to differentiate into gut or skeleton. While it has been proposed that lithium alters development by interfering with the inositol-tris phosphate-protein kinase C (IP₃-PKC) signaling pathway, the mechanism of action of lithium in sea urchins has remained elusive. Here we describe experiments that examine the hypothesis that lithium exerts its effect on sea urchin embryos via the IP₃-PKC pathway. We make use of methods developed to isolate epithelial precursor cells from the animal hemisphere of cleavage 16-cell stage embryos. Pairs of cells were isolated and one of each pair was injected with either myo-inositol or its inactive isomer, epi-inositol. Rhodamine dextran was co-injected as a lineage tracer to follow the fate of injected cells. We demonstrate that injected myo-inositol, but not epi-inositol, can reverse the effects of lithium on sea urchin blastomeres. This is direct evidence that lithium affects the IP₃-PKC pathway in sea urchins, and that this pathway plays an important role in cell fate determination.     

Polar effects of lithium in the heart of the zebrafish Danio rerio

 The molecular signalling mechanisms that are believed to govern the patterning of the heart early in embryonic development are not well understood. We have investigated the events which occur during patterning of the vertebrate heart by exposing gastrula stage zebrafish embryos to lithium, which is known to affect the phosphoinositol signalling pathway. Treatment of embryos at 50% epiboly (5.25 h after fertilization at 28.5°C) with 0.3 m LiCl for 5–15 min, results in embryos with defects which range from mild to severe, depending on the length of time the embryos are exposed to lithium. In the heart, defects appear progressively in the inflow tract, the sinus venosus and atrium. By using an antibody that recognizes an atrium-specific isoform of myosin, our results show that lithium treatment at gastrulation specifically affects the atrium and sinus venosus, and has little obvious effect on the ventricle. Defects induced by lithium differ from those induced by retinoic acid (RA) treatment of similarly staged embryos, and suggest that lithium and RA may affect the patterning signals important for establishment of the vertebrate heart by acting on different populations of cells or by influencing different patterning pathways. Received: 8 December 1995 / Accepted: 11 April 1996     

Marked Alteration at Midblastula Transition in the Effect of Lithium on Formation of the Larval Body Pattern of Xenopus laevis

Embryos of Xenopus laevis were exposed to 0.4 M LiCl for 5 min at various stages of development. The effect of lithium on the larval body pattern could be detected from the 2-cell to the late gastrula stage, but changed from reduction of posterior structures ("anteriorization") to reduction of anterior structures ("posteriorization") just after the 12th cleavage, the time of midblastula transition (MBT). Temporal coincidence of MBT with alteration of the effect of lithium was observed even with embryos derived from half-egg fragment, in which MBT occurs just after the 11th cleavage. These results suggest the existence of a mechanism for formation of the basic plan of the larval body whose function changes at MBT. Combinations of the pre- and post-MBT exposures to lithium induced marked posteriorization in most larvae, indicating that the basic plan is not irreversibly determined until MBT, but is fixed during post-MBT stages.     

The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

Many studies have shown that the ubiq-uitin-proteasome pathway (UPP) for the degradation of short-lived proteins plays a key role in regulating cell cycle progression[1—3]. At least two distinct prote-olytic pathways are required for cell cycle process. The first pathway promotes transition from G1 to S phase, and the second initiates the onset of anaphase and exit from mitosis. The inhibition of UPP will re-sult in the blockage of cell cycle process. The knowl-edge of the role of UPP in…     

The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

In order to study the effects of ubiquitin-proteasome pathway (UPP) on mouse oocyte meiosis and cleavage, oocytes undergoing maturation and parthenogenetic activation and 1-cell embryos were treated with lactacystin, a specific inhibitor of proteasome. The results indicated that the rate of GVBD was not influenced by the treatment, but polar body extrusion, parthenogenesis and first cleavage were inhibited. Immunofluorescent staining using anti β-tubulin antibody indicated that the continuous treatment of lactacystin from GV stage disorganized microtubules and spindle assembly. When metaphase stage oocytes were treated with the drug, the already formed spindle structure was not affected, but the oocytes were arrested at metaphases. The 1-cell embryos were arrested at interphase or metaphase of first mitosis when they were incubated in the drug. Proteasome regulatory subunit PA700 was located in the spindle region, as indicated by immunofluorescence. These results suggest that UPP has effects on the process of oocyte meiosis and early cleavage in many aspects, including normal organization of spindle at prophase and segregation of chromosomes at anaphase for normal meiosis.     

Phorbol esters alter cell fate during development of sea urchin embryos

Protein kinase C (PKC) has been implicated as important in controlling cell differentiation during embryonic development. We have examined the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, to alter the differentiation of cells during sea urchin development. Addition of TPA to embryos for 10-15 min during early cleavage caused dramatic changes in their development during gastrulation. Using tissue-specific antibodies, we have shown that TPA causes the number of cells that differentiate as endoderm and mesoderm to increase relative to the number that differentiate as ectoderm. cDNA probes show that treatment with TPA causes an increase in accumulation of RNAs specific to endoderm and mesoderm with a concomitant decrease in RNAs specific to ectoderm. Treatment of isolated prospective ectodermal cells with TPA causes them to differentiate into endoderm and mesoderm. The critical period for TPA to alter development is during early to mid cleavage, and treatment of embryos with TPA after that time has little effect. These results indicate that PKC may play a key role in determining the fate of cells during sea urchin development.     

Lithium induces changes in the plasma membrane protein pattern of early amphibian embryos

Summary— The plasma membrane protein pattern of Rana ridibunda embryos subjected to lithium (Li) treatment at various stages of development was examined by two-dimensional gel electrophoresis. Differences were observed at the neurula stage not only as compared to controls but among lithium-treated embryos as well. Of particular interest was the presence of proteins, specific for the gastrula stage, in lithium-treated embryos. The results are discussed in relation to the well-known effect of lithium on amphibian morphogenesis.     

Zygotic Wnt/beta-catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus

Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Here, we use transient exposure of embryos or explants to lithium, which mimics Wnt/beta-catenin signaling, as a tool to regulate the activation of this pathway at different times and places during early development. We show that activation of Wnt/beta-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after 2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly for an immediate-early response. In the early gastrula, inhibition of Wnt/beta-catenin signaling blocks the expression of XMyf5 and XMyoD, but not of Xbra. We further show that zygotic Wnt/beta-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/beta-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells.     

LiCl disrupts axial development in mouse but does not act through the beta-catenin/Lef-1 pathway

Chimera and cell marking studies suggest that axial determination in mouse embryos occurs at postimplantation stages. In contrast, Xenopus laevis axes are determined early due to the asymmetric distribution of maternally derived factors in the one-cell zygote. In our earlier study we used lithium chloride (LiCl) to perturb development of mouse axes. Here we investigate whether the lithium induced axial defects in mouse are being mediated by the beta-catenin/Lef-1 pathway as in Xenopus laevis. In lithium treated embryos we did not observe any changes in the amount or localization of beta-catenin protein. Furthermore, the lack of Lef-1 mRNA in treated and untreated embryos indicates the LiCl induced axial defects in the mouse are not mediated by the beta-catenin/Lef-1 pathway.     

Regulation of cleavage by protein kinase C in Chaetopterus

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC-gamma transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC-alpha and -beta transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC-gamma activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC-alpha and/or -beta from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down-regulation of this activity is essential for cleavage after fertilization.     

Differential Effects of Sodium Butyrate and Lithium Chloride on Rhesus Monkey Trophoblast Differentiation

Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O₂) and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of β-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of β-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/β-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNFα production. The results provide valuable tools to manipulate trophoblast differentiation in vitro and to better understand the differentiation pathways that occur during early gestation.     

Bcl2-independent chromatin cleavage is a very early event during induction of apoptosis in mouse thymocytes after treatment with either dexamethasone or ionizing radiation

We have quantified the emergence of early chromatin breaks during the signal transduction phase of apoptosis in mouse thymocytes after treatment with either ionizing radiation or dexamethasone. Dexamethasone at 1 microM can induce significant levels of DNA breaks (equivalent to the amount induced directly by 7.5 Gy ionizing radiation) within 0.5 h of treatment. The execution phase of apoptosis was not observed until 4-6 h after the same treatment. The presence of the Bcl2 transgene under the control of the p56lck promoter almost completely inhibited apoptosis up to 24 h after treatment, but it had virtually no effect on the early chromatin cleavage occurring in the first 6 h. Ionizing radiation induced chromatin cleavage both directly by damaging DNA and indirectly with kinetics similar to the induction of chromatin cleavage by dexamethasone. The presence of the Bcl2 transgene had no effect on the direct or indirect radiation-induced cleavage in the first 6 h, but after the first 6 h, the Bcl2 gene inhibited further radiation-induced chromatin cleavage. These results suggest that endonucleases are activated within minutes of treatment with either dexamethasone or ionizing radiation as part of the very early signal transduction phase of apoptosis, and prior to the irreversible commitment to cell death.     

Rho mediates cytokinesis and epiboly via ROCK in zebrafish

To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3-exoenzyme (C3) into zebrafish embryos. We found that C3 inhibited cytokinesis during early cleavages. C3 inhibition appeared to be specific on RhoA, since the constitutively active RhoA could partially rescued the C3-induced defects. Distributions of actin and the cleavage furrow associated beta-catenin were disrupted by C3. Belbbistatin, a myosin II inhibitor, also caused blastomeres disintegration. It suggested that Rho mediates cytokinesis via cleavage furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements during epiboly and gastrulation as evident by the impairment on no tail and goosecoid expression in blastoderm front runner cells and the dorsal lip of blastopore, respectively. Y-27632, an antagonist of Rho-associated kinase (ROK/ROCK), had the similar inhibitory effects on zebrafish development as the C3 treatments. Taken together, these results suggest that Rho mediates cleavage furrow protein assembly during cytokinesis and cellular migration during epiboly and gastrulation via a ROK/ROCK-dependent pathway.     

ROCK inhibition prevents early mouse embryo development

ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.     

Blood Levels and Management of Lithium Treatment

The limited value of plasma measurements in the management of treatment with lithium is discussed in the light of the mechanisms of its therapeutic actions and toxic effects.The plasma level of lithium usually rises twofold or threefold in the three to five hours after ingestion of each dose of delayed-release tablets and then gradually falls. The precise shape and height of the lithium curve depend on gastric emptying, which can be slowed with propantheline or speeded with metoclopramide. Depressed or demented patients may be irregular in taking their tablets and variable in food intake. Both the time of the blood test and this behaviour must be considered before changing the prescribed dose of lithium salt because of a laboratory result. A lithium tolerance curve may be a safer guide to treatment than single measures.Mild intermittent thirst is a common early side effect, and severe persistent thirst with polyuria is an uncommon later effect of daily intakes of at least 1,500 mg lithium carbonate. This diabetes insipidus is reversible, non-progressive, unrelated to plasma level, and distinct in attack from lithium-induced hypothyroidism, which may occur at low dosage but is also usually of late onset and reversible or treatable with thyroxine while lithium is continued. Obesity is another occasional effect of large doses. These side effects and the antimanic and prophylactic effects may have different mechanisms.     

In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing.

DNA oligonucleotide complementary to sequences in the 5' third of U3 snRNA were injected into Xenopus oocyte nuclei to disrupt endogenous U3 snRNA. The effect of this treatment on rRNA processing was examined. We found that some toads have a single rRNA processing pathway, whereas in other toads, two rRNA processing pathways can coexist in a single oocyte. U3 snRNA disruption in toads with the single rRNA processing pathway caused a reduction in 20S and '32S' pre-rRNA. In addition, in toads with two rRNA processing pathways, an increase in '36S' pre-rRNA of the second pathway is observed. This is the first in vivo demonstration that U3 snRNA plays a role in rRNA processing. Cleavage site #3 is at the boundary of ITS 1 and 5.8S and links all of the affected rRNA intermediates: 20S and '32S' are the products of site #3 cleavage in the first pathway and '36S' is the substrate for cleavage at site #3 in the second pathway. We postulate that U3 snRNP folds pre-rRNA into a conformation dictating correct cleavage at processing site #3.     

BACE overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo

Introducing mutations within the amyloid precursor protein (APP) that affect beta- and gamma-secretase cleavages results in amyloid plaque formation in vivo. However, the relationship between beta-amyloid deposition and the subcellular site of Abeta production is unknown. To determine the effect of increasing beta-secretase (BACE) activity on Abeta deposition, we generated transgenic mice overexpressing human BACE. Although modest overexpression enhanced amyloid deposition, high BACE overexpression inhibited amyloid formation despite increased beta-cleavage of APP. However, high BACE expression shifted the subcellular location of APP cleavage to the neuronal perikarya early in the secretory pathway. These results suggest that the production, clearance, and aggregation of Abeta peptides are highly dependent on the specific neuronal subcellular domain wherein Abeta is generated and highlight the importance of perikaryal versus axonal APP proteolysis in the development of Abeta amyloid pathology in Alzheimer's disease.