Developmental changes of potassium currents of embryonic leech ganglion cells in primary culture

The expression of calcium-activated potassium currents (IK(Ca)), delayed outward rectifier potassium currents (IK(slow)), and transient outward currents (IA) was studied during the development of the nervous system of the leech using the whole-cell patch-clamp recording technique. Dissociated cells were isolated from leech embryos between stage E7 and E16 and maintained in primary culture. K+ currents were recorded at E7, when only few anterior ganglia had formed beneath the primordial mouth. IK(slow) was present in all cells tested, while IK(Ca) was expressed in only 67% of the cells studied. Even as early as E7, different types of IK(Ca) have been found. Neither frequency of occurrence nor the charge density of IK(Ca) showed significant changes between E7 and E16. The density of IK(slow), however, increased by a factor of two between E7 and E8, which resulted in a significant increase in the total K+ current of these cells. This rise in potassium outward current developed in parallel with the appearance of Na+ and Ca2+ inward currents (Schirrmacher and Deitmer: J Exp Biol 155:435-453, 1991) during early development, shaping the electrical excitability in embryonic leech neurones. I(A) could be separated by its voltage-dependence and pharmacological properties. The current was detected at stage E9, when all 32 ganglia are formed in the embryo. The frequency of occurrence of I(A) increased from 16% at E9 to 70% at E15. The channel density, steady state inactivation, and kinetics showed no significant changes during development.     

Emergence of determined myotome precursor cells in the somite

Myotome and sclerotome precursor cells are derived, respectively, from cells in the dorsomedial and ventromedial regions of the somite. To assay changes in the specification of myotomal precursor cells during somite maturation, we implanted dorsomedial quadrant fragments, from staged quail somites, next to the notochords of host chick embryos, and superimposed two additional notochords on these implants. In this notochord signalling environment, dorsomedial quadrant cells that are developmentally plastic are expected to differentiate as cartilage, while cells determined to a myogenic fate are expected to differentiate as skeletal muscle. Large numbers of differentiated chondrocytes developed from dorsomedial quadrant grafts of all stages of paraxial mesoderm development tested, indicating that persistent chondrogenic potential in cells fated to form muscle and dermis can be elicited by notochord signals. Differentiated myocytes, however, appeared in two somite-stage-dependent phases. In the first phase, dorsomedial quadrants from segmental plate and early stage somites (II and IV) form small, disorganized clusters of individual myocytes. The frequency of first-phase myocluster formation increases as myogenic factor expression begins in the dorsomedial quadrant, indicating that myogenic determination assayed by this method is closely linked to the expression of myogenic factors in the dorsomedial quadrant. In the second phase, dorsomedial quadrants from somite stages XI-XIII consistently form morphologically organized muscle tissue containing large numbers of parallel-oriented, multinucleated myotubes. Mitotic labelling demonstrated that muscle precursors were determined to the muscle phenotype prior to withdrawal from the cell cycle. Thus, myogenic determination in cells of the dorsomedial quadrant is acquired at earlier stages of somite maturation than the ability to proliferate and form muscle tissue. These results are consistent with the hypothesis that successive lineages of myotome precursor cells with different mitotic and morphogenetic properties arise in the dorsomedial quadrant during somite maturation.     

Development and differentiation of glial precursor cells in the rat cerebellum

The development and differentiation of bipotential glial precursor cells has been studied extensively in tissue culture, but little is known about the distribution and fate of these cells within intact animals. To analyze the development of glial progenitor cells in the developing rat cerebellum, we utilized immunofluorescent, immunocytochemical, and autoradiographic techniques. Glial progenitor cells were identified with antibodies against the NG2 chondroitin-sulfate proteoglycan, a cell-surface antigen of 02A progenitor cells in vitro, and the distribution of this marker antigen was compared to that of marker antigens that identify immature astrocytes, mature astrocytes, oligodendrocyte precursors, and mature oligodendrocytes. Cells expressing the NG2 antigen appeared in the cerebellum during the last 3-4 days of embryonic life. Over the first 10 days of postnatal life, the NG2-labeled cells incorporated 3H-thymidine into their nuclei and their total number increased. At all ages examined, the NG2-labeled cells did not contain either vimentin-like or glial fibrillary acidic protein (GFAP)-like immunoreactivity, suggesting that they do not develop along an astrocytic pathway. NG2-labeled cells of embryonic animals expressed GD3 ganglioside antigens, a property of oligodendrocyte precursors, whereas NG2-positive cells of postnatal animals did not express GD3 immunoreactivity. Nevertheless, the NG2-labeled cells of the nascent white matter expressed oligodendrocyte-specific marker antigens. Cells lying outside of the white matter continued to express the NG2 antigen. In adult animals, the NG2-labeled cells incorporated 3H-thymidine. Glial cells isolated from adult animals and grown in tissue culture express the NG2 antigen and display the phenotypic plasticity characteristic of 02A progenitor cells. These findings demonstrate that a population of glial progenitor cells is extensive within both young and adult animals.     

Analysis of motile oligodendrocyte precursor cells in vitro and in brain slices

Oligodendrocyte precursor cells are purported to migrate over long distances into the various brain regions where they differentiate into oligodendrocytes and fulfill their appropriate tasks, i.e., myelination of axons. Here we characterize motile oligodendrocyte precursor cells in detail. Video-time lapse analysis was performed on isolated precursor cells in single cell cultures, in co-culture with cerebellar microexplants, and in living brain slices. Motility analysis of individual cells was combined with electrophysiological, immunological, and morphological characterizations. Translocation of the cell bodies was not continuous but occurred in waves. All motile cells exhibited a simple morphology and most, but not all, of them expressed the A2B5 epitope in vitro. Patch clamp analysis of the motile cells confirmed that they belong to the O-2A lineage. The percentage of motile cells, as well as their velocities, were enhanced on substrate-coated laminin in comparison to poly-L-lysine. Motility was not influenced by the presence of cerebellar microexplants. O-2A progenitor cells did not migrate strictly along neurite fascicles which were projected from the microexplants. Glial progenitor cells in situ also did not strictly migrate along the main direction of the axonal fibers of the corpus callosum but rather traversed the fibers with an overall direction toward the cortex. After Lucifer Yellow filling of the motile progenitor cells in situ, we could demonstrate that they were dye-coupled to yet unidentified cells of the corpus callosum.     

Proliferation of myeloid precursor cells in a microagar culture system

A microagar-culture system for the in vitro study of myelopoiesis committed stem cells has been reported. Stimulation was induced by a commercially available conditioned medium obtained from culture of histiocytoma cells. The effect of different concentrations of conditioned medium and a linear relation between the seeded cells and the formed colonies has been established. The granulocytic-monocytic character of cell aggregates has been shown by morphologic studies. Further possibilities for the use of the described method have been briefly discussed.     

Reconstitution of phagosome-lysosome fusion in streptolysin O-permeabilized cells

We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase pre-internalized into lysosomes to phagosomes containing biotinylated beta-glucuronidase-conjugated paramagnetic beads (1-2 microm). Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore, fusion was inhibited by GTPgammaS and Rab GDP dissociation inhibitor. These results suggest that rab proteins are involved in the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant NSF wild type partially restored the fusogenic activity, indicating that NSF is required for fusion between phagosomes and lysosomes.     

Cellular mechanisms of epiboly in leech embryos

Gastrulation in leech embryos is dominated by the epibolic movements of two tissues: germinal bands, composed of segmental precursor cells, and an overlying epithelium that is part of a provisional integument. During gastrulation, the germinal bands move over the surface of the embryo and coalesce along the prospective ventral midline. Concurrently, the epithelium spreads to cover the embryo. We have begun to analyze the mechanisms involved in gastrulation in the leech by assessing the independent contributions of the epithelium and the germinal bands to these cell movements. Here we describe cellular events during epiboly in normal embryos and in embryos perturbed by either reducing the number of cells in the epithelium, or by preventing the formation of the germinal bands, or both. These experiments indicate that both the germinal bands and the epithelium are able to undergo epibolic movements independently, although each is required for the other to behave as in control embryos.     

Molecular identity of hematopoietic precursor cells emerging in the human embryo

It is now accepted from studies in animal models that hematopoietic stem cells emerge in the para-aortic mesoderm-derived aorta-gonad-mesonephros region of the vertebrate embryo. We have previously identified the equivalent primitive hematogenous territory in the 4- to 6-week human embryo, under the form of CD34(+)CD45(+)Lin- high proliferative potential hematopoietic cells clustered on the ventral endothelium of the aorta. To characterize molecules involved in initial stem cell emergence, we first investigated the expression in that territory of known early hematopoietic regulators. We herein show that aorta-associated CD34(+) cells coexpress the tal-1/SCL, c-myb, GATA-2, GATA-3, c-kit, and flk-1/KDR genes, as do embryonic and fetal hematopoietic progenitors later present in the liver and bone marrow. Next, CD34(+)CD45(+) aorta-associated cells were sorted by flow cytometry from a 5-week embryo and a cDNA library was constructed therefrom. Differential screening of that library with total cDNA probes obtained from CD34(+) embryonic liver cells allowed the isolation of a kinase-related sequence previously identified in KG-1 cells. In addition to emerging blood stem cells, KG-1 kinase is also strikingly expressed in all developing endothelial cells in the yolk sac and embryo, which suggests its involvement in the genesis of both hematopoietic and vascular cell lineages in humans.     

Tests of significance in stepwise regression.

Tests of significance of the sample squared multiple correlation (R–2) in stepwise multiple regression have not been possible because its distribution is unknown. The present study used Monte Carlo simulation and least squares smoothing to construct tables of the upper 95th and 99th percentage points of the sample R–2 distribution in forward selection. A survey of published psychological research that used stepwise regression found a substantial inflation of reported significance levels when compared to the tabled values. Recommendations are given for use of these tables in evaluating results from forward selection and other stepwise methods. (19 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Formation and localization of cytoplasmic domains in leech and ascidian zygotes

Leech and ascidian embryos are well suited for the study of certain developmental processes. Although leeches and ascidians belong to different bilateralia groups (protostomes and deuterostomes, respectively) they share important developmental features and, in particular, the determinate character of their embryogenesis. In both types of embryos this property is related to the presence of specific cytoplasmic domains that are selectively allocated to different blastomeres during cleavage. In this review leech and ascidian eggs and zygotes are compared in terms of the structure of these cytoplasmic domains and of the cellular mechanisms involved in their formation and localization. During meiosis the zygote of leeches and ascidians undergo stereotypic actin-dependent contraction movements related to both the emission of the polar bodies and the formation and relocalization of cytoplasmic domains. After completion of meiosis, during first interphase, monaster microtubules nucleated from the sperm-derived centrosome play a key role in pronuclear migration. In addition, these astral microtubules direct the relocalization of cytoplasmic domains and the translocation and accumulation of organelles in the interior of the zygote. Microtubules and microfilaments, on the other hand, are involved in cortical reorganizations and organelle translocations in both zygote species during interphase and cleavage divisions. In the case of leech zygotes, this process leads to formation of characteristic polar cytoplasmic domains called teloplasms. These domains are selectively inherited by teloblasts, precursor stem cells of ectodermal and mesodermal tissues in the leech embryo. In the ascidian zygote, the cytoplasmic movements observed during interphase and mitosis lead to relocalization of the bulk of a mitochondria-rich domain, called the myoplasm, along with an endoplasmic reticulum-rich domain towards the future posterior pole of the embryo. The myoplasm is inherited by a subset of posterior blastomeres committed to become the primary muscle cells of the ascidian tadpole.     

Upregulation of actin gene expression in cells expressing exogenous beta-amyloid precursor protein

We investigated the effects of beta-amyloid peptide precursor (APP) overexpression upon the levels of other mRNAs. Using quantitative slot-blot hybridization and immunoblot analysis we observed that enhanced levels of APP elevated the levels of beta-actin and beta-actin mRNA. Our results also suggest that the cytoplasmic domain of APP is crucial for the elevation in beta-actin gene expression.     

A role for the mesoderm in endodermal migration and morphogenesis in Drosophila

The endodermal midgut arises from two primordia, the anterior midgut (AMG) primordium and the posterior midgut (PMG) primordium, which are separated by almost the entire length of the Drosophila embryo. To form the midgut, these two parts have to extend towards each other and to fuse laterally on both sides of the yolk. Shortly before and during that movement, AMG and PMG are arranged as mesenchymal cell masses, but later the midgut cells form an epithelium. We show that these two aspects of midgut development, migration of AMG and PMG and transition to an epithelium, depend on the mesoderm. The extension of the midgut primordia is achieved by cell migration along the visceral mesoderm which forms a continuous layer of cells within the germ band. In mutant embryos lacking the entire mesoderm or failing to differentiate the visceral mesoderm, AMG and PMG are formed but do not migrate properly. In addition, they fail to form an epithelium and instead either remain as compact cell masses anterior and posterior to the yolk (in twist and snail mutant embryos) or only occasionally wrap around the yolk before embryogenesis is completed (in tinman-deficient embryos). We conclude that the visceral mesoderm serves as a substratum for the migrating endodermal cells and that the contact between visceral mesoderm and endoderm is required for the latter to become an epithelium.     

Telomeric chromosome fusion in cells treated with colcemid and 5-bromdeoxyuridine

Specific chromosomes with two or more centromeres were found in the tetraploid metaphases of the second mitotic division after combined treatment with coldemid (0.05 mug/ml) and 5-bromodeoxyuridine (50 mug/ml) for 24 hours in the aneuploid Chinese hamster cell line. They were formed by telometric fusion of chromosomes and were absent in the cells treated with colcemid or 5-bromodeoxyuridine separately. A possible relation of this phenomenon to the telomeric association of chromosomes in the interphase nucleus, as well as to the chromosomal disturbances in the Louis Bar symdrome (ataxia -- telangiectasia) is discussed.     

Retinoic acid disrupts anterior ectodermal and endodermal development in ascidian larvae and postlarvae

In vertebrates, excess all-trans retinoic acid (RA) applied during axis formation leads to the apparent truncation of anterior structures. In this study we sought to determine the type of defects caused by ectopic RA on the development of the ascidian Herdmania curvata. We demonstrate that H. curvata embryos cultured in the presence of RA develop into larvae whose trunks are shortened and superficially resemble those of early metamorphosing postlarvae. Despite RA-treated larvae lacking papillar structures they respond normally to natural cues that induce metamorphosis, indicating that chemosensory functionality previously mapped to the most anterior region of normal larvae is unaffected by RA. Excess RA applied during postlarval development leads to a graded loss of the juvenile pharynx, apparently by respecifying anterior endoderm to a more posterior fate. This structure is considered homologous to the gill slits of amphioxus, which are also lost upon RA treatment. This suggests that RA may have had a role in the development of the pharynx of the ancestral chordate and that this function has been maintained in ascidians and cephalochordates and lost in vertebrates.     

The connective tissue coverings of leech peripheral nerves: anatomical evidence for the absence of cerebrospinal fluid in the leech

The central nervous system of Hirudo medicinalis is contained within a blood vessel, the ventral longitudinal sinus, but the nervous system is separated from the blood by the visceral endothelium. The visceral endothelium possesses many pinocytotic vesicles and basal infoldings and thus appears active whereas the parietal endothelium appears inactive. The junction between the visceral and parietal endothelia is abrupt. Peripheral nerves in this animal, as in vertebrates, are covered by endoneurium, perineurium, and epineurium. The endoneurium is continous with the fibrous tissue of the segmental ganglia. The perineurium, consisting of a single layer of flattened cells that surrounds the peripheral nerve like a sleeve, is not continuous with the endothelium of the ventral sinus, but is separated from it by 5-10 microns. Therefore, at the point where the peripheral nerve joins the segmental ganglion, the extracellular spaces of the central nervous system, the peripheral nervous system, and the body wall are all confluent. Thus, there are only two compartments of the extracellular space in the leech: the blood, which is enclosed by the endothelia of the coelomic sinuses, and the extracellular fluid of the body, which includes the extracellular fluid of the nervous system. There seems to be no equivalent of cerebrospinal fluid in the gnathobdellid leech.     

Localization of thoracic imaginal-disc precursor cells in the early embryo of Drosophila melanogaster

Our previous cell lineage analysis of the thoracic disc primordia of Drosophila showed that at the blastoderm and early gastrula stage, cells are not yet committed to form either larval or imaginal tissue (Meise and Janning, 1993). We have now refined our studies on the cell lineage and have mapped the imaginal primordia in the thoracic region. Homotopic transplantations of single cells within the thoracic region of blastoderm and early gastrula stages show that the precursor cells of thoracic imaginal discs are locally restricted to a small lateral area of the thoracic region. Clones labelling leg discs frequently included the Keilin's organs. Heterotopic transplantations along the dorsoventral axis indicate that cells within the thoracic region are not yet committed with respect to larval or imaginal tissue, their fate being dependent on the position where the transplanted cell had been deposited. On the other hand, cells taken from the abdominal anlagen and transplanted into the region of thoracic disc primordia could not participate in the formation of imaginal discs. This shows that, in contrast to the dorsoventral axis, determinative events had separated primordia along the anterior-posterior axis.     

Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells

The standard method for assessing the carcinogenicity of lubricating oil base stocks is the mouse skin-painting bioassay. This assay has the advantage of directly measuring the endpoint of interest, dermal carcinogenicity, but has the drawback of being time-consuming and expensive. For this reason, a variety of biological and chemical assays have been developed as predictive alternatives to the in vivo assay. This publication describes the application of three such methods to the assessment of carcinogenic potential of hydrotreated, re-refined oils: the modified Ames test, the analytical determination of 3-7-ring polycyclic aromatic compound content and the 32P-postlabeling assay for DNA adduct induction.     

Coactivation of putative octopamine- and serotonin-containing interneurons in the medicinal leech

Possible interactions between octopamine-immunoreactive (IR) and serotonergic neurons in the CNS of the medicinal leech were investigated. Simultaneous intracellular recordings of serotonin-containing neurons (either the Retzius neuron or cell 21) and the dorsolateral octopamine-IR (DLO) neuron demonstrated that both sets of neurons are coactive at times. Depolarization of either serotonergic cell 21 or the Retzius neuron did not alter the membrane potential of the DLO. Similarly, depolarization of the DLO did not affect the serotonergic neurons examined. Because it was found that the DLO and either the serotonergic cell 21 or Retzius neuron were at times coactive, we looked for possible sources of common excitatory inputs. The centrally located pressure (P)- and touch (T)-sensitive mechanosensory neurons excited the DLOs through a polysynaptic pathway. Stimulation of nociceptive (N) mechanosensory neurons did not cause a measurable depolarization in the membrane potential of the DLO. Through simultaneous recordings of the DLO, cell 21, and a particular identified mechanosensory neuron, it was demonstrated that activity in the T or P cells can excite both serotonergic cell 21 and the octopamine-IR DLO. These findings indicate that, in many instances, both serotonin and octopamine, biogenic amines with neuromodulatory actions in many different invertebrates, may be released simultaneously in the leech.