Inscuteable-independent apicobasally oriented asymmetric divisions in the Drosophila embryonic CNS

Inscuteable is the founding member of a protein complex localised to the apical cortex of Drosophila neural progenitors that controls their asymmetric division. Aspects of asymmetric divisions of all identified apicobasally oriented neural progenitors characterised to date, in both the central and peripheral nervous systems, require inscuteable. Here we examine the generality of this requirement. We show that many identified neuroblast lineages, in fact, do not require inscuteable for normal morphological development. To elucidate the requirements for apicobasal asymmetric divisions in a context where inscuteable is not essential, we focused on the MP2 > dMP2 + vMP2 division. We show that for MP2 divisions, asymmetric localisation and segregation of Numb and the specification of distinct dMP2 and vMP2 identities require bazooka but not inscuteable. We conclude that inscuteable is not required for all apicobasally oriented asymmetric divisions and that, in some cellular contexts, bazooka can mediate apicobasal asymmetric divisions without inscuteable.     

Membrane properties of identified embryonic nerve and glial cells of the leech central nervous system

Summary The membrane potential of identified nerve (Retzius) cells and neuropil glial cells from 11 (±1) day-old embryos of the leechHirudo medicinalis was recorded using conventional intracellular microelectrodes. At this stage all ganglia of the segmental nervous system are formed. The membrane potential of Retzius cells was –68±4 mV (±SD,n=8), and showed a slope of 42 mV between 10 mM and 100 mM external K concentration. Retzius cells were able to fire action potentials which had a fast Na-dependent component, and, under appropriate conditions, also generated slow Ca (Ba) action potentials. The mean membrane potential of the neuropil glial cell at physiological K concentration (4 mM) was –83±5 mV (±SD,n=10), and showed a dependence of 56 mV for a tenfold change in the external K concentration (> 4mM). Neuropil glial cells showed no signs of voltage-activated excitability, but they repeatedly depolarized in the presence of 0.1 mM 5-HT.     

Tre1 GPCR signaling orients stem cell divisions in the Drosophila central nervous system

During development, directional cell division is a major mechanism for establishing the orientation of tissue growth. Drosophila neuroblasts undergo asymmetric divisions perpendicular to the overlying epithelium to produce descendant neurons on the opposite side, thereby orienting initial neural tissue growth. However, the mechanism remains elusive. We provide genetic evidence that extrinsic GPCR signaling determines the orientation of cortical polarity underlying asymmetric divisions of neuroblasts relative to the epithelium. The GPCR Tre1 activates the G protein oα subunit in neuroblasts by interacting with the epithelium to recruit Pins, which regulates spindle orientation. Because Pins associates with the Par-complex via Inscuteable, Tre1 consequently recruits the polarity complex to orthogonally orient the polarity axis to the epithelium. Given the universal role of the Par complex in cellular polarization, we propose that the GPCR-Pins system is a comprehensive mechanism controlling tissue polarity by orienting polarized stem cells and their divisions.     

Tag team specification of a neural precursor in the Drosophila embryonic central nervous system

The development of vertebrate and invertebrate nervous systems requires the production of thousands to millions of uniquely specified neurons from progenitor neural stem cells. A central question focuses on the elucidation of the developmental mechanisms that function within neural stem cell lineages to impart unique identities to neurons. A recent report⁽¹⁾ details the roles that two genes, pdm-1 and pdm-2, play within an identified neural stem cell lineage in the Drosophila embryonic central nervous system. The results show that pdm-1 and pdm-2 are coexpressed in an identified neural precursor and function redundantly to specify the fate of this cell. As such this report offers an initial view of the genetic programs that create neural diversity.     

Programmed cell death in the embryonic central nervous system of Drosophila melanogaster

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. We provide here a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). We traced the spatio-temporal pattern of PCD and compared the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos. Furthermore, we have examined the clonal origin and fate of superfluous cells in H99 mutants by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. Our data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers we were able to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS.     

Circadian rhythms and glial cells of the central nervous system

Glial cells are the most abundant cells in the central nervous system and play crucial roles in neural development, homeostasis, immunity, and conductivity. Over the past few decades, glial cell activity in mammals has been linked to circadian rhythms, the 24-h chronobiological clocks that regulate many physiological processes. Indeed, glial cells rhythmically express clock genes that cell-autonomously regulate glial function. In addition, recent findings in rodents have revealed that disruption of the glial molecular clock could impact the entire organism. In this review, we discuss the impact of circadian rhythms on the function of the three major glial cell types – astrocytes, microglia, and oligodendrocytes – across different locations within the central nervous system. We also review recent evidence uncovering the impact of glial cells on the body's circadian rhythm. Together, this sheds new light on the involvement of glial clock machinery in various diseases.     

An antigen present in the Drosophila central nervous system only during embryonic and metamorphic stages

We report here about an antigen that is expressed in the central nervous system (CNS) of Drosophila only during the embryonic and metamorphic stages. In Drosophila, axonogenesis and synaptogenesis occur twice during the development: first in the embryonic and second in the metamorphic stages. We generated monoclonal antibodies (MAbs) in order to obtain molecular probes for analyzing axonogenesis or synaptogenesis in the CNS on the assumption that good candidates for molecules responsible for such phenomena must be present in the neuropil during those stages exclusively. As a result, we found MAb 66B2 whose intense immunoreactivity in the neuropil of the CNS was observed exclusively in the embryo and pupa, and not in the larva and adult. Immunoblot analyses showed that MAb 66B2 binds specifically to a protein with an apparent molecular weight of 350 K and neutral pl in the prepupal CNS. A significant amount of the antigen was isolated in forms that were soluble without detergent. Results of immunohistochemistry with MAb 66B2 in a primary culture of embryos showed that some live cells in the ganglion-like cluster were stained, and that neuronal cell bodies and neurites emanating from there were negative. These results strongly suggest that the 66B2 antigen observed in the CNS is an extracellular matrix component secreted from nonneuronal cells. These developmental changes in the 66B2 immuno-reactivity in the CNS presumably reflect dynamic changes of an extracellular matrix in the CNS that are accompanied by axonogenesis or synaptogenesis. © 1992 John Wiley & Sons, Inc.     

Labeling embryonic mouse central nervous system cells by in utero electroporation

During cerebral development, neurons are generated near the ventricle and then migrate toward the pial surface. In this review, we describe the method of in utero electroporation, this method allows the morphology of the migrating neurons to be visualized and the effect of overexpression or knock down of any gene to be examined. After electroporation of a green fluorescent protein (GFP) expression vector by this method, GFP-positive cells are first found in the ventricular zone, and their distribution then gradually shift toward the pial surface. A few days later, most of the GFP positive cells were aligned beneath the marginal zone, with the normal course of cortical neuronal migration.     

Electrogenic sodium-dependent bicarbonate secretion by glial cells of the leech central nervous system

The ability to move acid/base equivalents across the membrane of identified glial cells was investigated in isolated segmental ganglia of the leech Hirudo medicinalis. The intracellular pH (pHi) of the glial cells was measured with double-barreled, neutral-ligand, ion-sensitive microelectrodes during step changes of the external pH (pHo 7.4-7.0). The rate of intracellular acidification after the decrease in extracellular pH (pHo) was taken as a measure of the rate of acid/base transport across the glial membrane. Taking into account the total intracellular buffering power, the maximum rate of acid/base flux was 0.4 mM/min in CO2/HCO3-free saline, and 3.92 mM/min in the presence of 5% CO2/10 mM HCO-3, suggesting that the acid/base flux was dependent upon HCO3-. The rate of acid influx/base efflux increased both with the external HCO3- concentration and with increasing pHi (and hence HCO3-i). This suggested that the decrease in pHi was due to HCO3- efflux. The rapid decrease of pHi was accompanied by a HCO3--dependent depolarization of the glial membrane from -74 +/- 5 mV (n = 20) to -54 +/- 7 mV (n = 13). Both this depolarization and the rate of intracellular acidification were greatly reduced by the anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.3-0.5 mM), but were not affected by the removal of external Cl-. Reduction of the external Na+ concentration to one-tenth normal affected the rate of intracellular acidification only in the presence of CO2/HCO3-: the rate increased within the first 3-5 min after lowering external Na+; after longer exposures in low external Na+ the rate decreased, presumably due to depletion of intracellular Na+. Amiloride (1 mM), which inhibits the Na+-H+ exchange in these cells, had no effect on the rate of intracellular acidification. The intracellular Na activity (aNai) of the glial cells was measured to be 5.2 +/- 1.0 mM (n = 8) in CO2/HCO3-free saline; aNai increased to 7.3 +/- 2.2 mM (n = 8) after the addition of 5% CO2/24 mM HCO3-. Upon a change in pHo to 7.0 in the presence of CO2/HCO3-, aNai decreased by an average of 2 +/- 1.1 mM (n = 5); in CO2/HCO3--free saline external acidification produced a transient increase in aNai. It is concluded that, in the presence of CO2/HCO3-, the rate of intracellular acidification in glial cells is dominated by an outwardly directed, electrogenic Na+-HCO3-cotransport. Neurons, which do not possess this cotransporter, acidify at much lower rates under similar conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metamorphosis of the central nervous system of Drosophila

The study of the metamorphosis of the central nervous system of Drosophila focused on the ventral CNS. Many larval neurons are conserved through metamorphosis but they show pronounced remodeling of both central and peripheral processes. In general, transmitter expression appears to be conserved through metamorphosis but there are some examples of possible changes. Large numbers of new, adult-specific neurons are added to this basic complement of persisting larval cells. These cells are produced during larval life by embryonic neuroblasts that had persisted into the larval stage. These new neurons arrest their development soon after their birth but then mature into functional neurons during metamorphosis. Programmed cell death is also important for sculpting the adult CNS. One round of cell death occurs shortly after pupariation and a second one after the emergence of the adult fly.     

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.