A monoclonal antibody specific for diploid epithelial cells in Drosophila

Results from various experiments suggest that the cell surface has an important role in development. However, there is relatively little information on the specific surface molecules involved in developmental processes. In an effort to characterize cell-surface components that may be involved in Drosophila development, we have been making monoclonal antibodies against D. melanogaster imaginal disks. The holometabolous insects are unusual in that scattered among the larval tissues are groups of undifferentiated imaginal cells which, during metamorphosis, will form most of the adult insect. The imaginal disks, which we use as an immunogen, are hollow sacs of cells; each disk will form a specific part of the adult cuticle. Other imaginal cells are found as nests or rings in various larval organs. We describe here results indicating that one of the clones we have isolated, DA.1B6, makes an antibody against an antigen which, in larvae, is generally restricted to the undifferentiated sheets of imaginal epithelial cells. This and other results indicate that the antigen is specific for the diploid epithelia in Drosophila.     

Connectivity of chemosensory neurons is controlled by the gene poxn in Drosophila.

The function of the nervous system depends on the formation of a net of appropriate connections, but little is known of the genetic program underlying this process. In Drosophila two genes that specify different types of sense organs have been identified: cut (ct), which specifies the formation of external sense organs as opposed to chordotonal organs, and pox-neuro (poxn), which specifies the formation of poly-innervated (chemosensory) organs as opposed to mono-innervated (mechanosensory) organs. Whether these genes are also involved in specifying the connectivity of the corresponding neurons is not known. The larval sense organs are unsuitable for analysis of the axonal pathway and connections and so we have investigated the effect of poxn on the adult. Here we show that overexpression of poxn induces the morphological transformation of mechanosensory into chemosensory bristles on the legs and that the neurons innervating the morphologically transformed bristles follow pathways and establish connections that are appropriate for chemosensory bristles.     

Control of neuronal fate by the Drosophila segmentation gene even-skipped

The central nervous system (CNS) contains a remarkable diversity of cell types. The molecular basis for generating this neuronal diversity is poorly understood. Much is known, however, about the regulatory genes which control segmentation and segment identity during early Drosophila embryogenesis. Interestingly, most of the segmentation and homoeotic genes in Drosophila, as well as many of their vertebrate homologues, are expressed during the development of the nervous system (for example, ref. 3). Are these genes involved in specifying the identity of individual neurons during neurogenesis, just as they specify the identity of cells during segmentation? We previously described the CNS expression of the segmentation gene fushi tarazu (ftz) and showed that ftz CNS expression is involved in the determination of an identified neuron. Here we show that another segmentation gene, even-skipped (eve), is expressed in a different but overlapping subset of neurons. Temperature-sensitive inactivation of the eve protein during neurogenesis alters the fate of two of these neurons. Our results indicate that the nuclear protein products of the eve and ftz segmentation genes are components of the mechanism controlling cell fate during neuronal development.     

Astroglia induce neurogenesis from adult neural stem cells

During an investigation of the mechanisms through which the local environment controls the fate specification of adult neural stem cells, we discovered that adult astrocytes from hippocampus are capable of regulating neurogenesis by instructing the stem cells to adopt a neuronal fate. This role in fate specification was unexpected because, during development, neurons are generated before most of the astrocytes. Our findings, together with recent reports that astrocytes regulate synapse formation and synaptic transmission, reinforce the emerging view that astrocytes have an active regulatory role--rather than merely supportive roles traditionally assigned to them--in the mature central nervous system.     

En passant neurotrophic action of an intermediate axonal target in the developing mammalian CNS.

During development, neurons extend axons to their targets, then become dependent for their survival on trophic substances secreted by their target cells. Competition for limiting amounts of these substances is thought to account for much of the extensive naturally-occurring cell death that is seen throughout the nervous system. Here we show that spinal commissural neurons, a group of long projection neurons in the central nervous system (CNS), are also dependent for their survival on trophic support from one of their intermediate targets, the floor plate of the spinal cord. This dependence occurs during a several-day-long period when their axons extend along the floor plate, following which they develop additional trophic requirements. A dependence of neurons on trophic support derived en passant from their intermediate axonal targets provides a mechanism for rapidly eliminating misprojecting neurons, which may help to prevent the formation of aberrant neuronal circuits during the development of the nervous system.     

神经干细胞及其临床应用潜能

近年来神经干细胞已在成年哺乳动物中的中枢神经系统中分离成功。神经干细胞的最基本特征是具有分化为神经元、星状胶质细胞和少突胶质细胞的潜能，具有自我更新能力，并足以维持整个大脑所需。神经干细胞在修复受伤神经组织及治疗神经系统退行性疾病，如帕金森病、阿尔茨海默病、和亨庭顿病等方面有很好的应用前景。但在达到临床实际应用之前仍有一系列问题需要解决，最首要的是搞清神经干细胞的分化机制。     

BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra

Brain-derived neurotrophic factor (BDNF), present in minute amounts in the adult central nervous system, is a member of the nerve growth factor (NGF) family, which includes neurotrophin-3 (NT-3). NGF, BDNF and NT-3 all support survival of subpopulations of neural crest-derived sensory neurons; most sympathetic neurons are responsive to NGF, but not to BDNF; NT-3 and BDNF, but not NGF, promote survival of sensory neurons of the nodose ganglion. BDNF, but not NGF, supports the survival of cultured retinal ganglion cells but both NGF and BDNF promote the survival of septal cholinergic neurons in vitro. However, knowledge of their precise physiological role in development and maintenance of the nervous system neurons is still limited. The BDNF gene is expressed in many regions of the adult CNS, including the striatum. A protein partially purified from bovine striatum, a target of nigral dopaminergic neurons, with characteristics apparently similar to those of BDNF, can enhance the survival of dopaminergic neurons in mesencephalic cultures. BDNF seems to be a trophic factor for mesencephalic dopaminergic neurons, increasing their survival, including that of neuronal cells which degenerate in Parkinson's disease. Here we report the effects of BDNF on the survival of dopaminergic neurons of the developing substantia nigra.     

Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism

In degenerative disorders of the central nervous system (CNS), transplantation of neural multipotent (stem) precursor cells (NPCs) is aimed at replacing damaged neural cells. Here we show that in CNS inflammation, NPCs are able to promote neuroprotection by maintaining undifferentiated features and exerting unexpected immune-like functions. In a mouse model of chronic CNS inflammation, systemically injected adult syngeneic NPCs use constitutively activated integrins and functional chemokine receptors to selectively enter the inflamed CNS. These undifferentiated cells survive repeated episodes of CNS inflammation by accumulating within perivascular areas where reactive astrocytes, inflamed endothelial cells and encephalitogenic T cells produce neurogenic and gliogenic regulators. In perivascular CNS areas, surviving adult NPCs induce apoptosis of blood-borne CNS-infiltrating encephalitogenic T cells, thus protecting against chronic neural tissue loss as well as disease-related disability. These results indicate that undifferentiated adult NPCs have relevant therapeutic potential in chronic inflammatory CNS disorders because they display immune-like functions that promote long-lasting neuroprotection.     

Membrane conductance oscillations in astrocytes induced by phorbol ester

Glial cells in the central nervous systems (CNS) have complex functions which are difficult to decipher because of the intimate intertwining of glial cells with neurons. We have therefore developed an essentially neuron-free preparation of CNS astrocytes in the kainic acid lesioned hippocampal slice. With this preparation we have examined the effect of activating protein kinase C in astrocytes with a phorbol ester, TPA (12-O-tetradecanoyl-phorbol-13-acetate). In most cells, TPA induced rhythmic oscillations (0.1-3.0 Hz) of membrane potential which were typically 5-10 mV in amplitude and were associated with increases of up to eightfold in input resistance during the depolarizing phase. These large changes in membrane conductance are the first reported observations of endogenously generated conductance changes in astrocytes of the mammalian CNS and they could influence excitability of surrounding neurons, possibly by altering extracellular ion concentrations.     

Retinal pigmented epithelial cells induced to transdifferentiate to neurons by laminin

Although the regeneration of nervous tissue in the vertebrate is very limited, there are a few remarkable examples of this process. Understanding the factors that regulate CNS regeneration in those areas of the nervous system where it occurs, will doubtless provide generally applicable, essential information about the process. It has been known for some time that the amphibian retina regenerates following its destruction. Transplant studies, confirmed later by in vitro experiments, have shown that one source of new neurons in regenerating retina is the retinal pigmented epithelium (RPE). RPE cells can transdifferentiate to either neurons or lens cells in culture, but little is known about the factors that regulate this process. A recent study in vivo of retinal regeneration provided evidence that the association of RPE cells with the retinal vascular membrane is an important step in transdifferentiation. We report here that transdifferentiation in vitro is profoundly influenced by the substrate on which the cells are cultured; RPE cells plated on laminin-containing substrates frequently transdifferentiate into neurons. In addition, we have found a high concentration of laminin in the Rana retinal vascular membrane. Therefore, we propose that retinal regeneration is initiated by changes in the composition of the extracellular matrix that RPE cells contact early in the process.     

Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration

Nogo has been identified as a component of the central nervous system (CNS) myelin that prevents axonal regeneration in the adult vertebrate CNS. Analysis of Nogo-A has shown that an axon-inhibiting domain of 66 amino acids is expressed at the extracellular surface and at the endoplasmic reticulum lumen of transfected cells and oligodendrocytes. The acidic amino terminus of Nogo-A is detected at the cytosolic face of cellular membranes and may contribute to inhibition of axon regeneration at sites of oligodendrocyte injury. Here we show that the extracellular domain of Nogo (Nogo-66) inhibits axonal extension, but does not alter non-neuronal cell morphology. In contrast, a multivalent form of the N terminus of Nogo-A affects the morphology of both neurons and other cell types. Here we identify a brain-specific, leucine-rich-repeat protein with high affinity for soluble Nogo-66. Cleavage of the Nogo-66 receptor and other glycophosphatidylinositol-linked proteins from axonal surfaces renders neurons insensitive to Nogo-66. Nogo-66 receptor expression is sufficient to impart Nogo-66 axonal inhibition to unresponsive neurons. Disruption of the interaction between Nogo-66 and its receptor provides the potential for enhanced recovery after human CNS injury.     

Substrate phosphoprotein availability regulates eclosion hormone sensitivity in an insect CNS

The final step in the moulting of all insects is ecdysis, the shedding of the cuticle of the previous instar, which is triggered in Lepidoptera by the neurosecretory peptide eclosion hormone. This hormone acts directly on the nervous system to release the appropriate motor patterns for larval, pupal and adult ecdysis, but there are only brief periods near the end of each moult when the nervous system is competent to respond to the hormone. Previous experiments have shown that the action of eclosion hormone on the nervous system at pupal ecdysis in the tobacco hornworm, Manduca sexta, is mediated by the second messenger cyclic GMP. Here we report that the hormone-stimulated increase in cGMP results in the phosphorylation of two proteins, each with an apparent relative molecular mass (Mr) of 54,000. Moreover, the brief periods during which the central nervous system (CNS) is responsive to eclosion hormone seem to result from the transient presence of these substrate proteins within the nervous system. This provides a novel mechanism by which hormonal responsiveness can be regulated.     

Fruitless specifies sexually dimorphic neural circuitry in the Drosophila brain

The Drosophila fruitless (fru) gene product Fru has been postulated to be a neural sex determination factor that directs development of the central nervous system (CNS), thereby producing male-typical courtship behaviour and inducing male-specific muscle. Male-specific Fru protein is expressed in small groups of neurons scattered throughout the CNS of male, but not female, Drosophila. Collectively, these observations suggest that Fru 'masculinizes' certain neurons, thereby establishing neural substrates for male-typical behaviour. However, specific differences between neurons resulting from the presence or absence of Fru are unknown. Previous studies have suggested that Fru might result in sexual differences in the CNS at the functional level, as no overt sexual dimorphism in CNS structure was discernible. Here we identify a subset of fru-expressing interneurons in the brain that show marked sexual dimorphism in their number and projection pattern. We also demonstrate that Fru supports the development of neurons with male-specific dendritic fields, which are programmed to die during female development as a result of the absence of Fru. Thus, Fru expression can produce a male-specific neural circuit, probably used during heterosexual courtship, by preventing cell death in identifiable neurons.     

Astrocytes induce blood-brain barrier properties in endothelial cells

The highly impermeable tight junctions between endothelial cells forming the capillaries and venules in the central nervous system (CNS) of higher vertebrates are thought to be responsible for the blood-brain barrier that impedes the passive diffusion of solutes from the blood into the extracellular space of the CNS. The ability of CNS endothelial cells to form a blood-brain barrier is not intrinsic to these cells but instead is induced by the CNS environment: Stewart and Wiley demonstrated that when avascular tissue from 3-day-old quail brain is transplanted into the coelomic cavity of chick embryos, the chick endothelial cells that vascularize the quail brain grafts form a competent blood-brain barrier; on the other hand, when avascular embryonic quail coelomic grafts are transplanted into embryonic chick brain, the chick endothelial cells that invade the mesenchymal tissue grafts form leaky capillaries and venules. It is, however, not known which cells in the CNS are responsible for inducing endothelial cells to form the tight junctions characteristic of the blood-brain barrier. Astrocytes are the most likely candidates since their processes form endfeet that collectively surround CNS microvessels. In this report we provide direct evidence that astrocytes are capable of inducing blood-brain barrier properties in non-neural endothelial cells in vivo.     

Division and differentiation of isolated CNS blast cells in microculture

The mechanism of transformation of the overtly similar cells of the neural plate into the numerous and diverse cell types of the mature vertebrate central nervous system (CNS) can better be understood by studying the clonal development of isolated CNS precursor cells. Here I describe a culture system in which blast cells (cells capable of division) isolated from embryonic day 13.5-14.5 rat forebrain can divide and differentiate into a variety of clonal types. Most clones contain only neurons or glia; 22% contain both neurons and non-neuronal cells. For the division of blast cells, live conditioning cells need to be present indicating that environmental signals influence proliferation. Heterogeneous clones develop in homogeneous culture conditions, so factors intrinsic to the blast cells are probably important in determining the number and type of clonal progeny.     

Purification of a pluripotent neural stem cell from the adult mouse brain

The adult mammalian central nervous system (CNS) contains a population of neural stem cells (NSCs) with properties said to include the generation of non-neural progeny. However, the precise identity, location and potential of the NSC in situ remain unclear. We purified NSCs from the adult mouse brain by flow cytometry, and directly examined the cells' properties. Here we show that one type of NSC, which expresses the protein nestin but only low levels of PNA-binding and HSA proteins, is found in both ependymal and subventricular zones and accounts for about 63% of the total NSC activity. Furthermore, the selective depletion of the population of this stem cell in querkopf mutant mice (which are deficient in production of olfactory neurons) suggests that it acts as a major functional stem cell in vivo. Most freshly isolated NSCs, when co-cultured with a muscle cell line, rapidly differentiated in vitro into myocytes that contain myosin heavy chain (MyHC). This demonstrates that a predominant, functional type of stem cell exists in the periventricular region of the adult brain with the intrinsic ability to generate neural and non-neural cells.     

Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth

The inhibitory activity associated with myelin is a major obstacle for successful axon regeneration in the adult mammalian central nervous system (CNS). In addition to myelin-associated glycoprotein (MAG) and Nogo-A, available evidence suggests the existence of additional inhibitors in CNS myelin. We show here that a glycosylphosphatidylinositol (GPI)-anchored CNS myelin protein, oligodendrocyte-myelin glycoprotein (OMgp), is a potent inhibitor of neurite outgrowth in cultured neurons. Like Nogo-A, OMgp contributes significantly to the inhibitory activity associated with CNS myelin. To further elucidate the mechanisms that mediate this inhibitory activity of OMgp, we screened an expression library and identified the Nogo receptor (NgR) as a high-affinity OMgp-binding protein. Cleavage of NgR and other GPI-linked proteins from the cell surface renders axons of dorsal root ganglia insensitive to OMgp. Introduction of exogenous NgR confers OMgp responsiveness to otherwise insensitive neurons. Thus, OMgp is an important inhibitor of neurite outgrowth that acts through NgR and its associated receptor complex. Interfering with the OMgp/NgR pathway may allow lesioned axons to regenerate after injury in vivo.     

Neurons derived from radial glial cells establish radial units in neocortex

The neocortex of the adult brain consists of neurons and glia that are generated by precursor cells of the embryonic ventricular zone. In general, glia are generated after neurons during development, but radial glia are an exception to this rule. Radial glia are generated before neurogenesis and guide neuronal migration. Radial glia are mitotically active throughout neurogenesis, and disappear or become astrocytes when neuronal migration is complete. Although the lineage relationships of cortical neurons and glia have been explored, the clonal relationship of radial glia to other cortical cells remains unknown. It has been suggested that radial glia may be neuronal precursors, but this has not been demonstrated in vivo. We have used a retroviral vector encoding enhanced green fluorescent protein to label precursor cells in vivo and have examined clones 1-3 days later using morphological, immunohistochemical and electrophysiological techniques. Here we show that clones consist of mitotic radial glia and postmitotic neurons, and that neurons migrate along clonally related radial glia. Time-lapse images show that proliferative radial glia generate neurons. Our results support the concept that a lineage relationship between neurons and proliferative radial glia may underlie the radial organization of neocortex.