The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen.

The carboxy-terminal tail of nucleoplasmin, which specifies entry into the cell nucleus, contains four short sequences that are similar to previously identified nuclear location sequences. We show that none of these is able to locate chicken muscle pyruvate kinase to the cell nucleus. Deletion analysis was used to determine the limits of a nuclear location sequence and indicated that a 14-amino acid segment (RPAATKKAGQAKKK) should function as a minimal nuclear location sequence. When tested directly, however, this sequence was unable to locate pyruvate kinase to the cell nucleus. Restoration of three amino acids of nucleoplasmin sequence at either end of this sequence generated sequences that were able to locate pyruvate kinase to the cell nucleus. The 14-amino acid proposed minimal nuclear location sequence is present in the functional sequences, AVKRPAATKKAGQAKKK, RPAATKKAGQAKKKKLD, and the sequence AVKRPAATKKAGQAKKKKLD, which has additional amino acids at both ends. The minimal sequence element is therefore necessary but not sufficient for transport into the cell nucleus. This unusual feature of the nucleoplasmin nuclear location sequence suggests ways in which it could interact with the nuclear transport mechanism.

PDGF and intracellular signaling in the timing of oligodendrocyte differentiation.

In the rat optic nerve, bipotential O-2A progenitor cells give rise to oligodendrocytes and type 2 astrocytes on a precise schedule. Previous studies suggest that PDGF plays an important part in timing oligodendrocyte development by stimulating O-2A progenitor cells to proliferate until they become mitotically unresponsive to PDGF, stop dividing, and differentiate automatically into oligodendrocytes. Since the loss of mitotic responsiveness to PDGF has been shown not to be due to a loss of PDGF receptors, we have now examined the possibility that the unresponsiveness results from an uncoupling of these receptors from early intracellular signaling pathways. We show that (a) although PDGF does not stimulate newly formed oligodendrocytes to synthesize DNA, it induces an increase in cytosolic Ca2+ in these cells; (b) a combination of a Ca2+ ionophore plus a phorbol ester mimics the effect of PDGF, both in stimulating O-2A progenitor cell division and in reconstituting the normal timing of oligodendrocyte differentiation in culture; and (c) the same combination of drugs does not stimulate newly formed oligodendrocytes to proliferate, even in the presence of PDGF or dibutyryl cAMP. The most parsimonious explanation for these results is that O-2A progenitor cells become mitotically unresponsive to PDGF because the intracellular signaling pathways from the PDGF receptor to the nucleus are blocked downstream from the receptor and some of the early events that are triggered by receptor activation.

Ventral neurogenesis and the neuron-glial switch.

In the developing spinal cord, neuroepithelial precursors at different positions along the dorsal-ventral axis generate distinct neuronal and glial subtypes. For example, one group of ventral precursors generates neurons followed by oligodendrocytes. A spate of recent articles, including several in this issue of Neuron, are devoted to the mechanisms governing neuronal and glial subtype specification in the ventral cord. We review these studies and discuss the nature of the ventral neuron-oligodendrocyte switch.

Dorsal spinal cord neuroepithelium generates astrocytes but not oligodendrocytes.

There is evidence that oligodendrocytes in the spinal cord are derived from a restricted part of the ventricular zone near the floor plate. An alternative view is that oligodendrocytes are generated from all parts of the ventricular zone. We reinvestigated glial origins by constructing chick-quail chimeras in which dorsal or ventral segments of the embryonic chick neural tube were replaced with equivalent segments of quail neural tube. Ventral grafts gave rise to both oligodendrocytes and astrocytes. In contrast, dorsal grafts produced astrocytes but not oligodendrocytes. In mixed cultures of ventral and dorsal cells, only ventral cells generated oligodendrocytes, whereas both ventral and dorsal cells generated astrocytes. Therefore, oligodendrocytes are derived specifically from ventral neuroepithelium, and astrocytes from both dorsal and ventral.

Embryonic expression of myelin genes: evidence for a focal source of oligodendrocyte precursors in the ventricular zone of the neural tube.

2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) is an abundant protein of myelinating oligodendrocytes. We report that one of the alternatively spliced CNP mRNAs is also expressed in cultured oligodendrocyte progenitor cells. In situ hybridization revealed a thin longitudinal column of CNP-positive cells in the ventral ventricular zone of the embryonic day 14 rat spinal cord, coincident in time and space with cells that express the platelet-derived growth factor alpha receptor, another putative marker of the oligodendrocyte lineage. These data support the hypothesis that the oligodendrocyte lineage originates at a discrete location in the ventral ventricular zone of the embryonic day 14 rat spinal cord. We further report that transcripts encoding the myelin proteolipid protein (PLP/DM-20) are expressed in an unidentified population of neural progenitors in the ventricular zone abutting the floor plate. Our results support the idea that the ventricular zone is a mosaic of specialized progenitor cells.

PDGF mediates a neuron-astrocyte interaction in the developing retina.

Astrocytes invade the developing retina from the optic nerve head, over the axons of retinal ganglion cells (RGCs). RGCs express the platelet-derived growth factor A-chain (PDGF-A) and retinal astrocytes the PDGF alpha-receptor (PDGFR alpha), suggesting that PDGF mediates a paracrine interaction between these cells. To test this, we inhibited PDGF signaling in the eye with a neutralizing anti-PDGFR alpha antibody or a soluble extracellular fragment of PDGFR alpha. These treatments inhibited development of the astrocyte network. We also generated transgenic mice that overexpress PDGF-A in RGCs. This resulted in hyperproliferation of astrocytes, which in turn induced excessive vasculogenesis. Thus, PDGF appears to be a link in the chain of cell-cell interactions responsible for matching numbers of neurons, astrocytes, and blood vessels during retinal development.

Oligodendrocyte population dynamics and the role of PDGF in vivo.

Oligodendrocyte progenitors originate near the floor plate of the spinal cord, then proliferate and migrate throughout the cord before giving rise to oligodendrocytes. Progenitor cell proliferation stops before birth because the cell cycle slows down, linked to an increase in differentiation and death. Experiments with transgenic mice show that platelet-derived growth factor (PDGF) drives progenitor cell division and suggest that slowing of and exit from the cycle reflects a decline in PDGF signaling. Overexpressing PDGF induces hyperproliferation of progenitor cells and excessive, ectopic production of oligodendrocytes. However, the superfluous oligodendrocytes die at an immature stage of differentiation, leaving a normal complement of myelin-forming cells. Therefore, cell survival controls override proliferation controls for determining the final number and distribution of mature oligodendrocytes.

Platelet-derived growth factor is constitutively secreted from neuronal cell bodies but not from axons.

Neurons synthesise and secrete many growth and survival factors but it is not usually clear whether they are released locally at the cell body or further afield from axons or axon terminals. Without this information, we cannot predict the site(s) of action or the biological functions of many neuron-derived factors. For example, can neuronal platelet-derived growth factor (PDGF) be secreted from axons and reach glial cells in nerve-fibre (white-matter) tracts? To address this question, we expressed PDGF-A in retinal ganglion neurons in transgenic mice and tested for release of PDGF from cell bodies in the retina and from axons in the optic nerve. In both the retina and optic nerve, there are glial cells that express PDGF receptor alpha (PDGFR alpha) [1] and divide in response to PDGF [2-5], so we could detect functional PDGF indirectly through the mitogenic response of glia at both locations. Expressing PDGF-A in neurons under the control of the neuron-specific enolase promoter (NSE-PDGF-A) resulted in a striking hyperplasia of retinal astrocytes, demonstrating that PDGF is secreted from the cell bodies of neurons in the retina [4]. In contrast, glial proliferation in the optic nerve was unaffected, indicating that PDGF is not released from axons. When PDGF was expressed directly in the optic nerve under the control of an astrocyte-specific promoter (GFAP-PDGF-A), oligodendrocyte progenitors hyperproliferated, resulting in a hypertrophic optic nerve. We conclude that PDGF is constitutively secreted from neuronal cell bodies in vivo, but not from axons in white-matter tracts.

Control of progenitor cell number by mitogen supply and demand.

BACKGROUND: Much is known about how cell proliferation is controlled at the single cell level, but much less about the control of cell numbers in developing populations. Cell number might be determined by an intracellular division limiter or, alternatively, by the availability of mitogens or other factors outside the cell. We investigated the relative importance of intracellular and extracellular controls for one well-defined population of neural precursor cells, namely the glial progenitors that give rise to oligodendrocytes in the mouse spinal cord. RESULTS: We found by cumulative BrdU labeling in vivo that the progenitor cell division cycle slows down markedly as their numbers increase during embryogenesis. When cultured in saturating PDGF, the main mitogen for these cells, their cell cycle accelerated and was independent of their prior rate of division in vivo. This shows that mitogens are limiting in vivo, and suggests that division normally slows down because the PDGF concentration declines. In PDGF-transgenic mice, cell number was proportional to the PDGF supply and apparently unsaturable; at ten times the normal rate of supply, cell number was still increasing but the animals were no longer viable. CONCLUSIONS: Progenitor cell proliferation in the embryo is limited by environmental factors, not a cell-intrinsic mechanism. The linear relationship between PDGF supply and final cell number strongly suggests that cells deplete the mitogenic activity in their environment at a rate proportional to the total number of cells. The cells might simply consume the available PDGF or they might secrete autocrine inhibitors, or both.

Extensive mutagenesis of the nuclear location signal of simian virus 40 large-T antigen.

Site-directed mutagenesis was used to change Lys-128 of the simian virus 40 large-T nuclear location signal to Met, Ile, Arg, Gln, Asn, Leu, or His. Except for the large-T antigen of the Arg mutation, which was present in cytoplasmic and nuclear compartments, the resultant proteins were unable to enter the nucleus. By contrast, mutations at other sites within the signal were generally less severe in their effect. In some cases (Lys-128 to Gln, Asn, and His), the apparently cytoplasmic variants were able to support limited plasmid DNA replication, suggesting that low levels of large-T antigen undetectable by immunofluorescence were present in the nucleus. Such mutants did not support viral DNA replication. We conclude that there is a strong requirement for a basic residue at position 128 in the large-T nuclear location signal, with Lys the preferred residue.

The sacral autonomic outflow is sympathetic.

A kinship between cranial and pelvic visceral nerves of vertebrates has been accepted for a century. Accordingly, sacral preganglionic neurons are considered parasympathetic, as are their targets in the pelvic ganglia that prominently control rectal, bladder, and genital functions. Here, we uncover 15 phenotypic and ontogenetic features that distinguish pre- and postganglionic neurons of the cranial parasympathetic outflow from those of the thoracolumbar sympathetic outflow in mice. By every single one, the sacral outflow is indistinguishable from the thoracolumbar outflow. Thus, the parasympathetic nervous system receives input from cranial nerves exclusively and the sympathetic nervous system from spinal nerves, thoracic to sacral inclusively. This simplified, bipartite architecture offers a new framework to understand pelvic neurophysiology as well as development and evolution of the autonomic nervous system.

Schwann Cells Secrete a PDGF-like Factor: Evidence for an Autocrine Growth Mechanism involving PDGF.

We have investigated the influence of platelet-derived growth factor (PDGF) in peripheral nervous system gliogenesis using two types of Schwann cell cultures. Short-term Schwann cell cultures grow very slowly, but when maintained in culture for several months the division rate of some cells increases, and cell lines can be established. We show that Schwann cells in both short- and long-term culture possess PDGF receptors and synthesize DNA in response to PDGF. Competitive binding experiments show that Schwann cells express mainly PDGF beta-receptors and respond better to PDGF-BB than to PDGF-AA. Conditioned media from short- and long-term Schwann cell cultures contain PDGF-like mitogenic activity, and anti-PDGF immunoglobin partially inhibits DNA synthesis in long-term Schwann cell cultures. Antibody neutralization experiments and Northern blot analyses both indicate that the predominant PDGF isoform in these cultures is PDGF-BB. PDGF-like activity is also detected in extracts of rat sciatic nerve. Taken together, these results suggest that PDGF-BB may stimulate Schwann cell proliferation in an autocrine manner during normal development.

Meningococcal meningitis in northern Ghana: epidemiology and control measures.

Hospitalized meningococcal meningitis patients in northeastern Ghana during 1972-1973 were studied to provide baseline information about case clustering and age-specific attack rates to guide meningitis control programs. In 1973, group A meningococci were prevalent and 7% of isolates were sulfadiazine-resistant. In contrast to the age distribution of meningococcal meningitis in North and South America, peak attack rates occurred in 10- to 14-year-old Ghanaians. A mass immunization campaign using group A polysaccharide vaccine in heavily populated areas of the Bawku and Nalgerigu districts is recommended.

Oligodendrocyte development in the spinal cord and telencephalon: common themes and new perspectives.

There are clear parallels between oligodendrocyte development in the spinal cord and forebrain. However, there is new evidence that in both of these regions oligodendrocyte lineage development may be more complex than we earlier thought. This stems from the recent identification of three new transcription factor genes, Olig1, Olig2 and Sox10, that are expressed from the early stages of oligodendrocyte lineage development. In this article, we highlight the common themes underlying specification and early development of oligodendrocytes in the spinal cord and telencephalon. Then, we discuss recent studies of Sox10 and the Olig genes and their implications for oligodendrocyte specification. We conclude that although the mechanisms of oligodendrogenesis appear to be fundamentally similar at different rostro-caudal levels of the neuraxis, there are still many unanswered questions about the details of oligodendrocyte specification.

Oligodendrocyte progenitors: adult stem cells of the central nervous system?

Oligodendrocyte progenitors (OPs) are a major proliferating cell population within the adult CNS. In response to myelin loss or increasing demand, OPs have the capacity to differentiate into mature, myelinating oligodendrocytes. The name 'oligodendrocyte progenitor' suggests restriction to the oligodendrocyte cell lineage. However, with growing evidence of the lineage plasticity of OPs both in vitro and in vivo, we discuss whether they have potential beyond that expected of dedicated progenitor cells, and hence may justify categorization as adult stem cells.

The alternative-splice isoforms of the PDGF A-chain differ in their ability to associate with the extracellular matrix and to bind heparin in vitro.

Platelet-derived growth factor (PDGF) consists of disulfide-linked homo- or heterodimers of A and B chains. mRNA encoding the A chain (PDGF-A) occurs in two versions that differ by the presence or absence of a single short exon. These alternatively-spliced mRNAs encode polypeptides that differ in length by fifteen amino acids. The longer isoform (PDGF-AL) possesses a highly basic carboxy-terminal extension that is responsible for retaining PDGF-AL homodomers at the cell surface after secretion, while homodimers of the shorter isoform (PDGF-AS) are released into the extracellular medium. We have investigated the mechanism by which PDGF-AL remains in association with the cells that produce it. We expressed epitope-tagged versions of PDGF-AL and PDGF-AS in Cos cells and compared their intra- and extracellular distributions by immunofluorescence microscopy. PDGF-AL, but not PDGF-AS, was detected on and around cells in a diffuse pattern suggesting associated with the extracellular matrix (ECM). Metabolically radiolabelled PDGF-AL, but not PDGF-AS, could be eluted from ECM preparations by washing in high salt. Moreover, PDGF-AL bound reversibly to heparin-Sepharose in vitro at physiological salt concentrations, eluting at a salt concentration around 0.5 M. PDGF-AS did not bind to heparin under the same conditions. Thus, PDGF dimers that contain PDGF-AL may remain immobilized near the cells that secrete them by virtue of binding to heparin-like constituents of the ECM.

A singularity of PDGF alpha-receptor expression in the dorsoventral axis of the neural tube may define the origin of the oligodendrocyte lineage.

During rat embryogenesis, PDGF alpha receptor (PDGF-alpha R) mRNA is expressed in the ventral half of the spinal cord in two longitudinal columns, one each side of the central canal. Initially, these columns are only two cells wide but the cells subsequently appear to proliferate and disseminate throughout the spinal cord. Our previous studies of PDGF-alpha R expression in the developing CNS suggested that PDGF-alpha R may be a useful marker of the oligodendrocyte lineage in situ. The data presented here complement those studies and lead us to propose that the earliest oligodendrocyte precursors in the spinal cord originate in a very restricted region of the ventricular zone during a brief window of time around embryonic day 14 (E14). In the embryonic brain, migrating PDGF-alpha R+ cells appear to originate in a localized germinal zone in the ventral diencephalon (beneath the foramen of Monro). Our data demonstrate that gene expression and cell fate can be regulated with exquisite spatial resolution along the dorsoventral axis of the mammalian neural tube.