Evidence for migration of oligodendrocyte--type-2 astrocyte progenitor cells into the developing rat optic nerve

Formation of myelinated tracts in central nervous system (CNS) regions such as the optic nerve seems to depend on two glial cell types, both of which derive from a common progenitor cell. This oligodendrocyte--type-2 astrocyte (O-2A) progenitor cell gives rise to oligodendrocytes, which produce internodal myelin sheaths, and to type-2 astrocytes, which extend fine processes in the region of the nodal axolemma. The optic nerve also contains a third glial cell, the type-1 astrocyte, which derives from a separate precursor. These three glial cells develop in a fixed sequence over a two-week period: type-1 astrocytes appear at embryonic day 16 (E16), oligodendrocytes at the day of birth (E21 or postnatal day P0), and type-2 astrocytes between P8 and P10. Type-1 astrocytes secrete a potent mitogen which causes expansion of the O-2A progenitor cell population in vitro. Here, we report that dividing O-2A progenitor cells are highly motile and seem to migrate from the brain into the optic nerve, beginning at its chiasmal end. Our results indicate that long-distance migration along the neural axis is characteristic only of progenitors of the O-2A lineage and may serve to distribute these cells to regions of the CNS that will become myelinated. These results also suggest that the intrinsic neuroepithelial cells of the optic stalk may be even more restricted than previously thought, giving rise only to type-1 astrocytes.     

Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture

The various cell types in a multicellular animal differentiate on a predictable schedule but the mechanisms responsible for timing cell differentiation are largely unknown. We have studied a population of bipotential glial (O-2A) progenitor cells in the developing rat optic nerve that gives rise to oligodendrocytes beginning at birth and to type-2 astrocytes beginning in the second postnatal week. Whereas, in vivo, these O-2A progenitor cells proliferate and give rise to postimitotic oligodendrocytes over several weeks, in serum-free (or low-serum) culture they stop dividing prematurely and differentiate into oligodendrocytes within two or three days. The normal timing of oligodendrocyte development can be restored if embryonic optic-nerve cells are cultured in medium conditioned by type-1 astrocytes, the first glial cells to differentiate in the nerve: in this case the progenitor cells continue to proliferate, the first oligodendrocytes appear on the equivalent of the day of birth, and new oligodendrocytes continue to develop over several weeks, just as in vivo. Here we show that platelet-derived growth factor (PDGF) can replace type-1-astrocyte-conditioned medium in restoring the normal timing of oligodendrocyte differentiation in vitro and that anti-PDGF antibodies inhibit this property of the appropriately conditioned medium. We also show that PDGF is present in the developing optic nerve. These findings suggest that type-1-astrocyte-derived PDGF drives the clock that times oligodendrocyte development.     

The oligodendrocyte-type-2 astrocyte cell lineage is specialized for myelination

Astrocytes are one of the most numerous cell types in the vertebrate central nervous system (CNS) and yet their functions are largely unknown. In the rat optic nerve there are two distinct types of astrocyte: type-1 astrocytes develop from one type of precursor cell, and type-2 astrocytes develop from bipotential, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, that initially give rise to oligodendrocytes (which make myelin in the CNS), and then to type-2 astrocytes. Type-1 astrocytes form the glial limiting membrane at the periphery of the optic nerve and are probably responsible for glial scar formation following nerve transection. The functions of type-2 astrocytes, which, like oligodendrocytes, are found mainly in tracts of myelinated axons throughout the CNS, are unknown. In this report we provide evidence that processes from type-2 astrocytes contribute to the structure of nodes of Ranvier, suggesting that the O-2A cell lineage is specialized for constructing myelin sheaths and nodes in the mammalian CNS.     

Differentiation of a bipotential glial progenitor cell in a single cell microculture

Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.     

Proliferating bipotential glial progenitor cells in adult rat optic nerve

We have shown previously that the rat optic nerve contains three types of macroglial cells--oligodendrocytes and two types of astrocytes--which develop as two distinct lineages. Type-1 astrocytes develop from one type of precursor cell beginning at embryonic day 16 (E16), while oligodendrocytes and then type-2 astrocytes develop from a common, bipotential progenitor cell beginning at birth (E21) and postnatal days 7-10 (P7-10), respectively. Here we report that proliferating bipotential oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells are present in the adult rat optic nerve, raising the possibility that these cells are produced continually from self-renewing stem cells throughout life.     

A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium

We have identified a cell type in 7-day-old rat optic nerve that differentiates into a fibrous astrocyte if cultured in the presence of fetal calf serum and into an oligodendrocyte if cultured in the absence of serum. In certain culture conditions some of these cells acquire a mixed phenotype, displaying properties of both astrocytes and oligodendrocytes. These observations suggest that fibrous astrocytes and oligodendrocytes develop from a common progenitor cell and provide a striking example of developmental plasticity and environmental influence in the differentiation of CNS glial cells.     

Facilitation of voltage-gated ion channels in frog neuroglia by nerve impulses

The functions of glial cells in the nervous system are not well defined, with the exception of myelin production by oligodendrocytes, uptake of amino-acid synaptic transmitters, and a contribution to extracellular potassium homeostasis. Neuroglia have receptors for neurotransmitters which may be involved in neuron-glia interactions. Recent studies have demonstrated voltage-gated ion channels in glial membranes. In a study of the optic nerve of the frog, small areas of the surface were examined with the loose patch-clamp method, and voltage-gated Na+ and K+ channels, presumably located in the membranes of the astrocytes forming the glia limitans, were identified. We now report that nerve impulses in the axons of the frog optic nerve transiently alter the properties of the voltage-dependent membrane channels of the surface glial cells (astrocytes), a demonstration of a new form of neuron-glia interaction.     

Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids

L-GLUTAMATE and L-aspartate are thought to have a widespread function as synaptic transmitters in the mammalian central nervous system and there are at least three types of neuronal glutamate receptors, which can be activated by the selective agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate. Recent experiments indicate that glutamate receptors also occur in astrocytes. We have used patch-clamp methods to determine whether one type of macroglial cell, the type-2 astrocyte, possesses glutamate receptors, as previously proposed from neurochemical studies. We find that glutamate and related amino acids can evoke whole-cell and single-channel currents in type-2 astrocytes from rat cerebellum. Although these cells are found mainly in white matter, where neurotransmission does not occur, their processes are closely associated with axons at nodes of Ranvier, suggesting that such receptors are involved in neuronal-glial signalling at the node. Our experiments show that glial cells possess quisqualate- and kainate-receptor channels but lack receptors for NMDA. Interestingly, these glutamate channels exhibit multiple conductance levels that are similar in amplitude to the neuronal glutamate channels.     

Local generation of glia is a major astrocyte source in postnatal cortex

Glial cells constitute nearly 50% of the cells in the human brain. Astrocytes, which make up the largest glial population, are crucial to the regulation of synaptic connectivity during postnatal development. Because defects in astrocyte generation are associated with severe neurological disorders such as brain tumours, it is important to understand how astrocytes are produced. Astrocytes reportedly arise from two sources: radial glia in the ventricular zone and progenitors in the subventricular zone, with the contribution from each region shifting with time. During the first three weeks of postnatal development, the glial cell population, which contains predominantly astrocytes, expands 6-8-fold in the rodent brain. Little is known about the mechanisms underlying this expansion. Here we show that a major source of glia in the postnatal cortex in mice is the local proliferation of differentiated astrocytes. Unlike glial progenitors in the subventricular zone, differentiated astrocytes undergo symmetric division, and their progeny integrate functionally into the existing glial network as mature astrocytes that form endfeet with blood vessels, couple electrically to neighbouring astrocytes, and take up glutamate after neuronal activity.     

Effect of ciliary neurotrophic factor on the reactive astrogliosis of cultured astrocytes from newborn rat brain

The effect of ciliary neurotrophic factor (CNTF) on reactive astrogliosis was studied on a mechanical scratch model of the confluent astrocytic cultures from newborn rat brain. Following injury, the astrocytes at the edge of the injured area displayed a typical process of the reactive astrogliosis. This process included apparently hyperplastic change and significantly increased GFAP expression of the flat astrocytes, and migration to the injured area of the O-2A progenitor cells and their differentiation into process-bearing astrocytes. Exogenous CNTF applied to the cell cultures significantly promoted the hyperplasia and GFAP expression of the flat astrocytes. The results suggest that CNTF can enhance the reactive astrogliosis in the injured area.     

Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity

Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon-glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon-glia metabolic coupling serves a physiological function.     

Glial cells express N-CAM/D2-CAM-like polypeptides in vitro

The joining together of neurites to form fascicles and the growth of axons along glial surfaces during early development suggest that neurone-neurone and neurone-glial adhesion interactions are of considerable importance for defining nerve tracts. In vitro studies have indicated that adhesion between neurones involves a glycoprotein that has been independently studied under the names of N-CAM (for neural cell adhesion molecule), D2-CAM and BSP-2 (refs 10, 11). As N-CAM/D2-CAM appears to be a homophilic ligand that binds to N-CAM/D2-CAM polypeptide on adjacent cells, this glycoprotein is potentially important in adhesion interactions between any two N-CAM/D2-CAM-expressing cells. While it has been suggested that neurone-glial adhesion involves molecules other than N-CAM/D2-CAM, it is known that N-CAM/D2-CAM antigenic determinants are expressed by glial cells in vivo and that injection of anti-N-CAM antibodies into the eye-cup of chick embryos disrupts normal patterns of neuritic apposition to glial endfeet in the developing optic stalk. Do the molecules expressed by glia share restricted antigenic determinants, or binding domains, with N-CAM/D2-CAM, or are N-CAM/D2-CAM polypeptides expressed by glia? Here we present immunocytochemical evidence which suggests that all classes of macroglia express N-CAM/D2-CAM antigenic determinants on their surfaces and immunochemical analyses which indicate that the molecules expressed by purified astrocytes are closely similar, or identical, to at least some forms of N-CAM/D2-CAM obtained from whole brain or purified neurones. However, our results also suggest that different N-CAM/D2-CAM polypeptides may be separately expressed by neurones and astrocytes.     

Stimulation of oligodendroglial proliferation and maturation by interleukin-2

There exists considerable evidence that the growth of glial cells can be influenced by T-cell-derived lymphokines and monokines. Astrocytes proliferate in the presence of mitogen- or antigen-stimulated T-cell supernatants, supernatants from human T-lymphotropic virus (HTLV)-transformed T cells, and purified human interleukin-1 (IL-1; ref. 4). Oligodendrocytes proliferate and differentiate when incubated with supernatants from mitogen-activated or HTLV-transformed T cells. In addition, we have recently purified a T-cell-derived lymphokine of relative molecular mass 30,000, termed glial growth promoting factor (GGPF), which specifically stimulates the proliferation of oligodendrocytes. The traditional role of interleukins 1 and 2 is in the initiation, propagation and regulation of the immune response. IL-1, released by a variety of cells including monocytes, stimulates T cells to produce IL-2; IL-2 in turn induces the expansion of T cells that is critical for immune responsiveness. Recently, IL-2 has been shown to induce B-cell proliferation and immunoglobulin secretion, indicating that its action is not restricted to T cells. We now report that recombinant human IL-2 influences the growth of glial cells--specifically, the proliferation and differentiation of oligodendrocytes. IL-2 may have a role in the inflammatory neural lesions of multiple sclerosis patients and in the growth of brain glia during injury or disease.     

Differential Notch signalling distinguishes neural stem cells from intermediate progenitors

During brain development, neurons and glia are generated from a germinal zone containing both neural stem cells (NSCs) and more limited intermediate neural progenitors (INPs). The signalling events that distinguish between these two proliferative neural cell types remain poorly understood. The Notch signalling pathway is known to maintain NSC character and to inhibit neurogenesis, although little is known about the role of Notch signalling in INPs. Here we show that both NSCs and INPs respond to Notch receptor activation, but that NSCs signal through the canonical Notch effector C-promoter binding factor 1 (CBF1), whereas INPs have attenuated CBF1 signalling. Furthermore, whereas knockdown of CBF1 promotes the conversion of NSCs to INPs, activation of CBF1 is insufficient to convert INPs back to NSCs. Using both transgenic and transient in vivo reporter assays we show that NSCs and INPs coexist in the telencephalic ventricular zone and that they can be prospectively separated on the basis of CBF1 activity. Furthermore, using in vivo transplantation we show that whereas NSCs generate neurons, astrocytes and oligodendrocytes at similar frequencies, INPs are predominantly neurogenic. Together with previous work on haematopoietic stem cells, this study suggests that the use or blockade of the CBF1 cascade downstream of Notch is a general feature distinguishing stem cells from more limited progenitors in a variety of tissues.     

Guidance of optic nerve fibres by N-cadherin adhesion molecules

The dendritic branches (neurites) of developing neurons migrate along specific pathways to reach their targets. It has been suggested that this migration is guided by factors present on the surface of other neurons or glial cells. The molecular nature of such factors, however, remains to be elucidated. N-cadherin is a cell-surface glycoprotein which belongs to the cadherin family of cell-cell adhesion molecules. This adhesion molecule is expressed in various neuronal cells as well as in glial cells of the central and peripheral nervous systems in vertebrate embryos and recent immunological studies suggested that N-cadherin may play a role in guiding the migration of neurites on myotubes or astrocytes. To further examine this possibility, we used a molecular-genetic approach; that is, we examined the outgrowth of chicken embryonic optic axons on monolayer cultures of Neuro 2a or L cells transfected with the complementary DNA encoding chicken N-cadherin. The data indicate that N-cadherin is used as a guide molecule for the migration of optic axons on cell surfaces.     

Clustering of voltage-dependent sodium channels on axons depends on Schwann cell contact.

In myelinated nerves, segregation of voltage-dependent sodium channels to nodes of Ranvier is crucial for saltatory conduction along axons. As sodium channels associate and colocalize with ankyrin at nodes of Ranvier, one possibility is that sodium channels are recruited and immobilized at axonal sites which are specified by the subaxolemmal cytoskeleton, independent of glial cell contact. Alternatively, segregation of channels at distinct sites along the axon may depend on glial cell contact. To resolve this question, we have examined the distribution of sodium channels, ankyrin and spectrin in myelination-competent cocultures of sensory neurons and Schwann cells by immunofluorescence, using sodium channel-, ankyrin- and spectrin-specific antibodies. In the absence of Schwann cells, sodium channels, ankyrin and spectrin are homogeneously distributed on sensory axons. When Schwann cells are introduced into these cultures, the distribution of sodium channels dramatically changes so that channel clusters on axons are abundant, but ankyrin and spectrin remain homogeneously distributed. Addition of latex beads or Schwann cell membranes does not induce channel clustering. Our results suggest that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to organize and maintain channel distribution during differentiation of myelinated axons.     

Distinct contribution of stem and progenitor cells to epidermal maintenance

The skin interfollicular epidermis (IFE) is the first barrier against the external environment and its maintenance is critical for survival. Two seemingly opposite theories have been proposed to explain IFE homeostasis. One posits that IFE is maintained by long-lived slow-cycling stem cells that give rise to transit-amplifying cell progeny, whereas the other suggests that homeostasis is achieved by a single committed progenitor population that balances stochastic fate. Here we probe the cellular heterogeneity within the IFE using two different inducible Cre recombinase–oestrogen receptor constructs targeting IFE progenitors in mice. Quantitative analysis of clonal fate data and proliferation dynamics demonstrate the existence of two distinct proliferative cell compartments arranged in a hierarchy involving slow-cycling stem cells and committed progenitor cells. After wounding, only stem cells contribute substantially to the repair and long-term regeneration of the tissue, whereas committed progenitor cells make a limited contribution.     

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease.     

De novo cardiomyocytes from within the activated adult heart after injury

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.